One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase.

PubMed

Acevedo, Juan Pablo; Reetz, Manfred T; Asenjo, Juan A; Parra, Loreto P

2017-05-01

Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry. This paper describes the recombinant expression and characterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), and the use of a novel one step combined directed evolution technique that comprises saturation mutagenesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-L enzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2-17, recombinantly expressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulations using a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions and residues, which are considered to be the possible structural elements that define the thermolability of this enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutations in some of the flexible regions and residues identified. Twelve positive mutant clones were found to improve the T 50 15 value of the enzyme, in some cases without affecting the activity at 25°C. The best mutant showed a 4.3°C increase in its T 50 15 . The efficiency of the directed evolution approach can also be expected to work in the protein engineering of stereoselectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution.

PubMed

Renata, Hans; Wang, Z Jane; Arnold, Frances H

2015-03-09

High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature's known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been exploited for chemical synthesis, with an emphasis on reactions that do not have natural counterparts. Non-natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non-native catalytic functions that may provide future opportunities for the expansion of the enzyme universe. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Strategies for design of improved biocatalysts for industrial applications.

PubMed

Madhavan, Aravind; Sindhu, Raveendran; Binod, Parameswaran; Sukumaran, Rajeev K; Pandey, Ashok

2017-12-01

Biocatalysts are creating increased interest among researchers due to their unique properties. Several enzymes are efficiently produced by microorganisms. However, the use of natural enzymes as biocatalysts is hindered by low catalytic efficiency and stability during various industrial processes. Many advanced enzyme technologies have been developed to reshape the existing natural enzymes to reduce these limitations and prospecting of novel enzymes. Frequently used enzyme technologies include protein engineering by directed evolution, immobilisation techniques, metagenomics etc. This review summarizes recent and emerging advancements in the area of enzyme technologies for the development of novel biocatalysts and further discusses the future directions in this field. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expanding the Nucleotide and Sugar 1-Phosphate Promiscuity of Nucleotidyltransferase RmlA via Directed Evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moretti, Rocco; Chang, Aram; Peltier-Pain, Pauline

2012-03-15

Directed evolution is a valuable technique to improve enzyme activity in the absence of a priori structural knowledge, which can be typically enhanced via structure-guided strategies. In this study, a combination of both whole-gene error-prone polymerase chain reaction and site-saturation mutagenesis enabled the rapid identification of mutations that improved RmlA activity toward non-native substrates. These mutations have been shown to improve activities over 10-fold for several targeted substrates, including non-native pyrimidine- and purine-based NTPs as well as non-native d- and l-sugars (both a- and b-isomers). This study highlights the first broadly applicable high throughput sugar-1-phosphate nucleotidyltransferase screen and the firstmore » proof of concept for the directed evolution of this enzyme class toward the identification of uniquely permissive RmlA variants.« less

Assessing directed evolution methods for the generation of biosynthetic enzymes with potential in drug biosynthesis

PubMed Central

Nannemann, David P; Birmingham, William R; Scism, Robert A; Bachmann, Brian O

2011-01-01

To address the synthesis of increasingly structurally diverse small-molecule drugs, methods for the generation of efficient and selective biological catalysts are becoming increasingly important. ‘Directed evolution’ is an umbrella term referring to a variety of methods for improving or altering the function of enzymes using a nature-inspired twofold strategy of mutagenesis followed by selection. This article provides an objective assessment of the effectiveness of directed evolution campaigns in generating enzymes with improved catalytic parameters for new substrates from the last decade, excluding studies that aimed to select for only improved physical properties and those that lack kinetic characterization. An analysis of the trends of methodologies and their success rates from 81 qualifying examples in the literature reveals the average fold improvement for kcat (or Vmax), Km and kcat/Km to be 366-, 12- and 2548-fold, respectively, whereas the median fold improvements are 5.4, 3 and 15.6. Further analysis by enzyme class, library-generation methodology and screening methodology explores relationships between successful campaigns and the methodologies employed. PMID:21644826

Compartmentalized self-replication: a novel method for the directed evolution of polymerases and other enzymes.

PubMed

Ghadessy, Farid J; Holliger, Philipp

2007-01-01

Compartmentalized self-replication (CSR) is a novel method for the directed evolution of enzymes and, in particular, polymerases. In its simplest form, CSR consists of a simple feedback loop involving a polymerase that replicates only its own encoding gene (self-replication). Self-replication occurs in discrete, spatially separate, noncommunicating compartments formed by a heat-stable water-in-oil emulsion. Compartmentalization ensures the linkage of phenotype and genotype (i.e., it ensures that each polymerase replicates only its own encoding gene to the exclusion of those in the other compartments). As a result, adaptive gains by the polymerase directly (and proportionally) translate into genetic amplification of the encoding polymerase gene. CSR has proven to be a useful strategy for the directed evolution of polymerases directly from diverse repertoires of polymerase genes. In this chapter, we describe some of the CSR protocols used successfully to evolve variants of T. aquaticus Pol I (Taq) polymerase with novel and useful properties, such as increased thermostability or resistance to the potent inhibitor, heparin, from a repertoire of randomly mutated Taq polymerase genes.

A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

PubMed

Belsare, Ketaki D; Andorfer, Mary C; Cardenas, Frida S; Chael, Julia R; Park, Hyun June; Lewis, Jared C

2017-03-17

Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450 BM3 , pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.

Expanding the metabolic engineering toolbox with directed evolution.

PubMed

Abatemarco, Joseph; Hill, Andrew; Alper, Hal S

2013-12-01

Cellular systems can be engineered into factories that produce high-value chemicals from renewable feedstock. Such an approach requires an expanded toolbox for metabolic engineering. Recently, protein engineering and directed evolution strategies have started to play a growing and critical role within metabolic engineering. This review focuses on the various ways in which directed evolution can be applied in conjunction with metabolic engineering to improve product yields. Specifically, we discuss the application of directed evolution on both catalytic and non-catalytic traits of enzymes, on regulatory elements, and on whole genomes in a metabolic engineering context. We demonstrate how the goals of metabolic pathway engineering can be achieved in part through evolving cellular parts as opposed to traditional approaches that rely on gene overexpression and deletion. Finally, we discuss the current limitations in screening technology that hinder the full implementation of a metabolic pathway-directed evolution approach. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

PubMed Central

Reetz, Manfred T.

2004-01-01

A fundamentally new approach to asymmetric catalysis in organic chemistry is described based on the in vitro evolution of enantioselective enzymes. It comprises the appropriate combination of gene mutagenesis and expression coupled with an efficient high-throughput screening system for evaluating enantioselectivity (enantiomeric excess assay). Several such cycles lead to a “Darwinistic” process, which is independent of any knowledge concerning the structure or the mechanism of the enzyme being evolved. The challenge is to choose the optimal mutagenesis methods to navigate efficiently in protein sequence space. As a first example, the combination of error-prone mutagenesis, saturation mutagenesis, and DNA-shuffling led to a dramatic enhancement of enantioselectivity of a lipase acting as a catalyst in the kinetic resolution of a chiral ester. Mutations at positions remote from the catalytically active center were identified, a surprising finding, which was explained on the basis of a novel relay mechanism. The scope and limitations of the method are discussed, including the prospect of directed evolution of stereoselective hybrid catalysts composed of robust protein hosts in which transition metal centers have been implanted. PMID:15079053

Biocatalysts for the pharmaceutical industry created by structure-guided directed evolution of stereoselective enzymes.

PubMed

Li, Guangyue; Wang, Jian-Bo; Reetz, Manfred T

2018-04-01

Enzymes have been used for a long time as catalysts in the asymmetric synthesis of chiral intermediates needed in the production of therapeutic drugs. However, this alternative to man-made catalysts has suffered traditionally from distinct limitations, namely the often observed wrong or insufficient enantio- and/or regioselectivity, low activity, narrow substrate range, and insufficient thermostability. With the advent of directed evolution, these problems can be generally solved. The challenge is to develop and apply the most efficient mutagenesis methods which lead to highest-quality mutant libraries requiring minimal screening. Structure-guided saturation mutagenesis and its iterative form have emerged as the method of choice for evolving stereo- and regioselective mutant enzymes needed in the asymmetric synthesis of chiral intermediates. The number of (industrial) applications in the preparation of chiral pharmaceuticals is rapidly increasing. This review features and analyzes typical case studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

The nature of chemical innovation: new enzymes by evolution.

PubMed

Arnold, Frances H

2015-11-01

I describe how we direct the evolution of non-natural enzyme activities, using chemical intuition and information on structure and mechanism to guide us to the most promising reaction/enzyme systems. With synthetic reagents to generate new reactive intermediates and just a few amino acid substitutions to tune the active site, a cytochrome P450 can catalyze a variety of carbene and nitrene transfer reactions. The cyclopropanation, N-H insertion, C-H amination, sulfimidation, and aziridination reactions now demonstrated are all well known in chemical catalysis but have no counterparts in nature. The new enzymes are fully genetically encoded, assemble and function inside of cells, and can be optimized for different substrates, activities, and selectivities. We are learning how to use nature's innovation mechanisms to marry some of the synthetic chemists' favorite transformations with the exquisite selectivity and tunability of enzymes.

Engineering Biosynthesis of Non-ribosomal Peptides and Polyketides by Directed Evolution.

PubMed

Rui, Zhe; Zhang, Wenjun

2016-01-01

Non-ribosomal peptides (NRPs) and polyketides (PKs) play key roles in pharmaceutical industry due to their promising biological activities. The structural complexity of NRPs and PKs, however, creates significant synthetic challenges for producing these natural products and their analogues by purely chemical means. Alternatively, difficult syntheses can be achieved by using biosynthetic enzymes with improved efficiency and altered selectivity that are acquired from directed evolution. Key to the successful directed evolution is the methodology of screening/selection. This review summarizes the screening/selection strategies that have been employed to improve or modify the functions of non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), in the hope of triggering the wide adoption of the directed evolution approaches in the engineered biosynthesis of NRPs and PKs for drug discovery.

Selection platforms for directed evolution in synthetic biology

PubMed Central

Tizei, Pedro A.G.; Csibra, Eszter; Torres, Leticia; Pinheiro, Vitor B.

2016-01-01

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules–gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function–be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code. PMID:27528765

Selection platforms for directed evolution in synthetic biology.

PubMed

Tizei, Pedro A G; Csibra, Eszter; Torres, Leticia; Pinheiro, Vitor B

2016-08-15

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules-gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function-be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code. © 2016 The Author(s).

Enzyme Recruitment and Its Role in Metabolic Expansion

PubMed Central

2015-01-01

Although more than 109 years have passed since the existence of the last universal common ancestor, proteins have yet to reach the limits of divergence. As a result, metabolic complexity is ever expanding. Identifying and understanding the mechanisms that drive and limit the divergence of protein sequence space impact not only evolutionary biologists investigating molecular evolution but also synthetic biologists seeking to design useful catalysts and engineer novel metabolic pathways. Investigations over the past 50 years indicate that the recruitment of enzymes for new functions is a key event in the acquisition of new metabolic capacity. In this review, we outline the genetic mechanisms that enable recruitment and summarize the present state of knowledge regarding the functional characteristics of extant catalysts that facilitate recruitment. We also highlight recent examples of enzyme recruitment, both from the historical record provided by phylogenetics and from enzyme evolution experiments. We conclude with a look to the future, which promises fruitful consequences from the convergence of molecular evolutionary theory, laboratory-directed evolution, and synthetic biology. PMID:24483367

Evolution of catalytic function

NASA Technical Reports Server (NTRS)

Joyce, G. F.

1993-01-01

An RNA-based evolution system was constructed in the laboratory and used to develop RNA enzymes with novel catalytic function. By controlling the nature of the catalytic task that the molecules must perform in order to survive, it is possible to direct the evolving population toward the expression of some desired catalytic behavior. More recently, this system has been coupled to an in vitro translation procedure, raising the possibility of evolving protein enzymes in the laboratory to produce novel proteins with desired catalytic properties. The aim of this line of research is to reduce darwinian evolution, the fundamental process of biology, to a laboratory procedure that can be made to operate in the service of organic synthesis.

Evolution of a designed retro-aldolase leads to complete active site remodeling

PubMed Central

Giger, Lars; Caner, Sami; Obexer, Richard; Kast, Peter; Baker, David; Ban, Nenad; Hilvert, Donald

2013-01-01

Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes. PMID:23748672

Thermoadaptation-Directed Enzyme Evolution in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

PubMed Central

Kobayashi, Jyumpei; Wada, Keisuke; Furukawa, Megumi; Doi, Katsumi

2014-01-01

Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · T→G · C and C · G→T · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes. PMID:25326311

Thermoadaptation-directed enzyme evolution in an error-prone thermophile derived from Geobacillus kaustophilus HTA426.

PubMed

Suzuki, Hirokazu; Kobayashi, Jyumpei; Wada, Keisuke; Furukawa, Megumi; Doi, Katsumi

2015-01-01

Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · T→G · C and C · G→T · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Directed evolution of an RNA enzyme

NASA Technical Reports Server (NTRS)

Beaudry, Amber A.; Joyce, Gerald F.

1992-01-01

An in vitro evolution procedures was used to obtain RNA enzymes with a particular catalytic function. A population of 10 exp 13 variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce 'progeny' ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.

A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents.

PubMed

Hao, Jijun; Berry, Alan

2004-09-01

Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following four rounds of directed evolution using DNA shuffling of the fda genes from Escherichia coli and Edwardsiella ictaluri. Variants from all four generations of evolution have been purified and characterized. The variants show increased thermostability with no loss of catalytic function at room temperature. The temperature at which 50% of the initial enzyme activity is lost after incubation for 10 min (T50) of the most stable variant, 4-43D6, is increased by 11-12 degrees C over the wild-type enzymes and the half-life of activity at 53 degrees C is increased approximately 190-fold. In addition, variant 4-43D6 shows increased stability to treatment with organic solvents. DNA sequencing of the evolved variants has identified the mutations which have been introduced and which lead to increased thermostability, and the role of the mutations introduced is discussed.

Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution

PubMed Central

Renata, Hans; Wang, Z. Jane

2015-01-01

High selectivities and exquisite control over reaction outcomes entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. We will use this review to outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progressions have been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been discovered and exploited for chemical synthesis, emphasizing reactions that do not have natural counterparts. The new functions have mechanistic parallels to the native reaction mechanisms that often manifest as catalytic promiscuity and the ability to convert from one function to the other with minimal mutation. We present examples of how non-natural activities have been improved by directed evolution, mimicking the process used by nature to create new catalysts. Examples of new enzyme functions include epoxide opening reactions with non-natural nucleophiles catalyzed by a laboratory-evolved halohydrin dehalogenase, cyclopropanation and other carbene transfer reactions catalyzed by cytochrome P450 variants, and non-natural modes of cyclization by a modified terpene synthase. Lastly, we describe discoveries of non-native catalytic functions that may provide future opportunities for expanding the enzyme universe. PMID:25649694

Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species.

PubMed

Plummer, Scott M; Plummer, Mark A; Merkel, Patricia A; Hagen, Moira; Biddle, Jennifer F; Waidner, Lisa A

2016-11-01

Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe-Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production. Two of the improved mutants have a logarithmic increase in hydrogen production in our in vitro assay. Furthermore, the model predicts hydrogenase sequences with hydrogen productions as high as 540-fold over the positive control. Taken together, these results demonstrate the potential of directed evolution to improve the native bacterial hydrogenases as a first step for improvement of hydrogenase activity, further in silico prediction, and finally, construction and demonstration of an improved algal hydrogenase in an in vivo assay of C. reinhardtii hydrogen production. Copyright © 2016 Elsevier Inc. All rights reserved.

Analytical method development for directed enzyme evolution research: a high throughput high-performance liquid chromatography method for analysis of ribose and ribitol and a capillary electrophoresis method for the separation of ribose enantiomers.

PubMed

Sun, Baoguo; Miller, Gregory; Lee, Wan Yee; Ho, Kelvin; Crowe, Michael A; Partridge, Leslie

2013-01-04

Analytical methods were developed for a directed enzyme evolution research programme, which pursued high performance enzymes to produce high quality L-ribose using large scale biocatalytic reaction. A high throughput HPLC method with evaporative light-scattering detection was developed to test ribose and ribitol in the enzymatic reaction, a β-cyclobond 2000 analytical column separated ribose and ribitol in 2.3 min, a C(18) guard column was used as an on-line filter to clean up the enzyme sample matrix and a short gradient was applied to wash the column, the enzymatic reaction solution can be directly injected after quenching. Total run time of each sample was approx. 4 min which provided capability of screening 4×96-well plates/day/instrument. Meanwhile, a capillary electrophoresis method was developed for the separation of ribose enantiomers, while 7-aminonaphthalene-1,3-disulfonic acid was used as derivatisation reagent and 25 mM tetraborate with 5 mM β-cyclodextrin was used as electrolyte. 0.35%of D-ribose in L-ribose can be detected which can be translated into 99.3% ee of L-ribose. Derivatisation reagent and sample matrix did not interfere with the measurement. Copyright © 2012 Elsevier B.V. All rights reserved.

Engineering thermal stability of L-asparaginase by in vitro directed evolution.

PubMed

Kotzia, Georgia A; Labrou, Nikolaos E

2009-03-01

L-asparaginase (EC 3.5.1.1, L-ASNase) catalyses the hydrolysis of l-Asn, producing L-Asp and ammonia. This enzyme is an anti-neoplastic agent; it is used extensively in the chemotherapy of acute lymphoblastic leukaemia. In this study, we describe the use of in vitro directed evolution to create a new enzyme variant with improved thermal stability. A library of enzyme variants was created by a staggered extension process using the genes that code for the L-ASNases from Erwinia chrysanthemi and Erwinia carotovora. The amino acid sequences of the parental L-ASNases show 77% identity, but their half-inactivation temperature (T(m)) differs by 10 degrees C. A thermostable variant of the E. chrysamthemi enzyme was identified that contained a single point mutation (Asp133Val). The T(m) of this variant was 55.8 degrees C, whereas the wild-type enzyme has a T(m) of 46.4 degrees C. At 50 degrees C, the half-life values for the wild-type and mutant enzymes were 2.7 and 159.7 h, respectively. Analysis of the electrostatic potential of the wild-type enzyme showed that Asp133 is located at a neutral region on the enzyme surface and makes a significant and unfavourable electrostatic contribution to overall stability. Site-saturation mutagenesis at position 133 was used to further analyse the contribution of this position on thermostability. Screening of a library of random Asp133 mutants confirmed that this position is indeed involved in thermostability and showed that the Asp133Leu mutation confers optimal thermostability.

Presidential Green Chemistry Challenge: 2006 Greener Reaction Conditions Award

EPA Pesticide Factsheets

Presidential Green Chemistry Challenge 2006 award winner, Codexis, directed the evolution of three designer enzymes to produce the key chiral building block for atorvastatin, the active ingredient in the drug Lipitor.

Directed evolution of stereoselective enzymes based on genetic selection as opposed to screening systems.

PubMed

Acevedo-Rocha, Carlos G; Agudo, Ruben; Reetz, Manfred T

2014-12-10

Directed evolution of stereoselective enzymes provides a means to generate useful biocatalysts for asymmetric transformations in organic chemistry and biotechnology. Almost all of the numerous examples reported in the literature utilize high-throughput screening systems based on suitable analytical techniques. Since the screening step is the bottleneck of the overall procedure, researchers have considered the use of genetic selection systems as an alternative to screening. In principle, selection would be the most elegant and efficient approach because it is based on growth advantage of host cells harboring stereoselective mutants, but devising such selection systems is very challenging. They must be designed so that the host organism profits from the presence of an enantioselective variant. Progress in this intriguing research area is summarized in this review, which also includes some examples of display systems designed for enantioselectivity as assayed by fluorescence-activated cell sorting (FACS). Although the combination of display systems and FACS is a powerful approach, we also envision innovative ideas combining metabolic engineering and genetic selection systems with protein directed evolution for the development of highly selective and efficient biocatalysts. Copyright © 2014 Elsevier B.V. All rights reserved.

Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily*

PubMed Central

Chow, Jeng Yeong; Xue, Bo; Lee, Kang Hao; Tung, Alvin; Wu, Long; Robinson, Robert C.; Yew, Wen Shan

2010-01-01

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of kcat/Km of 72-fold toward 3-oxo-N-dodecanoyl-l-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-l-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis. PMID:20980257

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily.

PubMed

Chow, Jeng Yeong; Xue, Bo; Lee, Kang Hao; Tung, Alvin; Wu, Long; Robinson, Robert C; Yew, Wen Shan

2010-12-24

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k(cat)/K(m) of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

PubMed Central

Currin, Andrew; Swainston, Neil; Day, Philip J.

2015-01-01

The amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions. However, the number of possible proteins is far too large to test individually, so we need means for navigating the ‘search space’ of possible protein sequences efficiently and reliably in order to find desirable activities and other properties. Enzymologists distinguish binding (K d) and catalytic (k cat) steps. In a similar way, judicious strategies have blended design (for binding, specificity and active site modelling) with the more empirical methods of classical directed evolution (DE) for improving k cat (where natural evolution rarely seeks the highest values), especially with regard to residues distant from the active site and where the functional linkages underpinning enzyme dynamics are both unknown and hard to predict. Epistasis (where the ‘best’ amino acid at one site depends on that or those at others) is a notable feature of directed evolution. The aim of this review is to highlight some of the approaches that are being developed to allow us to use directed evolution to improve enzyme properties, often dramatically. We note that directed evolution differs in a number of ways from natural evolution, including in particular the available mechanisms and the likely selection pressures. Thus, we stress the opportunities afforded by techniques that enable one to map sequence to (structure and) activity in silico, as an effective means of modelling and exploring protein landscapes. Because known landscapes may be assessed and reasoned about as a whole, simultaneously, this offers opportunities for protein improvement not readily available to natural evolution on rapid timescales. Intelligent landscape navigation, informed by sequence-activity relationships and coupled to the emerging methods of synthetic biology, offers scope for the development of novel biocatalysts that are both highly active and robust. PMID:25503938

A general strategy for the evolution of bond-forming enzymes using yeast display

PubMed Central

Chen, Irwin; Dorr, Brent M.; Liu, David R.

2011-01-01

The ability to routinely generate efficient protein catalysts of bond-forming reactions chosen by researchers, rather than nature, is a long-standing goal of the molecular life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A–catalyzed transpeptidation activity, resulting in enrichment factors of 6,000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods. PMID:21697512

Enzymatic Functionalization of Carbon-Hydrogen Bonds1

PubMed Central

Lewis, Jared C.; Coelho, Pedro S.

2010-01-01

The development of new catalytic methods to functionalize carbon-hydrogen (C-H) bonds continues to progress at a rapid pace due to the significant economic and environmental benefits of these transformations over traditional synthetic methods. In nature, enzymes catalyze regio- and stereoselective C-H bond functionalization using transformations ranging from hydroxylation to hydroalkylation under ambient reaction conditions. The efficiency of these enzymes relative to analogous chemical processes has led to their increased use as biocatalysts in preparative and industrial applications. Furthermore, unlike small molecule catalysts, enzymes can be systematically optimized via directed evolution for a particular application and can be expressed in vivo to augment the biosynthetic capability of living organisms. While a variety of technical challenges must still be overcome for practical application of many enzymes for C-H bond functionalization, continued research on natural enzymes and on novel artificial metalloenzymes will lead to improved synthetic processes for efficient synthesis of complex molecules. In this critical review, we discuss the most prevalent mechanistic strategies used by enzymes to functionalize non-acidic C-H bonds, the application and evolution of these enzymes for chemical synthesis, and a number of potential biosynthetic capabilities uniquely enabled by these powerful catalysts. PMID:21079862

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

PubMed Central

Fischbach, Michael A.; Lai, Jonathan R.; Roche, Eric D.; Walsh, Christopher T.; Liu, David R.

2007-01-01

Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced with a domain from a foreign NRPS to create a chimeric assembly line that produces a new variant of a natural NRP. However, such chimeric NRPS modules are often heavily impaired, impeding efforts to create novel NRP variants by swapping domains from different modules or organisms. Here we show that impaired chimeric NRPSs can be functionally restored by directed evolution. Using rounds of mutagenesis coupled with in vivo screens for NRP production, we rapidly isolated variants of two different chimeric NRPSs with ≈10-fold improvements in enzyme activity and product yield, including one that produces new derivatives of the potent NRP/polyketide antibiotic andrimid. Because functional restoration in these examples required only modest library sizes (103 to 104 clones) and three or fewer rounds of screening, our approach may be widely applicable even for NRPSs from genetically challenging hosts. PMID:17620609

Identification of a missing link in the evolution of an enzyme into a transcriptional regulator.

PubMed

Durante-Rodríguez, Gonzalo; Mancheño, José Miguel; Rivas, Germán; Alfonso, Carlos; García, José Luis; Díaz, Eduardo; Carmona, Manuel

2013-01-01

The evolution of transcriptional regulators through the recruitment of DNA-binding domains by enzymes is a widely held notion. However, few experimental approaches have directly addressed this hypothesis. Here we report the reconstruction of a plausible pathway for the evolution of an enzyme into a transcriptional regulator. The BzdR protein is the prototype of a subfamily of prokaryotic transcriptional regulators that controls the expression of genes involved in the anaerobic degradation of benzoate. We have shown that BzdR consists of an N-terminal DNA-binding domain connected through a linker to a C-terminal effector-binding domain that shows significant identity to the shikimate kinase (SK). The construction of active synthetic BzdR-like regulators by fusing the DNA-binding domain of BzdR to the Escherichia coli SKI protein strongly supports the notion that an ancestral SK domain could have been involved in the evolutionary origin of BzdR. The loss of the enzymatic activity of the ancestral SK domain was essential for it to evolve as a regulatory domain in the current BzdR protein. This work also supports the view that enzymes precede the emergence of the regulatory systems that may control their expression.

The evolution of renin-angiotensin blockade: angiotensin-converting enzyme inhibitors as the starting point.

PubMed

Sica, Domenic A

2010-04-01

The renin-angiotensin system has been a target in the treatment of hypertension for close to three decades. Several medication classes that block specific aspects of this system have emerged as useful therapies, including angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and, most recently, direct renin inhibitors. There has been a natural history to the development of each of these three drug classes, starting with their use as antihypertensive agents; thereafter, in each case they have been employed as end-organ protective agents. To date, there has been scant evidence to favor angiotensin receptor blockers or direct renin inhibitors over angiotensin-converting enzyme inhibitors in treating hypertension or in affording end-organ protection; thus, angiotensin-converting enzyme inhibitors remain the standard of care when renin-angiotensin system blockade is warranted.

Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas

DOE PAGES

Alvizo, Oscar; Nguyen, Luan J.; Savile, Christopher K.; ...

2014-11-03

Carbonic anhydrase (CA) is one of nature’s fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme’s sensitivity to the harsh process conditions. Using directed evolution, the properties of a β-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability andmore » alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. In conclusion, at pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.« less

Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas

PubMed Central

Alvizo, Oscar; Nguyen, Luan J.; Savile, Christopher K.; Bresson, Jamie A.; Lakhapatri, Satish L.; Solis, Earl O. P.; Fox, Richard J.; Broering, James M.; Benoit, Michael R.; Zimmerman, Sabrina A.; Novick, Scott J.; Liang, Jack; Lalonde, James J.

2014-01-01

Carbonic anhydrase (CA) is one of nature’s fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme’s sensitivity to the harsh process conditions. Using directed evolution, the properties of a β-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction. PMID:25368146

Screening glycosynthase libraries with a fluoride chemosensor assay independently of enzyme specificity: identification of a transitional hydrolase to synthase mutant.

PubMed

Andrés, Eduardo; Aragunde, Hugo; Planas, Antoni

2014-03-01

Glycosynthases have become efficient tools for the enzymatic synthesis of oligosaccharides, glycoconjugates and polysaccharides. Enzyme-directed evolution approaches are applied to improve the performance of current glycosynthases and engineer specificity for non-natural substrates. However, simple and general screening methods are required since most of the reported assays are specific for each particular enzyme. In the present paper, we report a general screening assay that is independent of enzyme specificity, and implemented in an HTS (high-throughput screening) format for the screening of cell extracts in directed evolution experiments. Fluoride ion is a general by-product released in all glycosynthase reactions with glycosyl fluoride donors. The new assay is based on the use of a specific chemical sensor (a silyl ether of a fluorogenic methylumbelliferone) to transduce fluoride concentration into a fluorescence signal. As a proof-of-concept, it has been applied to a nucleophile saturation mutant library of Bacillus licheniformis 1,3-1,4-β-glucanase. Beyond the expected mutations at the glutamic acid (catalytic) nucleophile, other variants have been shown to acquire glycosynthase activity. Surprisingly, an aspartic acid for glutamic acid replacement renders a highly active glycosynthase, but still retains low hydrolase activity. It appears as an intermediate state between glycosyl hydrolase and glycosynthase.

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

DOE PAGES

Standaert, Robert F.; Giannone, Richard J.; Michener, Joshua K.

2018-04-18

Here, metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neithermore » enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.« less

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Standaert, Robert F.; Giannone, Richard J.; Michener, Joshua K.

Here, metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neithermore » enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.« less

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes.

PubMed

Standaert, Robert F; Giannone, Richard J; Michener, Joshua K

2018-06-01

Metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI , from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neither enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA , duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI , growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.

Computer Aided Enzyme Design and Catalytic Concepts

PubMed Central

Frushicheva, Maria P.; Mills, Matthew J. L.; Schopf, Patrick; Singh, Manoj K.; Warshel, Arieh

2014-01-01

Gaining a deeper understanding of enzyme catalysis is of great practical and fundamental importance. Over the years it has become clear that despite advances made in experimental mutational studies, a quantitative understanding of enzyme catalysis will not be possible without the use of computer modeling approaches. While we believe that electrostatic preorganization is by far the most important catalytic factor, convincing the wider scientific community of this may require the demonstration of effective rational enzyme design. Here we make the point that the main current advances in enzyme design are basically advances in directed evolution and that computer aided enzyme design must involve approaches that can reproduce catalysis in well-defined test cases. Such an approach is provided by the empirical valence bond method. PMID:24814389

Microbial Enzymes: Tools for Biotechnological Processes

PubMed Central

Adrio, Jose L.; Demain, Arnold L.

2014-01-01

Microbial enzymes are of great importance in the development of industrial bioprocesses. Current applications are focused on many different markets including pulp and paper, leather, detergents and textiles, pharmaceuticals, chemical, food and beverages, biofuels, animal feed and personal care, among others. Today there is a need for new, improved or/and more versatile enzymes in order to develop more novel, sustainable and economically competitive production processes. Microbial diversity and modern molecular techniques, such as metagenomics and genomics, are being used to discover new microbial enzymes whose catalytic properties can be improved/modified by different strategies based on rational, semi-rational and random directed evolution. Most industrial enzymes are recombinant forms produced in bacteria and fungi. PMID:24970208

Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution.

PubMed

Shivange, Amol V; Hoeffken, Hans Wolfgang; Haefner, Stefan; Schwaneberg, Ulrich

2016-12-01

Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: ( i ) identification of conserved and highly variable regions; ( ii ) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; ( iii ) in vitro DNA recombination of synthetic genes; and ( iv ) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)

PubMed Central

Gielen, Fabrice; Hours, Raphaelle; Emond, Stephane; Fischlechner, Martin; Schell, Ursula

2016-01-01

Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD+-dependent amino acid dehydrogenases. The detection limit (10 μM in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ∼100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12 °C higher). The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows. PMID:27821774

Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution.

PubMed

Kwan, David H; Constantinescu, Iren; Chapanian, Rafi; Higgins, Melanie A; Kötzler, Miriam P; Samain, Eric; Boraston, Alisdair B; Kizhakkedathu, Jayachandran N; Withers, Stephen G

2015-05-06

Blood transfusions are critically important in many medical procedures, but the presence of antigens on red blood cells (RBCs, erythrocytes) means that careful blood-typing must be carried out prior to transfusion to avoid adverse and sometimes fatal reactions following transfusion. Enzymatic removal of the terminal N-acetylgalactosamine or galactose of A- or B-antigens, respectively, yields universal O-type blood, but is inefficient. Starting with the family 98 glycoside hydrolase from Streptococcus pneumoniae SP3-BS71 (Sp3GH98), which cleaves the entire terminal trisaccharide antigenic determinants of both A- and B-antigens from some of the linkages on RBC surface glycans, through several rounds of evolution, we developed variants with vastly improved activity toward some of the linkages that are resistant to cleavage by the wild-type enzyme. The resulting enzyme effects more complete removal of blood group antigens from cell surfaces, demonstrating the potential for engineering enzymes to generate antigen-null blood from donors of various types.

Directed evolution of a β-1,3-1,4-glucanase from Bacillus subtilis MA139 for improving thermal stability and other characteristics.

PubMed

Pei, Honglei; Guo, Xiaojing; Yang, Wenhan; Lv, Junnan; Chen, Yiqun; Cao, Yunhe

2015-07-01

In order to improve some characteristics of a β-1,3-1,4-glucanase from Bacillus subtilis MA139, directed evolution was conducted in this study. After error-prone PCR, the β-1,3-1,4-glucanase gene, glu-opt, was cloned into the vector pBGP1 and transformed into Pichia pastoris X-33 to construct a mutant library. Three variants named as 7-32, 7-87, and 7-115 were screened from 8000 colonies. Amino-acid sequence analysis showed that these mutants had one or two amino-acid substitutions (7-32: T113S, 7-87: M44V/N53H, and 7-115: N157D). The variants were over-expressed in P. pastoris by methanol induction. After purification of the enzyme proteins, the characteristics of the variants were analyzed in detail. It indicated that these mutant enzymes had broader ranges of pH value and better pH stability than the wild-type enzyme. The mutant enzyme 7-87 had the best ability to tolerate an acid environment (pH 2.0), while the wild-type enzyme had no activity under this condition. Moreover, all these mutants demonstrated improved thermal stability. In particular, the mutant enzyme 7-32 had residual enzymatic activity of 60% and 40% after being incubated at 80 °C and 90 °C for 10 min. While, the wild-type enzyme had no residual enzymatic activity after being incubated at 80 °C for 4 min. In addition, the mutant enzymes had better tolerance to some chemicals than the wild-type enzyme. The improved stability could enhance the prospects for this enzyme to have use in the feed industry to reduce the effects of the anti-nutritional factor β-glucan. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase

PubMed Central

Preiswerk, Nathalie; Beck, Tobias; Schulz, Jessica D.; Milovník, Peter; Mayer, Clemens; Siegel, Justin B.; Baker, David; Hilvert, Donald

2014-01-01

By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme–product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions. PMID:24847076

Engineering Novel and Improved Biocatalysts by Cell Surface Display

PubMed Central

Smith, Mason R.; Khera, Eshita; Wen, Fei

2017-01-01

Biocatalysts, especially enzymes, have the ability to catalyze reactions with high product selectivity, utilize a broad range of substrates, and maintain activity at low temperature and pressure. Therefore, they represent a renewable, environmentally friendly alternative to conventional catalysts. Most current industrial-scale chemical production processes using biocatalysts employ soluble enzymes or whole cells expressing intracellular enzymes. Cell surface display systems differ by presenting heterologous enzymes extracellularly, overcoming some of the limitations associated with enzyme purification and substrate transport. Additionally, coupled with directed evolution, cell surface display is a powerful platform for engineering enzymes with enhanced properties. In this review, we will introduce the molecular and cellular principles of cell surface display and discuss how it has been applied to engineer enzymes with improved properties as well as to develop surface-engineered microbes as whole-cell biocatalysts. PMID:29056821

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution.

PubMed

Yonemoto, Isaac T; Matteri, Christopher W; Nguyen, Thao Amy; Smith, Hamilton O; Weyman, Philip D

2013-07-02

Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "Deep ecotype" that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

Directing enzyme devolution for biosynthesis of alkanols and 1,n-alkanediols from natural polyhydroxy compounds.

PubMed

Dai, Lu; Tao, Fei; Tang, Hongzhi; Guo, Yali; Shen, Yaling; Xu, Ping

2017-11-01

Primordial enzymes are proposed to possess broad specificities. Through divergence and evolution, enzymes have been refined to exhibit specificity towards one reaction or substrate, and are thus commonly assumed as "specialists". However, some enzymes are "generalists" that catalyze a range of substrates and reactions. This property has been defined as enzyme promiscuity and is of great importance for the evolution of new functions. The promiscuities of two enzymes, namely glycerol dehydratase and diol dehydratase, were herein exploited for catalyzing long-chain polyols, including 1,2-butanediol, 1,2,4-butanetriol, erythritol, 1,2-pentanediol, 1,2,5-pentanetriol, and 1,2,6-hexanetriol. The specific activities required for catalyzing these six long-chain polyols were studied via in vitro enzyme assays, and the catalytic efficiencies were increased through protein engineering. The promiscuous functions were subsequently applied in vivo to establish 1,4-butanediol pathways from lignocellulose derived compounds, including xylose and erythritol. In addition, a pathway for 1-pentanol production from 1,2-pentanediol was also constructed. The results suggest that exploiting enzyme promiscuity is promising for exploring new catalysts, which would expand the repertoire of genetic elements available to synthetic biology and may provide a starting point for designing and engineering novel pathways for valuable chemicals. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Combinatorial pathway optimization in Escherichia coli by directed co-evolution of rate-limiting enzymes and modular pathway engineering.

PubMed

Lv, Xiaomei; Gu, Jiali; Wang, Fan; Xie, Wenping; Liu, Min; Ye, Lidan; Yu, Hongwei

2016-12-01

Metabolic engineering of microorganisms for heterologous biosynthesis is a promising route to sustainable chemical production which attracts increasing research and industrial interest. However, the efficiency of microbial biosynthesis is often restricted by insufficient activity of pathway enzymes and unbalanced utilization of metabolic intermediates. This work presents a combinatorial strategy integrating modification of multiple rate-limiting enzymes and modular pathway engineering to simultaneously improve intra- and inter-pathway balance, which might be applicable for a range of products, using isoprene as an example product. For intra-module engineering within the methylerythritol-phosphate (MEP) pathway, directed co-evolution of DXS/DXR/IDI was performed adopting a lycopene-indicated high-throughput screening method developed herein, leading to 60% improvement of isoprene production. In addition, inter-module engineering between the upstream MEP pathway and the downstream isoprene-forming pathway was conducted via promoter manipulation, which further increased isoprene production by 2.94-fold compared to the recombinant strain with solely protein engineering and 4.7-fold compared to the control strain containing wild-type enzymes. These results demonstrated the potential of pathway optimization in isoprene overproduction as well as the effectiveness of combining metabolic regulation and protein engineering in improvement of microbial biosynthesis. Biotechnol. Bioeng. 2016;113: 2661-2669. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution▿ †

PubMed Central

Trefzer, Axel; Jungmann, Volker; Molnár, István; Botejue, Ajit; Buckel, Dagmar; Frey, Gerhard; Hill, D. Steven; Jörg, Mario; Ligon, James M.; Mason, Dylan; Moore, David; Pachlatko, J. Paul; Richardson, Toby H.; Spangenberg, Petra; Wall, Mark A.; Zirkle, Ross; Stege, Justin T.

2007-01-01

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4″-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4″-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin. PMID:17483257

Directed evolution of artificial metalloenzymes for in vivo metathesis

NASA Astrophysics Data System (ADS)

Jeschek, Markus; Reuter, Raphael; Heinisch, Tillmann; Trindler, Christian; Klehr, Juliane; Panke, Sven; Ward, Thomas R.

2016-09-01

The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions. For example, artificial metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist: repurposing natural metalloenzymes for abiotic transformations; in silico metalloenzyme (re-)design; and incorporation of abiotic cofactors into proteins. The third strategy offers the opportunity to design a wide variety of artificial metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein. This limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction without equivalent in nature. Building on previous work on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the ‘metathase’ because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial metalloenzyme that catalyses an abiotic reaction in vivo, with potential applications in, for example, non-natural metabolism.

Biocatalysts: application and engineering for industrial purposes.

PubMed

Jemli, Sonia; Ayadi-Zouari, Dorra; Hlima, Hajer Ben; Bejar, Samir

2016-01-01

Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application.

[Biosensor development in clinical analysis].

PubMed

Boitieux, J L; Desmet, G; Thomas, D

1985-01-01

The use of enzymes immobilized or as markers formed the subject of more than thousand publications in the field of industry or biomedical applications, during the last five years. Recently, some authors published works concerning immobilization of total microorganisms for catalytic purposes, others use the enzymatic activity for marking molecules involved in immunological analysis processes. Together industrial biotechnology and medical analysis laboratory are interested with the evolution of these procedures involving the activity of immobilized enzymes. Enzyme immobilization allowed the lowering of analysis costs for, in this case, the enzyme can be used several times. We take account of the two main cases which are encountered during utilization of immobilized enzymes of analytical purposes. The enzyme is used directly for the catalysed reaction or it is used as enzymatic marker. These both aspects are developed mainly for the elaboration of enzymatic and immunoenzymatic electrodes and the realization of automatic computerized devices allowing continuous estimation of numerous biological blood parameters. From these two precise examples, glucose and antigen determination, the authors show the evolution of these technologies in the field of immobilized enzymes or captors and the analysis of signals given by these electrodes requiring a computerized treatment. This new technology opens to important potentialities in the analytical field. The automatization of these devices allowing the control in real time, will probably make easier the optimization steps of procedures actually used in the biomedical sphere.

Regioselective alkane hydroxylation with a mutant CYP153A6 enzyme

DOEpatents

Koch, Daniel J.; Arnold, Frances H.

2013-01-29

Cytochrome P450 CYP153A6 from Myobacterium sp. strain HXN1500 was engineered using in-vivo directed evolution to hydroxylate small-chain alkanes regioselectively. Mutant CYP153A6-BMO1 selectively hydroxylates butane and pentane at the terminal carbon to form 1-butanol and 1-pentanol, respectively, at rates greater than wild-type CYP153A6 enzymes. This biocatalyst is highly active for small-chain alkane substrates and the regioselectivity is retained in whole-cell biotransformations.

Change, exchange, and rearrange: protein engineering for the biotechnological production of fuels, pharmaceuticals, and other chemicals.

PubMed

Fisher, Michael A; Tullman-Ercek, Danielle

2013-12-01

Enzymes are indispensable in the effort to produce chemicals from fuels to pharmaceuticals in an ecologically friendly manner. They have the potential to catalyze reactions with high specificity and efficiency without the use of hazardous chemicals. Nature provides an extensive collection of enzymes, but often these must be altered to perform desired functions under required conditions. Advances in protein engineering permit the design and/or directed evolution of enzymes specifically tailored for such industrial applications. Recent years have seen the development of improved enzymes to assist in both the conversion of biomass into fuels and chemicals, and the creation of key intermediates in pharmaceutical production. Copyright © 2013 Elsevier Ltd. All rights reserved.

Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase.

PubMed

Obexer, Richard; Godina, Alexei; Garrabou, Xavier; Mittl, Peer R E; Baker, David; Griffiths, Andrew D; Hilvert, Donald

2017-01-01

Designing catalysts that achieve the rates and selectivities of natural enzymes is a long-standing goal in protein chemistry. Here, we show that an ultrahigh-throughput droplet-based microfluidic screening platform can be used to improve a previously optimized artificial aldolase by an additional factor of 30 to give a >10 9 rate enhancement that rivals the efficiency of class I aldolases. The resulting enzyme catalyses a reversible aldol reaction with high stereoselectivity and tolerates a broad range of substrates. Biochemical and structural studies show that catalysis depends on a Lys-Tyr-Asn-Tyr tetrad that emerged adjacent to a computationally designed hydrophobic pocket during directed evolution. This constellation of residues is poised to activate the substrate by Schiff base formation, promote mechanistically important proton transfers and stabilize multiple transition states along a complex reaction coordinate. The emergence of such a sophisticated catalytic centre shows that there is nothing magical about the catalytic activities or mechanisms of naturally occurring enzymes, or the evolutionary process that gave rise to them.

A 13-Week Research-Based Biochemistry Laboratory Curriculum

ERIC Educational Resources Information Center

Lefurgy, Scott T.; Mundorff, Emily C.

2017-01-01

Here, we present a 13-week research-based biochemistry laboratory curriculum designed to provide the students with the experience of engaging in original research while introducing foundational biochemistry laboratory techniques. The laboratory experience has been developed around the directed evolution of an enzyme chosen by the instructor, with…

Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells

USDA-ARS?s Scientific Manuscript database

Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high ...

Optimization of the in silico designed Kemp eliminase KE70 by computational design and directed evolution

PubMed Central

Khersonsky, Olga; Röthlisberger, Daniela; Wollacott, Andrew M.; Murphy, Paul; Dym, Orly; Albeck, Shira; Kiss, Gert; Houk, K. N.; Baker, David; Tawfik, Dan S.

2013-01-01

Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with >200-fold higher catalytic efficiency, and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorporated into the originally designed KE70 along with random mutations, and the resulting libraries were screened for improved eliminase activity. Nine rounds of mutation and selection resulted in >400-fold improvement in the catalytic efficiency of the original KE70 design, reflected in both higher kcat and lower KM values, with the best variants exhibiting kcat/KM values of >5x104 s−1M−1. The optimized KE70 variants were characterized structurally and biochemically providing insights into the origins of the improvements in catalysis. Three primary contributions were identified: first, the reshaping of the active site cavity to achieve tighter substrate binding; second, the fine-tuning of the electrostatics around the catalytic His-Asp dyad; and third, stabilization of the active-site dyad in a conformation optimal for catalysis. PMID:21277311

Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae.

PubMed

Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

2012-08-01

The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

PubMed Central

2013-01-01

Background Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. Results We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Conclusions Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement. PMID:23819621

Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes

PubMed Central

Rodríguez-Bolaños, Monica; Cabrera, Nallely

2016-01-01

The reactivation of triosephosphate isomerase (TIM) from unfolded monomers induced by guanidine hydrochloride involves different amino acids of its sequence in different stages of protein refolding. We describe a systematic mutagenesis method to find critical residues for certain physico-chemical properties of a protein. The two similar TIMs of Trypanosoma brucei and Trypanosoma cruzi have different reactivation velocities and efficiencies. We used a small number of chimeric enzymes, additive mutants and planned site-directed mutants to produce an enzyme from T. brucei with 13 mutations in its sequence, which reactivates fast and efficiently like wild-type (WT) TIM from T. cruzi, and another enzyme from T. cruzi, with 13 slightly altered mutations, which reactivated slowly and inefficiently like the WT TIM of T. brucei. Our method is a shorter alternative to random mutagenesis, saturation mutagenesis or directed evolution to find multiple amino acids critical for certain properties of proteins. PMID:27733588

Ecological transition predictably associated with gene degeneration.

PubMed

Wessinger, Carolyn A; Rausher, Mark D

2015-02-01

Gene degeneration or loss can significantly contribute to phenotypic diversification, but may generate genetic constraints on future evolutionary trajectories, potentially restricting phenotypic reversal. Such constraints may manifest as directional evolutionary trends when parallel phenotypic shifts consistently involve gene degeneration or loss. Here, we demonstrate that widespread parallel evolution in Penstemon from blue to red flowers predictably involves the functional inactivation and degeneration of the enzyme flavonoid 3',5'-hydroxylase (F3'5'H), an anthocyanin pathway enzyme required for the production of blue floral pigments. Other types of genetic mutations do not consistently accompany this phenotypic shift. This pattern may be driven by the relatively large mutational target size of degenerative mutations to this locus and the apparent lack of associated pleiotropic effects. The consistent degeneration of F3'5'H may provide a mechanistic explanation for the observed asymmetry in the direction of flower color evolution in Penstemon: Blue to red transitions are common, but reverse transitions have not been observed. Although phenotypic shifts in this system are likely driven by natural selection, internal constraints may generate predictable genetic outcomes and may restrict future evolutionary trajectories. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A self-triggered picoinjector in microfluidics

NASA Astrophysics Data System (ADS)

Yang, Yiming; Liu, Songsheng; Jia, Chunping; Mao, Hongju; Jin, Qinghui; Zhao, Jianlong; Zhou, Hongbo

2016-12-01

Droplet-based microfluidics has recently emerged as a potential platform for studies of single-cell, directed evolution, and genetic sequencing. In droplet-based microfluidics, adding reagents into drops is one of the most important functions. In this paper, we develop a new self-triggered picoinjector to add controlled volumes of reagent into droplets at kilohertz rates. In the picoinjector, the reagent injecting is triggered by the coming droplet itself, without needing a droplet detection module. Meanwhile, the dosing volume can be precisely controlled. These features make the system more practical and reliable. We expect the new picoinjector will find important applications of droplet-based microfluidics in automated biological assay, directed evolution, enzyme assay, and so on.

Directed evolution of a model primordial enzyme provides insights into the development of the genetic code.

PubMed

Müller, Manuel M; Allison, Jane R; Hongdilokkul, Narupat; Gaillon, Laurent; Kast, Peter; van Gunsteren, Wilfred F; Marlière, Philippe; Hilvert, Donald

2013-01-01

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function.

Directional selection causes decanalization in a group I ribozyme.

PubMed

Hayden, Eric J; Weikert, Christian; Wagner, Andreas

2012-01-01

A canalized genotype is robust to environmental or genetic perturbations. Canalization is expected to result from stabilizing selection on a well-adapted phenotype. Decanalization, the loss of robustness, might follow periods of directional selection toward a new optimum. The evolutionary forces causing decanalization are still unknown, in part because it is difficult to determine the fitness effects of mutations in populations of organisms with complex genotypes and phenotypes. Here, we report direct experimental measurements of robustness in a system with a simple genotype and phenotype, the catalytic activity of an RNA enzyme. We find that the robustness of a population of RNA enzymes decreases during a period of directional selection in the laboratory. The decrease in robustness is primarily caused by the selective sweep of a genotype that is decanalized relative to the wild-type, both in terms of mutational robustness and environmental robustness (thermodynamic stability). Our results experimentally demonstrate that directional selection can cause decanalization on short time scales, and demonstrate co-evolution of mutational and environmental robustness.

Continuous directed evolution of aminoacyl-tRNA synthetases

PubMed Central

Bryson, David I.; Fan, Chenguang; Guo, Li-Tao; Miller, Corwin; Söll, Dieter; Liu, David R.

2017-01-01

Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of non-canonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing non-canonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity. PMID:29035361

Improvement of operational stability of Ogataea minuta carbonyl reductase for chiral alcohol production.

PubMed

Honda, Kohsuke; Inoue, Mizuha; Ono, Tomohiro; Okano, Kenji; Dekishima, Yasumasa; Kawabata, Hiroshi

2017-06-01

Directed evolution of enantio-selective carbonyl reductase from Ogataea minuta was conducted to improve the operational stability of the enzyme. A mutant library was constructed by an error-prone PCR and screened using a newly developed colorimetric assay. The stability of a mutant with two amino acid substitutions was significantly higher than that of the wild type at 50°C in the presence of dimethyl sulfoxide. Site-directed mutagenesis analysis showed that the improved stability of the enzyme can be attributed to the amino acid substitution of V166A. The half-lives of the V166A mutant were 11- and 6.1-times longer than those of the wild type at 50°C in the presence and absence, respectively, of 20% (v/v) dimethyl sulfoxide. No significant differences in the substrate specificity and enantio-selectivity of the enzyme were observed. The mutant enzyme converted 60 mM 2,2,2-trifluoroacetophenone to (R)-(-)-α-(trifluoromethyl)benzyl alcohol in a molar yield of 71% whereas the conversion yield with an equivalent concentration of the wild-type enzyme was 27%. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakayasu, Ernesto S.; Burnet, Meagan C.; Walukiewicz, Hanna E.

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggestmore » that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCEPost-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.« less

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakayasu, Ernesto S.; Burnet, Meagan C.; Walukiewicz, Hanna E.

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggestmore » that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCE Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.« less

Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

DOE PAGES

Nakayasu, Ernesto S.; Burnet, Meagan C.; Walukiewicz, Hanna E.; ...

2017-11-28

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggestmore » that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCE Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.« less

Easy preparation of a large-size random gene mutagenesis library in Escherichia coli.

PubMed

You, Chun; Percival Zhang, Y-H

2012-09-01

A simple and fast protocol for the preparation of a large-size mutant library for directed evolution in Escherichia coli was developed based on the DNA multimers generated by prolonged overlap extension polymerase chain reaction (POE-PCR). This protocol comprised the following: (i) a linear DNA mutant library was generated by error-prone PCR or shuffling, and a linear vector backbone was prepared by regular PCR; (ii) the DNA multimers were generated based on these two DNA templates by POE-PCR; and (iii) the one restriction enzyme-digested DNA multimers were ligated to circular plasmids, followed by transformation to E. coli. Because the ligation efficiency of one DNA fragment was several orders of magnitude higher than that of two DNA fragments for typical mutant library construction, it was very easy to generate a mutant library with a size of more than 10(7) protein mutants per 50 μl of the POE-PCR product. Via this method, four new fluorescent protein mutants were obtained based on monomeric cherry fluorescent protein. This new protocol was simple and fast because it did not require labor-intensive optimizations in restriction enzyme digestion and ligation, did not involve special plasmid design, and enabled constructing a large-size mutant library for directed enzyme evolution within 1 day. Copyright © 2012 Elsevier Inc. All rights reserved.

Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach

PubMed Central

Abi-Ghanem, Josephine; Chusainow, Janet; Karimova, Madina; Spiegel, Christopher; Hofmann-Sieber, Helga; Hauber, Joachim; Buchholz, Frank; Pisabarro, M. Teresa

2013-01-01

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre’s enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine. PMID:23275541

The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization.

PubMed

Noor, Elad; Flamholz, Avi; Bar-Even, Arren; Davidi, Dan; Milo, Ron; Liebermeister, Wolfram

2016-11-01

Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell's capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified.

What are the Limitations of Enzymes in Synthetic Organic Chemistry?

PubMed

Reetz, Manfred T

2016-12-01

Enzymes have been used in organic chemistry and biotechnology for 100 years, but their widespread application has been prevented by a number of limitations, including the often-observed limited thermostability, narrow substrate scope, and low or wrong stereo- and/or regioselectivity. Directed evolution provides a means to address and generally solve these problems, especially since recent methodology development has made this protein engineering method faster, more efficient, and more reliable than in the past. This Darwinian approach to asymmetric catalysis has led to a number of industrial applications. Metabolic-pathway engineering, mutasynthesis, and fermentation are likewise enzyme-based techniques that enrich chemistry. This account outlines the scope, and particularly, the limitations, of biocatalysis. The complementary nature of enzymes and man-made catalysts is emphasized. © 2016 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

PubMed Central

Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

2015-01-01

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

DOE PAGES

Sunden, Fanny; Peck, Ariana; Salzman, Julia; ...

2015-04-22

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modesmore » of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.« less

Subcellular Relocalization and Positive Selection Play Key Roles in the Retention of Duplicate Genes of Populus Class III Peroxidase Family[W][OPEN

PubMed Central

Ren, Lin-Ling; Liu, Yan-Jing; Liu, Hai-Jing; Qian, Ting-Ting; Qi, Li-Wang; Wang, Xiao-Ru; Zeng, Qing-Yin

2014-01-01

Gene duplication is the primary source of new genes and novel functions. Over the course of evolution, many duplicate genes lose their function and are eventually removed by deletion. However, some duplicates have persisted and evolved diverse functions. A particular challenge is to understand how this diversity arises and whether positive selection plays a role. In this study, we reconstructed the evolutionary history of the class III peroxidase (PRX) genes from the Populus trichocarpa genome. PRXs are plant-specific enzymes that play important roles in cell wall metabolism and in response to biotic and abiotic stresses. We found that two large tandem-arrayed clusters of PRXs evolved from an ancestral cell wall type PRX to vacuole type, followed by tandem duplications and subsequent functional specification. Substitution models identified seven positively selected sites in the vacuole PRXs. These positively selected sites showed significant effects on the biochemical functions of the enzymes. We also found that positive selection acts more frequently on residues adjacent to, rather than directly at, a critical active site of the enzyme, and on flexible regions rather than on rigid structural elements of the protein. Our study provides new insights into the adaptive molecular evolution of plant enzyme families. PMID:24934172

Molecular dynamics explorations of active site structure in designed and evolved enzymes.

PubMed

Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

2015-04-21

This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP, a noncatalytic arrangement of the catalytic triad is dominant. Unnatural truncated substrates are inactive because of the lack of protein-protein interactions provided by the ACP. Directed evolution is able to gradually restore the catalytic organization of the active site by motion of the protein backbone that alters the active site geometry. In the third case, we demonstrate the key role of MD in combination with crystallography to identify the origins of substrate-dependent stereoselectivities in a number of Codexis-engineered ketoreductases, one of which is used commercially for the production of the antibiotic sulopenem. Here, mutations alter the shape of the active site as well as the accessibility of water to different regions of it. Each of these examples reveals something different about how mutations can influence enzyme activity and shows that directed evolution, like natural evolution, can increase catalytic activity in a variety of remarkable and often subtle ways.

Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface.

PubMed

Limsakul, Praopim; Peng, Qin; Wu, Yiqian; Allen, Molly E; Liang, Jing; Remacle, Albert G; Lopez, Tyler; Ge, Xin; Kay, Brian K; Zhao, Huimin; Strongin, Alex Y; Yang, Xiang-Lei; Lu, Shaoying; Wang, Yingxiao

2018-04-19

Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Classification and Evolution of Enzyme Function

PubMed Central

Martínez Cuesta, Sergio; Rahman, Syed Asad; Furnham, Nicholas; Thornton, Janet M.

2015-01-01

Enzymes are the proteins responsible for the catalysis of life. Enzymes sharing a common ancestor as defined by sequence and structure similarity are grouped into families and superfamilies. The molecular function of enzymes is defined as their ability to catalyze biochemical reactions; it is manually classified by the Enzyme Commission and robust approaches to quantitatively compare catalytic reactions are just beginning to appear. Here, we present an overview of studies at the interface of the evolution and function of enzymes. PMID:25986631

Integrated cellulosic enzymes hydrolysis and fermentative advanced yeast bioconversion solution ready for biomass biorefineries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2011-05-04

These are slides from this conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme,more » as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Development of a Commerical Enzyme System for Lignocellulosic Biomass Saccharification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2011-02-14

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Fully Integrated Lignocellulosic Biorefinery with Onsite Production of Enzymes and Yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2010-06-14

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Microbial production of natural and non-natural flavonoids: Pathway engineering, directed evolution and systems/synthetic biology.

PubMed

Pandey, Ramesh Prasad; Parajuli, Prakash; Koffas, Mattheos A G; Sohng, Jae Kyung

2016-01-01

In this review, we address recent advances made in pathway engineering, directed evolution, and systems/synthetic biology approaches employed in the production and modification of flavonoids from microbial cells. The review is divided into two major parts. In the first, various metabolic engineering and system/synthetic biology approaches used for production of flavonoids and derivatives are discussed broadly. All the manipulations/engineering accomplished on the microorganisms since 2000 are described in detail along with the biosynthetic pathway enzymes, their sources, structures of the compounds, and yield of each product. In the second part of the review, post-modifications of flavonoids by four major reactions, namely glycosylations, methylations, hydroxylations and prenylations using recombinant strains are described. Copyright © 2016 Elsevier Inc. All rights reserved.

High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

PubMed Central

Rodríguez-Escribano, David; de Salas, Felipe; Camarero, Susana

2017-01-01

Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin–Ciocalteau reagent (FCR), to detect the decrease in phenolic content produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). The method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates. PMID:28820431

High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez-Escribano, David; de Salas, Felipe; Pardo, Isabel

Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin-Ciocalteau reagent (FCR), to detect the decrease in phenolic contentmore » produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). As a result, the method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates.« less

High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

DOE PAGES

Rodriguez-Escribano, David; de Salas, Felipe; Pardo, Isabel; ...

2017-08-18

Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin-Ciocalteau reagent (FCR), to detect the decrease in phenolic contentmore » produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). As a result, the method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates.« less

The Classification and Evolution of Enzyme Function.

PubMed

Martínez Cuesta, Sergio; Rahman, Syed Asad; Furnham, Nicholas; Thornton, Janet M

2015-09-15

Enzymes are the proteins responsible for the catalysis of life. Enzymes sharing a common ancestor as defined by sequence and structure similarity are grouped into families and superfamilies. The molecular function of enzymes is defined as their ability to catalyze biochemical reactions; it is manually classified by the Enzyme Commission and robust approaches to quantitatively compare catalytic reactions are just beginning to appear. Here, we present an overview of studies at the interface of the evolution and function of enzymes. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Can enzyme engineering benefit from the modulation of protein motions? Lessons learned from NMR relaxation dispersion experiments.

PubMed

Doucet, Nicolas

2011-04-01

Despite impressive progress in protein engineering and design, our ability to create new and efficient enzyme activities remains a laborious and time-consuming endeavor. In the past few years, intricate combinations of rational mutagenesis, directed evolution and computational methods have paved the way to exciting engineering examples and are now offering a new perspective on the structural requirements of enzyme activity. However, these structure-function analyses are usually guided by the time-averaged static models offered by enzyme crystal structures, which often fail to describe the functionally relevant 'invisible states' adopted by proteins in space and time. To alleviate such limitations, NMR relaxation dispersion experiments coupled to mutagenesis studies have recently been applied to the study of enzyme catalysis, effectively complementing 'structure-function' analyses with 'flexibility-function' investigations. In addition to offering quantitative, site-specific information to help characterize residue motion, these NMR methods are now being applied to enzyme engineering purposes, providing a powerful tool to help characterize the effects of controlling long-range networks of flexible residues affecting enzyme function. Recent advancements in this emerging field are presented here, with particular attention to mutagenesis reports highlighting the relevance of NMR relaxation dispersion tools in enzyme engineering.

Opposing effects of folding and assembly chaperones on evolvability of Rubisco.

PubMed

Durão, Paulo; Aigner, Harald; Nagy, Péter; Mueller-Cajar, Oliver; Hartl, F Ulrich; Hayer-Hartl, Manajit

2015-02-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the fixation of CO2 in photosynthesis. Despite its pivotal role, Rubisco is an inefficient enzyme and thus is a key target for directed evolution. Rubisco biogenesis depends on auxiliary factors, including the GroEL/ES-type chaperonin for folding and the chaperone RbcX for assembly. Here we performed directed evolution of cyanobacterial form I Rubisco using a Rubisco-dependent Escherichia coli strain. Overexpression of GroEL/ES enhanced Rubisco solubility and tended to expand the range of permissible mutations. In contrast, the specific assembly chaperone RbcX had a negative effect on evolvability by preventing a subset of mutants from forming holoenzyme. Mutation F140I in the large Rubisco subunit, isolated in the absence of RbcX, increased carboxylation efficiency approximately threefold without reducing CO2 specificity. The F140I mutant resulted in a ∼55% improved photosynthesis rate in Synechocystis PCC6803. The requirement of specific biogenesis factors downstream of chaperonin may have retarded the natural evolution of Rubisco.

Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

PubMed

Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

2011-07-01

The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

DOE PAGES

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.; ...

2017-07-07

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization

PubMed Central

Noor, Elad; Flamholz, Avi; Bar-Even, Arren; Davidi, Dan; Milo, Ron; Liebermeister, Wolfram

2016-01-01

Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell’s capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified. PMID:27812109

Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution

NASA Astrophysics Data System (ADS)

Kille, Sabrina; Zilly, Felipe E.; Acevedo, Juan P.; Reetz, Manfred T.

2011-09-01

A current challenge in synthetic organic chemistry is the development of methods that allow the regio- and stereoselective oxidative C-H activation of natural or synthetic compounds with formation of the corresponding alcohols. Cytochrome P450 enzymes enable C-H activation at non-activated positions, but the simultaneous control of both regio- and stereoselectivity is problematic. Here, we demonstrate that directed evolution using iterative saturation mutagenesis provides a means to solve synthetic problems of this kind. Using P450 BM3(F87A) as the starting enzyme and testosterone as the substrate, which results in a 1:1 mixture of the 2β- and 15β-alcohols, mutants were obtained that are 96-97% selective for either of the two regioisomers, each with complete diastereoselectivity. The mutants can be used for selective oxidative hydroxylation of other steroids without performing additional mutagenesis experiments. Molecular dynamics simulations and docking experiments shed light on the origin of regio- and stereoselectivity.

Directed divergent evolution of a thermostable D-tagatose epimerase towards improved activity for two hexose substrates.

PubMed

Bosshart, Andreas; Hee, Chee Seng; Bechtold, Matthias; Schirmer, Tilman; Panke, Sven

2015-03-02

Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D-tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shells around the substrate-binding site of Var8 yielded the eight-site mutant IDF8 (ninefold improved kcat for the epimerization of D-fructose) and the six-site mutant ILS6 (14-fold improved epimerization of L-sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chromobacterium violaceum ω-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4'-substituted acetophenones, and follows Swain-Lupton parameterisation.

PubMed

Cassimjee, Karim Engelmark; Humble, Maria Svedendahl; Land, Henrik; Abedi, Vahak; Berglund, Per

2012-07-28

For biocatalytic production of pharmaceutically important chiral amines the ω-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum ω-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (∼5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.

Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution

DOE PAGES

Sunden, Fanny; AlSadhan, Ishraq; Lyubimov, Artem; ...

2017-10-25

Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. Here, we mutated distinguishing active-site residues to generate enzymes that had a common Zn 2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of thesemore » pruned enzymes with a series of substrates. A substantial rate enhancement of ~1011-fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn 2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 10 7–10 8-fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.« less

Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunden, Fanny; AlSadhan, Ishraq; Lyubimov, Artem

Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. Here, we mutated distinguishing active-site residues to generate enzymes that had a common Zn 2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of thesemore » pruned enzymes with a series of substrates. A substantial rate enhancement of ~1011-fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn 2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 10 7–10 8-fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.« less

Vertical Integration of Biomass Saccharification of Enzymes for Sustainable Cellulosic Biofuel Production in a Biorefinery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2011-05-09

These are a set of slides from this conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for eachmore » cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

A Microplate Format Assay for Real-Time Screening for New Aldolases that Accept Aryl-Substituted Acceptor Substrates.

PubMed

Ma, Huan; Enugala, Thilak Reddy; Widersten, Mikael

2015-12-01

Aldolases are potentially important biocatalysts for asymmetric synthesis of polyhydroxylated compounds. Fructose 6-phosphate aldolase (FSA) is of particular interest by virtue of its unusually relaxed dependency on phosphorylated substrates. FSA has been reported to be a promising catalyst of aldol addition involving aryl-substituted acceptors such as phenylacetaldehyde that can react with donor ketones such as hydroxyacetone. Improvement of the low intrinsic activity with bulky acceptor substrates of this type is of great interest but has been hampered by the lack of powerful screening protocols applicable in directed evolution strategies. Here we present a new screen allowing for direct spectrophotometric recording of retro-aldol cleavage. The assay utilizes an aldehyde reductase produced in vitro by directed evolution; it reduces the aldehyde product formed after cleavage of the aldol by FSA. The assay is suitable both for steady-state enzyme kinetics and for real-time activity screening in a 96-well format. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Trade-offs with stability modulate innate and mutationally acquired drug-resistance in bacterial dihydrofolate reductase enzymes.

PubMed

Matange, Nishad; Bodkhe, Swapnil; Patel, Maitri; Shah, Pooja

2018-06-05

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In this study, we show that trimethoprim-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harbouring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild type making them prone to aggregation and proteolysis. This destabilization is associated with lower expression level resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis we show that perturbation of critical stabilizing hydrophobic interactions in wild type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial DHFRs. Mutational and computational analyses in EcDHFR as well as in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues, modulates TMP-resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to trimethoprim. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme. ©2018 The Author(s).

Insights into the evolution of enzyme substrate promiscuity after the discovery of (βα)₈ isomerase evolutionary intermediates from a diverse metagenome.

PubMed

Noda-García, Lianet; Juárez-Vázquez, Ana L; Ávila-Arcos, María C; Verduzco-Castro, Ernesto A; Montero-Morán, Gabriela; Gaytán, Paul; Carrillo-Tripp, Mauricio; Barona-Gómez, Francisco

2015-06-10

Current sequence-based approaches to identify enzyme functional shifts, such as enzyme promiscuity, have proven to be highly dependent on a priori functional knowledge, hampering our ability to reconstruct evolutionary history behind these mechanisms. Hidden Markov Model (HMM) profiles, broadly used to classify enzyme families, can be useful to distinguish between closely related enzyme families with different specificities. The (βα)8-isomerase HisA/PriA enzyme family, involved in L-histidine (HisA, mono-substrate) biosynthesis in most bacteria and plants, but also in L-tryptophan (HisA/TrpF or PriA, dual-substrate) biosynthesis in most Actinobacteria, has been used as model system to explore evolutionary hypotheses and therefore has a considerable amount of evolutionary, functional and structural knowledge available. We searched for functional evolutionary intermediates between the HisA and PriA enzyme families in order to understand the functional divergence between these families. We constructed a HMM profile that correctly classifies sequences of unknown function into the HisA and PriA enzyme sub-families. Using this HMM profile, we mined a large metagenome to identify plausible evolutionary intermediate sequences between HisA and PriA. These sequences were used to perform phylogenetic reconstructions and to identify functionally conserved amino acids. Biochemical characterization of one selected enzyme (CAM1) with a mutation within the functionally essential N-terminus phosphate-binding site, namely, an alanine instead of a glycine in HisA or a serine in PriA, showed that this evolutionary intermediate has dual-substrate specificity. Moreover, site-directed mutagenesis of this alanine residue, either backwards into a glycine or forward into a serine, revealed the robustness of this enzyme. None of these mutations, presumably upon functionally essential amino acids, significantly abolished its enzyme activities. A truncated version of this enzyme (CAM2) predicted to adopt a (βα)6-fold, and thus entirely lacking a C-terminus phosphate-binding site, was identified and shown to have HisA activity. As expected, reconstruction of the evolution of PriA from HisA with HMM profiles suggest that functional shifts involve mutations in evolutionarily intermediate enzymes of otherwise functionally essential residues or motifs. These results are in agreement with a link between promiscuous enzymes and intragenic epistasis. HMM provides a convenient approach for gaining insights into these evolutionary processes.

Cyanide hydratases and cyanide dihydratases: emerging tools in the biodegradation and biodetection of cyanide.

PubMed

Martínková, Ludmila; Veselá, Alicja Barbara; Rinágelová, Anna; Chmátal, Martin

2015-11-01

The purpose of this study is to summarize the current knowledge of the enzymes which are involved in the hydrolysis of cyanide, i.e., cyanide hydratases (CHTs; EC 4.2.1.66) and cyanide dihydratases (CynD; EC 3.5.5.1). CHTs are probably exclusively produced by filamentous fungi and widely occur in these organisms; in contrast, CynDs were only found in a few bacterial genera. CHTs differ from CynDs in their reaction products (formamide vs. formic acid and ammonia, respectively). Several CHTs were also found to transform nitriles but with lower relative activities compared to HCN. Mutants of CynDs and CHTs were constructed to study the structure-activity relationships in these enzymes or to improve their catalytic properties. The effect of the C-terminal part of the protein on the enzyme activity was determined by constructing the corresponding deletion mutants. CynDs are less active at alkaline pH than CHTs. To improve its bioremediation potential, CynD from Bacillus pumilus was engineered by directed evolution combined with site-directed mutagenesis, and its operation at pH 10 was thus enabled. Some of the enzymes have been tested for their potential to eliminate cyanide from cyanide-containing wastewaters. CynDs were also used to construct cyanide biosensors.

Functional Roles of Slow Enzyme Conformational Changes in Network Dynamics

PubMed Central

Wu, Zhanghan; Xing, Jianhua

2012-01-01

Extensive studies from different fields reveal that many macromolecules, especially enzymes, show slow transitions among different conformations. This phenomenon is named such things as dynamic disorder, heterogeneity, hysteretic or mnemonic enzymes across these different fields, and has been directly demonstrated by single molecule enzymology and NMR studies recently. We analyzed enzyme slow conformational changes in the context of regulatory networks. A single enzymatic reaction with slow conformational changes can filter upstream network noises, and can either resonantly respond to the system stimulus at certain frequencies or respond adaptively for sustained input signals of the network fluctuations. It thus can serve as a basic functional motif with properties that are normally for larger intermolecular networks in the field of systems biology. We further analyzed examples including enzymes functioning against pH fluctuations, metabolic state change of Artemia embryos, and kinetic insulation of fluctuations in metabolic networks. The study also suggests that hysteretic enzymes may be building blocks of synthetic networks with various properties such as narrow-banded filtering. The work fills the missing gap between studies on enzyme biophysics and network level dynamics, and reveals that the coupling between the two is functionally important; it also suggests that the conformational dynamics of some enzymes may be evolutionally selected. PMID:23009855

Carbon-sulfur bond-forming reaction catalysed by the radical SAM enzyme HydE.

PubMed

Rohac, Roman; Amara, Patricia; Benjdia, Alhosna; Martin, Lydie; Ruffié, Pauline; Favier, Adrien; Berteau, Olivier; Mouesca, Jean-Marie; Fontecilla-Camps, Juan C; Nicolet, Yvain

2016-05-01

Carbon-sulfur bond formation at aliphatic positions is a challenging reaction that is performed efficiently by radical S-adenosyl-L-methionine (SAM) enzymes. Here we report that 1,3-thiazolidines can act as ligands and substrates for the radical SAM enzyme HydE, which is involved in the assembly of the active site of [FeFe]-hydrogenase. Using X-ray crystallography, in vitro assays and NMR spectroscopy we identified a radical-based reaction mechanism that is best described as the formation of a C-centred radical that concomitantly attacks the sulfur atom of a thioether. To the best of our knowledge, this is the first example of a radical SAM enzyme that reacts directly on a sulfur atom instead of abstracting a hydrogen atom. Using theoretical calculations based on our high-resolution structures we followed the evolution of the electronic structure from SAM through to the formation of S-adenosyl-L-cysteine. Our results suggest that, at least in this case, the widely proposed and highly reactive 5'-deoxyadenosyl radical species that triggers the reaction in radical SAM enzymes is not an isolable intermediate.

Carbon-sulfur bond-forming reaction catalysed by the radical SAM enzyme HydE

NASA Astrophysics Data System (ADS)

Rohac, Roman; Amara, Patricia; Benjdia, Alhosna; Martin, Lydie; Ruffié, Pauline; Favier, Adrien; Berteau, Olivier; Mouesca, Jean-Marie; Fontecilla-Camps, Juan C.; Nicolet, Yvain

2016-05-01

Carbon-sulfur bond formation at aliphatic positions is a challenging reaction that is performed efficiently by radical S-adenosyl-L-methionine (SAM) enzymes. Here we report that 1,3-thiazolidines can act as ligands and substrates for the radical SAM enzyme HydE, which is involved in the assembly of the active site of [FeFe]-hydrogenase. Using X-ray crystallography, in vitro assays and NMR spectroscopy we identified a radical-based reaction mechanism that is best described as the formation of a C-centred radical that concomitantly attacks the sulfur atom of a thioether. To the best of our knowledge, this is the first example of a radical SAM enzyme that reacts directly on a sulfur atom instead of abstracting a hydrogen atom. Using theoretical calculations based on our high-resolution structures we followed the evolution of the electronic structure from SAM through to the formation of S-adenosyl-L-cysteine. Our results suggest that, at least in this case, the widely proposed and highly reactive 5‧-deoxyadenosyl radical species that triggers the reaction in radical SAM enzymes is not an isolable intermediate.

Evolutionary dynamics of enzymes.

PubMed

Demetrius, L

1995-08-01

This paper codifies and rationalizes the large diversity in reaction rates and substrate specificity of enzymes in terms of a model which postulates that the kinetic properties of present-day enzymes are the consequence of the evolutionary force of mutation and selection acting on a class of primordial enzymes with poor catalytic activity and broad substrate specificity. Enzymes are classified in terms of their thermodynamic parameters, activation enthalpy delta H* and activation entropy delta S*, in their kinetically significant transition states as follows: type 1, delta H* > 0, delta S* < 0; type 2, delta H* < or = 0, delta S* < or = 0; type 3, delta H* > 0, delta S* > 0. We study the evolutionary dynamics of these three classes of enzymes subject to mutation, which acts at the level of the gene which codes for the enzyme and selection, which acts on the organism that contains the enzyme. Our model predicts the following evolutionary trends in the reaction rate and binding specificity for the three classes of molecules. In type 1 enzymes, evolution results in random, non-directional changes in the reaction rate and binding specificity. In type 2 and 3 enzymes, evolution results in a unidirectional increase in both the reaction rate and binding specificity. We exploit these results in order to codify the diversity in functional properties of present-day enzymes. Type 1 molecules will be described by intermediate reaction rates and broad substrate specificity. Type 2 enzymes will be characterized by diffusion-controlled rates and absolute substrate specificity. The type 3 catalysts can be further subdivided in terms of their activation enthalpy into two classes: type 3a (delta H* small) and type 3b (delta H* large). We show that type 3a will be represented by the same functional properties that identify type 2, namely, diffusion-controlled rates and absolute substrate specificity, whereas type 3b will be characterized by non-diffusion-controlled rates and absolute substrate specificity. We infer from this depiction of the three classes of enzymes, a general relation between the two functional properties, reaction rate and substrate specificity, namely, enzymes with diffusion-controlled rates have absolute substrate specificity. By appealing to energetic considerations, we furthermore show that enzymes with diffusion-controlled rates (types 2 and 3a) form a small subset of the class of all enzymes. This codification of present-day enzymes derived from an evolutionary model, essentially relates the structural properties of enzymes, as described by their thermodynamic parameters, to their functional properties, as represented by the reaction rate and substrate specificity.

Methodology Development in Directed Evolution: Exploring Options when Applying Triple-Code Saturation Mutagenesis.

PubMed

Qu, Ge; Lonsdale, Richard; Yao, Peiyuan; Li, Guangyue; Liu, Beibei; Reetz, Manfred T; Sun, Zhoutong

2018-02-02

Directed evolution of stereo- or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building "small but smart" mutant libraries have been developed. Herein, we compared two different strategies regarding the application of triple-code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino-acid alphabets. By using the synthetically difficult-to-reduce prochiral ketone tetrahydrofuran-3-one as a substrate, highly R- and S-selective variants were obtained (92-99 % ee) with minimal screening. The origin of stereoselectivity was provided by molecular dynamics analyses, which is discussed in terms of the Bürgi-Dunitz trajectory. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular evolution of the Li/li chemical defence polymorphism in white clover (Trifolium repens L.).

PubMed

Olsen, K M; Sutherland, B L; Small, L L

2007-10-01

White clover (Trifolium repens) is naturally polymorphic for cyanogenesis (hydrogen cyanide release following tissue damage). The ecological factors favouring cyanogenic and acyanogenic plants have been examined in numerous studies over the last half century, making this one of the best-documented examples of an adaptive polymorphism in plants. White clover cyanogenesis is controlled by two, independently segregating Mendelian genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolysing enzyme, linamarase. In this study, we examine the molecular evolution and population genetics of Li as it relates to the cyanogenesis polymorphism. We report here that Li exists as a single-copy gene in plants possessing linamarase activity, and that the absence of enzyme activity in li/li plants is correlated with the absence of much or all of the gene from the white clover genome. Consistent with this finding, we confirm by reverse transcription-polymerase chain reaction that Li gene expression is absent in plants lacking enzyme activity. In a molecular population genetic analysis of Li and three unlinked genes using a worldwide sample of clover plants, we find an absence of nucleotide variation and statistically significant deviations from neutrality at Li; these findings are consistent with recent positive directional selection at this cyanogenesis locus.

Expanding P450 catalytic reaction space through evolution and engineering

PubMed Central

McIntosh, John A.; Farwell, Christopher C.; Arnold, Frances H.

2014-01-01

Advances in protein and metabolic engineering have led to wider use of enzymes to synthesize important molecules. However, many desirable transformations are not catalyzed by any known enzyme, driving interest in understanding how new enzymes can be created. The cytochrome P450 enzyme family, whose members participate in xenobiotic metabolism and natural products biosynthesis, catalyzes an impressive range of difficult chemical reactions that continues to grow as new enzymes are characterized. Recent work has revealed that P450-derived enzymes can also catalyze useful reactions previously accessible only to synthetic chemistry. The evolution and engineering of these enzymes provides an excellent case study for how to genetically encode new chemistry and expand biology’s reaction space. PMID:24658056

From inanimate matter to living systems

NASA Technical Reports Server (NTRS)

Fox, S. W.

1980-01-01

Since the early part of this century, the Genesis account of the origin and evolution of life has been explained as an extrapolation of astronomical and geochemical processes. The essence of the answer to date is a protoreproductive protocell of much biochemical and cytophysical competance. The processes of its origin, molecular ordering, and its functions are described. A crucial understanding is that of the nonrandomness of evolutionary processes at all stages (with perhaps a minor statistical component). In this way, evolution conflicts with statistical randomness; the latter is a favorite assumption of both scientific and creationistic critics of the proteinoid theory. The principle contribution of the proteinoid theory to the understanding of general biology is to particularize the view that evolutionary direction is rooted in the shapes of molecules, in stereochemistry. After molecules of the right kind first assembled to protocells, life in its various stages of evolution was an inevitable consequence. It is molecules that continue to assemble as part of living process and, in the role of enzymes, continue to direct life cycle of the cell.

Characterization of Function of the GlgA2 Glycogen/Starch Synthase in Cyanobacterium sp. Clg1 Highlights Convergent Evolution of Glycogen Metabolism into Starch Granule Aggregation1

PubMed Central

Kadouche, Derifa; Arias, Maria Cecilia

2016-01-01

At variance with the starch-accumulating plants and most of the glycogen-accumulating cyanobacteria, Cyanobacterium sp. CLg1 synthesizes both glycogen and starch. We now report the selection of a starchless mutant of this cyanobacterium that retains wild-type amounts of glycogen. Unlike other mutants of this type found in plants and cyanobacteria, this mutant proved to be selectively defective for one of the two types of glycogen/starch synthase: GlgA2. This enzyme is phylogenetically related to the previously reported SSIII/SSIV starch synthase that is thought to be involved in starch granule seeding in plants. This suggests that, in addition to the selective polysaccharide debranching demonstrated to be responsible for starch rather than glycogen synthesis, the nature and properties of the elongation enzyme define a novel determinant of starch versus glycogen accumulation. We show that the phylogenies of GlgA2 and of 16S ribosomal RNA display significant congruence. This suggests that this enzyme evolved together with cyanobacteria when they diversified over 2 billion years ago. However, cyanobacteria can be ruled out as direct progenitors of the SSIII/SSIV ancestral gene found in Archaeplastida. Hence, both cyanobacteria and plants recruited similar enzymes independently to perform analogous tasks, further emphasizing the importance of convergent evolution in the appearance of starch from a preexisting glycogen metabolism network. PMID:27208262

Characterization of Function of the GlgA2 Glycogen/Starch Synthase in Cyanobacterium sp. Clg1 Highlights Convergent Evolution of Glycogen Metabolism into Starch Granule Aggregation.

PubMed

Kadouche, Derifa; Ducatez, Mathieu; Cenci, Ugo; Tirtiaux, Catherine; Suzuki, Eiji; Nakamura, Yasunori; Putaux, Jean-Luc; Terrasson, Amandine Durand; Diaz-Troya, Sandra; Florencio, Francisco Javier; Arias, Maria Cecilia; Striebeck, Alexander; Palcic, Monica; Ball, Steven G; Colleoni, Christophe

2016-07-01

At variance with the starch-accumulating plants and most of the glycogen-accumulating cyanobacteria, Cyanobacterium sp. CLg1 synthesizes both glycogen and starch. We now report the selection of a starchless mutant of this cyanobacterium that retains wild-type amounts of glycogen. Unlike other mutants of this type found in plants and cyanobacteria, this mutant proved to be selectively defective for one of the two types of glycogen/starch synthase: GlgA2. This enzyme is phylogenetically related to the previously reported SSIII/SSIV starch synthase that is thought to be involved in starch granule seeding in plants. This suggests that, in addition to the selective polysaccharide debranching demonstrated to be responsible for starch rather than glycogen synthesis, the nature and properties of the elongation enzyme define a novel determinant of starch versus glycogen accumulation. We show that the phylogenies of GlgA2 and of 16S ribosomal RNA display significant congruence. This suggests that this enzyme evolved together with cyanobacteria when they diversified over 2 billion years ago. However, cyanobacteria can be ruled out as direct progenitors of the SSIII/SSIV ancestral gene found in Archaeplastida. Hence, both cyanobacteria and plants recruited similar enzymes independently to perform analogous tasks, further emphasizing the importance of convergent evolution in the appearance of starch from a preexisting glycogen metabolism network. © 2016 American Society of Plant Biologists. All Rights Reserved.

Accessing Nature’s diversity through metabolic engineering and synthetic biology

PubMed Central

King, Jason R.; Edgar, Steven; Qiao, Kangjian; Stephanopoulos, Gregory

2016-01-01

In this perspective, we highlight recent examples and trends in metabolic engineering and synthetic biology that demonstrate the synthetic potential of enzyme and pathway engineering for natural product discovery. In doing so, we introduce natural paradigms of secondary metabolism whereby simple carbon substrates are combined into complex molecules through “scaffold diversification”, and subsequent “derivatization” of these scaffolds is used to synthesize distinct complex natural products. We provide examples in which modern pathway engineering efforts including combinatorial biosynthesis and biological retrosynthesis can be coupled to directed enzyme evolution and rational enzyme engineering to allow access to the “privileged” chemical space of natural products in industry-proven microbes. Finally, we forecast the potential to produce natural product-like discovery platforms in biological systems that are amenable to single-step discovery, validation, and synthesis for streamlined discovery and production of biologically active agents. PMID:27081481

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunden, Fanny; Peck, Ariana; Salzman, Julia

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modesmore » of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.« less

Neofunctionalization of a duplicate hatching enzyme gene during the evolution of teleost fishes.

PubMed

Sano, Kaori; Kawaguchi, Mari; Watanabe, Satoshi; Yasumasu, Shigeki

2014-10-19

Duplication and subsequent neofunctionalization of the teleostean hatching enzyme gene occurred in the common ancestor of Euteleostei and Otocephala, producing two genes belonging to different phylogenetic clades (clade I and II). In euteleosts, the clade I enzyme inherited the activity of the ancestral enzyme of swelling the egg envelope by cleavage of the N-terminal region of egg envelope proteins. The clade II enzyme gained two specific cleavage sites, N-ZPd and mid-ZPd but lost the ancestral activity. Thus, euteleostean clade II enzymes assumed a new function; solubilization of the egg envelope by the cooperative action with clade I enzyme. However, in Otocephala, the clade II gene was lost during evolution. Consequently, in a late group of Otocephala, only the clade I enzyme is present to swell the egg envelope. We evaluated the egg envelope digestion properties of clade I and II enzymes in Gonorynchiformes, an early diverging group of Otocephala, using milkfish, and compared their digestion with those of other fishes. Finally, we propose a hypothesis of the neofunctionalization process. The milkfish clade II enzyme cleaved N-ZPd but not mid-ZPd, and did not cause solubilization of the egg envelope. We conclude that neofunctionalization is incomplete in the otocephalan clade II enzymes. Comparison of clade I and clade II enzyme characteristics implies that the specificity of the clade II enzymes gradually changed during evolution after the duplication event, and that a change in substrate was required for the addition of the mid-ZPd site and loss of activity at the N-terminal region. We infer the process of neofunctionalization of the clade II enzyme after duplication of the gene. The ancestral clade II gene gained N-ZPd cleavage activity in the common ancestral lineage of the Euteleostei and Otocephala. Subsequently, acquisition of cleavage activity at the mid-ZPd site and loss of cleavage activity in the N-terminal region occurred during the evolution of Euteleostei, but not of Otocephala. The clade II enzyme provides an example of the development of a neofunctional gene for which the substrate, the egg envelope protein, has adapted to a gradual change in the specificity of the corresponding enzyme.

Positive Selection in Rapidly Evolving Plastid–Nuclear Enzyme Complexes

PubMed Central

Rockenbach, Kate; Havird, Justin C.; Monroe, J. Grey; Triant, Deborah A.; Taylor, Douglas R.; Sloan, Daniel B.

2016-01-01

Rates of sequence evolution in plastid genomes are generally low, but numerous angiosperm lineages exhibit accelerated evolutionary rates in similar subsets of plastid genes. These genes include clpP1 and accD, which encode components of the caseinolytic protease (CLP) and acetyl-coA carboxylase (ACCase) complexes, respectively. Whether these extreme and repeated accelerations in rates of plastid genome evolution result from adaptive change in proteins (i.e., positive selection) or simply a loss of functional constraint (i.e., relaxed purifying selection) is a source of ongoing controversy. To address this, we have taken advantage of the multiple independent accelerations that have occurred within the genus Silene (Caryophyllaceae) by examining phylogenetic and population genetic variation in the nuclear genes that encode subunits of the CLP and ACCase complexes. We found that, in species with accelerated plastid genome evolution, the nuclear-encoded subunits in the CLP and ACCase complexes are also evolving rapidly, especially those involved in direct physical interactions with plastid-encoded proteins. A massive excess of nonsynonymous substitutions between species relative to levels of intraspecific polymorphism indicated a history of strong positive selection (particularly in CLP genes). Interestingly, however, some species are likely undergoing loss of the native (heteromeric) plastid ACCase and putative functional replacement by a duplicated cytosolic (homomeric) ACCase. Overall, the patterns of molecular evolution in these plastid–nuclear complexes are unusual for anciently conserved enzymes. They instead resemble cases of antagonistic coevolution between pathogens and host immune genes. We discuss a possible role of plastid–nuclear conflict as a novel cause of accelerated evolution. PMID:27707788

On the Temperature Dependence of Enzyme-Catalyzed Rates.

PubMed

Arcus, Vickery L; Prentice, Erica J; Hobbs, Joanne K; Mulholland, Adrian J; Van der Kamp, Marc W; Pudney, Christopher R; Parker, Emily J; Schipper, Louis A

2016-03-29

One of the critical variables that determine the rate of any reaction is temperature. For biological systems, the effects of temperature are convoluted with myriad (and often opposing) contributions from enzyme catalysis, protein stability, and temperature-dependent regulation, for example. We have coined the phrase "macromolecular rate theory (MMRT)" to describe the temperature dependence of enzyme-catalyzed rates independent of stability or regulatory processes. Central to MMRT is the observation that enzyme-catalyzed reactions occur with significant values of ΔCp(‡) that are in general negative. That is, the heat capacity (Cp) for the enzyme-substrate complex is generally larger than the Cp for the enzyme-transition state complex. Consistent with a classical description of enzyme catalysis, a negative value for ΔCp(‡) is the result of the enzyme binding relatively weakly to the substrate and very tightly to the transition state. This observation of negative ΔCp(‡) has important implications for the temperature dependence of enzyme-catalyzed rates. Here, we lay out the fundamentals of MMRT. We present a number of hypotheses that arise directly from MMRT including a theoretical justification for the large size of enzymes and the basis for their optimum temperatures. We rationalize the behavior of psychrophilic enzymes and describe a "psychrophilic trap" which places limits on the evolution of enzymes in low temperature environments. One of the defining characteristics of biology is catalysis of chemical reactions by enzymes, and enzymes drive much of metabolism. Therefore, we also expect to see characteristics of MMRT at the level of cells, whole organisms, and even ecosystems.

Strategies for discovery and improvement of enzyme function: state of the art and opportunities

PubMed Central

Kaul, Praveen; Asano, Yasuhisa

2012-01-01

Summary Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non‐conventional media, using new substrates for old enzymes, active‐site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities. PMID:21883976

Strategies for discovery and improvement of enzyme function: state of the art and opportunities.

PubMed

Kaul, Praveen; Asano, Yasuhisa

2012-01-01

Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non-conventional media, using new substrates for old enzymes, active-site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities. © 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Role of conformational dynamics in the evolution of novel enzyme function.

PubMed

Maria-Solano, Miguel A; Serrano-Hervás, Eila; Romero-Rivera, Adrian; Iglesias-Fernández, Javier; Osuna, Sílvia

2018-05-21

The free energy landscape concept that describes enzymes as an ensemble of differently populated conformational sub-states in dynamic equilibrium is key for evaluating enzyme activity, enantioselectivity, and specificity. Mutations introduced in the enzyme sequence can alter the populations of the pre-existing conformational states, thus strongly modifying the enzyme ability to accommodate alternative substrates, revert its enantiopreferences, and even increase the activity for some residual promiscuous reactions. In this feature article, we present an overview of the current experimental and computational strategies to explore the conformational free energy landscape of enzymes. We provide a series of recent publications that highlight the key role of conformational dynamics for the enzyme evolution towards new functions and substrates, and provide some perspectives on how conformational dynamism should be considered in future computational enzyme design protocols.

Characterizing the roles of changing population size and selection on the evolution of flux control in metabolic pathways.

PubMed

Orlenko, Alena; Chi, Peter B; Liberles, David A

2017-05-25

Understanding the genotype-phenotype map is fundamental to our understanding of genomes. Genes do not function independently, but rather as part of networks or pathways. In the case of metabolic pathways, flux through the pathway is an important next layer of biological organization up from the individual gene or protein. Flux control in metabolic pathways, reflecting the importance of mutation to individual enzyme genes, may be evolutionarily variable due to the role of mutation-selection-drift balance. The evolutionary stability of rate limiting steps and the patterns of inter-molecular co-evolution were evaluated in a simulated pathway with a system out of equilibrium due to fluctuating selection, population size, or positive directional selection, to contrast with those under stabilizing selection. Depending upon the underlying population genetic regime, fluctuating population size was found to increase the evolutionary stability of rate limiting steps in some scenarios. This result was linked to patterns of local adaptation of the population. Further, during positive directional selection, as with more complex mutational scenarios, an increase in the observation of inter-molecular co-evolution was observed. Differences in patterns of evolution when systems are in and out of equilibrium, including during positive directional selection may lead to predictable differences in observed patterns for divergent evolutionary scenarios. In particular, this result might be harnessed to detect differences between compensatory processes and directional processes at the pathway level based upon evolutionary observations in individual proteins. Detecting functional shifts in pathways reflects an important milestone in predicting when changes in genotypes result in changes in phenotypes.

Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts.

PubMed

Bernal, Claudia; Rodríguez, Karen; Martínez, Ronny

2018-06-09

Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, -thus not reusable- and their performance under real operational conditions is uncertain. Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts. In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel "immobilized biocatalyst engineering" research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications. Copyright © 2018. Published by Elsevier Inc.

Evolution of the ubiquitin-activating enzyme Uba1 (E1)

NASA Astrophysics Data System (ADS)

Allan, Douglas C.; Phillips, J. C.

2017-10-01

Ubiquitin tags diseased proteins and initiates an enzyme conjugation cascade, which has three stages. The first-stage enzyme Uba1 (E1) has evolved only modestly from slime mold to humans, and is > 14 times larger than Ub. Here we use critical point thermodynamic scaling theory to connect Uba1 (E1) evolution from yeast and slime mold to fruit flies and humans to subtle changes in its amino acid sequences.

Role of Petal-Specific Orcinol O-Methyltransferases in the Evolution of Rose Scent1

PubMed Central

Scalliet, Gabriel; Lionnet, Claire; Le Bechec, Mickaël; Dutron, Laurence; Magnard, Jean-Louis; Baudino, Sylvie; Bergougnoux, Véronique; Jullien, Frédéric; Chambrier, Pierre; Vergne, Philippe; Dumas, Christian; Cock, J. Mark; Hugueney, Philippe

2006-01-01

Orcinol O-methyltransferase (OOMT) 1 and 2 catalyze the last two steps of the biosynthetic pathway leading to the phenolic methyl ether 3,5-dimethoxytoluene (DMT), the major scent compound of many rose (Rosa x hybrida) varieties. Modern roses are descended from both European and Chinese species, the latter being producers of phenolic methyl ethers but not the former. Here we investigated why phenolic methyl ether production occurs in some but not all rose varieties. In DMT-producing varieties, OOMTs were shown to be localized specifically in the petal, predominanty in the adaxial epidermal cells. In these cells, OOMTs become increasingly associated with membranes during petal development, suggesting that the scent biosynthesis pathway catalyzed by these enzymes may be directly linked to the cells' secretory machinery. OOMT gene sequences were detected in two non-DMT-producing rose species of European origin, but no mRNA transcripts were detected, and these varieties lacked both OOMT protein and enzyme activity. These data indicate that up-regulation of OOMT gene expression may have been a critical step in the evolution of scent production in roses. PMID:16361520

Facile Construction of Random Gene Mutagenesis Library for Directed Evolution Without the Use of Restriction Enzyme in Escherichia coli.

PubMed

Kim, Jae-Eung; Huang, Rui; Chen, Hui; You, Chun; Zhang, Y-H Percival

2016-09-01

A foolproof protocol was developed for the construction of mutant DNA library for directed protein evolution. First, a library of linear mutant gene was generated by error-prone PCR or molecular shuffling, and a linear vector backbone was prepared by high-fidelity PCR. Second, the amplified insert and vector fragments were assembled by overlap-extension PCR with a pair of 5'-phosphorylated primers. Third, full-length linear plasmids with phosphorylated 5'-ends were self-ligated with T4 ligase, yielding circular plasmids encoding mutant variants suitable for high-efficiency transformation. Self-made competent Escherichia coli BL21(DE3) showed a transformation efficiency of 2.4 × 10(5) cfu/µg of the self-ligated circular plasmid. Using this method, three mutants of mCherry fluorescent protein were found to alter their colors and fluorescent intensities under visible and UV lights, respectively. Also, one mutant of 6-phosphorogluconate dehydrogenase from a thermophilic bacterium Moorella thermoacetica was found to show the 3.5-fold improved catalytic efficiency (kcat /Km ) on NAD(+) as compared to the wild-type. This protocol is DNA-sequence independent, and does not require restriction enzymes, special E. coli host, or labor-intensive optimization. In addition, this protocol can be used for subcloning the relatively long DNA sequences into any position of plasmids. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Large-Scale Analysis Exploring Evolution of Catalytic Machineries and Mechanisms in Enzyme Superfamilies.

PubMed

Furnham, Nicholas; Dawson, Natalie L; Rahman, Syed A; Thornton, Janet M; Orengo, Christine A

2016-01-29

Enzymes, as biological catalysts, form the basis of all forms of life. How these proteins have evolved their functions remains a fundamental question in biology. Over 100 years of detailed biochemistry studies, combined with the large volumes of sequence and protein structural data now available, means that we are able to perform large-scale analyses to address this question. Using a range of computational tools and resources, we have compiled information on all experimentally annotated changes in enzyme function within 379 structurally defined protein domain superfamilies, linking the changes observed in functions during evolution to changes in reaction chemistry. Many superfamilies show changes in function at some level, although one function often dominates one superfamily. We use quantitative measures of changes in reaction chemistry to reveal the various types of chemical changes occurring during evolution and to exemplify these by detailed examples. Additionally, we use structural information of the enzymes active site to examine how different superfamilies have changed their catalytic machinery during evolution. Some superfamilies have changed the reactions they perform without changing catalytic machinery. In others, large changes of enzyme function, in terms of both overall chemistry and substrate specificity, have been brought about by significant changes in catalytic machinery. Interestingly, in some superfamilies, relatives perform similar functions but with different catalytic machineries. This analysis highlights characteristics of functional evolution across a wide range of superfamilies, providing insights that will be useful in predicting the function of uncharacterised sequences and the design of new synthetic enzymes. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

[Application of bioinformatics in researches of industrial biocatalysis].

PubMed

Yu, Hui-Min; Luo, Hui; Shi, Yue; Sun, Xu-Dong; Shen, Zhong-Yao

2004-05-01

Industrial biocatalysis is currently attracting much attention to rebuild or substitute traditional producing process of chemicals and drugs. One of key focuses in industrial biocatalysis is biocatalyst, which is usually one kind of microbial enzyme. In the recent, new technologies of bioinformatics have played and will continue to play more and more significant roles in researches of industrial biocatalysis in response to the waves of genomic revolution. One of the key applications of bioinformatics in biocatalysis is the discovery and identification of the new biocatalyst through advanced DNA and protein sequence search, comparison and analyses in Internet database using different algorithm and software. The unknown genes of microbial enzymes can also be simply harvested by primer design on the basis of bioinformatics analyses. The other key applications of bioinformatics in biocatalysis are the modification and improvement of existing industrial biocatalyst. In this aspect, bioinformatics is of great importance in both rational design and directed evolution of microbial enzymes. Based on the successful prediction of tertiary structures of enzymes using the tool of bioinformatics, the undermentioned experiments, i.e. site-directed mutagenesis, fusion protein construction, DNA family shuffling and saturation mutagenesis, etc, are usually of very high efficiency. On all accounts, bioinformatics will be an essential tool for either biologist or biological engineer in the future researches of industrial biocatalysis, due to its significant function in guiding and quickening the step of discovery and/or improvement of novel biocatalysts.

Ancient origin and recent innovations of RNA polymerase IV and V

DOE PAGES

Huang, Yi; Kendall, Timmy; Forsythe, Evan S.; ...

2015-03-12

Small RNA-mediated chromatin modification is a conserved feature of eukaryotes. In flowering plants, the short interfering (si)RNAs that direct transcriptional silencing are abundant and subfunctionalization has led to specialized machinery responsible for synthesis and action of these small RNAs. In particular, plants possess polymerase (Pol) IV and Pol V, multi-subunit homologs of the canonical DNA-dependent RNA Pol II, as well as specialized members of the RNA-dependent RNA Polymerase (RDR), Dicer-like (DCL), and Argonaute (AGO) families. Together these enzymes are required for production and activity of Pol IV-dependent (p4-)siRNAs, which trigger RNA-directed DNA methylation (RdDM) at homologous sequences. p4-siRNAs accumulate highlymore » in developing endosperm, a specialized tissue found only in flowering plants, and are rare in nonflowering plants, suggesting that the evolution of flowers might coincide with the emergence of specialized RdDM machinery. Through comprehensive identification of RdDM genes from species representing the breadth of the land plant phylogeny, we describe the ancient origin of Pol IV and Pol V, suggesting that a nearly complete and functional RdDM pathway could have existed in the earliest land plants. We also uncover innovations in these enzymes that are coincident with the emergence of seed plants and flowering plants, and recent duplications that might indicate additional subfunctionalization. Phylogenetic analysis reveals rapid evolution of Pol IV and Pol V subunits relative to their Pol II counterparts and suggests that duplicates were retained and subfunctionalized through Escape from Adaptive Conflict. Finally, evolution within the carboxy-terminal domain of the Pol V largest subunit is particularly striking, where illegitimate recombination facilitated extreme sequence divergence.« less

Ancient origin and recent innovations of RNA polymerase IV and V

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Yi; Kendall, Timmy; Forsythe, Evan S.

Small RNA-mediated chromatin modification is a conserved feature of eukaryotes. In flowering plants, the short interfering (si)RNAs that direct transcriptional silencing are abundant and subfunctionalization has led to specialized machinery responsible for synthesis and action of these small RNAs. In particular, plants possess polymerase (Pol) IV and Pol V, multi-subunit homologs of the canonical DNA-dependent RNA Pol II, as well as specialized members of the RNA-dependent RNA Polymerase (RDR), Dicer-like (DCL), and Argonaute (AGO) families. Together these enzymes are required for production and activity of Pol IV-dependent (p4-)siRNAs, which trigger RNA-directed DNA methylation (RdDM) at homologous sequences. p4-siRNAs accumulate highlymore » in developing endosperm, a specialized tissue found only in flowering plants, and are rare in nonflowering plants, suggesting that the evolution of flowers might coincide with the emergence of specialized RdDM machinery. Through comprehensive identification of RdDM genes from species representing the breadth of the land plant phylogeny, we describe the ancient origin of Pol IV and Pol V, suggesting that a nearly complete and functional RdDM pathway could have existed in the earliest land plants. We also uncover innovations in these enzymes that are coincident with the emergence of seed plants and flowering plants, and recent duplications that might indicate additional subfunctionalization. Phylogenetic analysis reveals rapid evolution of Pol IV and Pol V subunits relative to their Pol II counterparts and suggests that duplicates were retained and subfunctionalized through Escape from Adaptive Conflict. Finally, evolution within the carboxy-terminal domain of the Pol V largest subunit is particularly striking, where illegitimate recombination facilitated extreme sequence divergence.« less

Research and Application of Marine Microbial Enzymes: Status and Prospects

PubMed Central

Zhang, Chen; Kim, Se-Kwon

2010-01-01

Over billions of years, the ocean has been regarded as the origin of life on Earth. The ocean includes the largest range of habitats, hosting the most life-forms. Competition amongst microorganisms for space and nutrients in the marine environment is a powerful selective force, which has led to evolution. The evolution prompted the marine microorganisms to generate multifarious enzyme systems to adapt to the complicated marine environments. Therefore, marine microbial enzymes can offer novel biocatalysts with extraordinary properties. This review deals with the research and development work investigating the occurrence and bioprocessing of marine microbial enzymes. PMID:20631875

Directed evolution for thermostabilization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus.

PubMed

Sugimoto, Naohisa; Takakura, Yasuaki; Shiraki, Kentaro; Honda, Shinya; Takaya, Naoki; Hoshino, Takayuki; Nakamura, Akira

2013-01-01

To obtain a selection marker gene functional in a thermophilic bacterium, Thermus thermophilus, an in vivo-directed evolutionary strategy was conducted on a hygromycin B phosphotransferase gene (hyg) from Streptomyces hygroscopicus. The expression of wild-type hyg in T. thermophilus provided hygromycin B (HygB) resistance up to 60 °C. Through selection of mutants showing HygB resistance at higher temperatures, eight amino acid substitutions and the duplication of three amino acids were identified. A variant containing seven substitutions and the duplication (HYG10) showed HygB resistance at a highest temperature of 74 °C. Biochemical and biophysical analyses of recombinant HYG and HYG10 revealed that HYG10 was in fact thermostabilized. Modeling of the three-dimensional structure of HYG10 suggests the possible roles of the various substitutions and the duplication on thermostabilization, of which three substitutions and the duplication located at the enzyme surface suggested that these mutations made the enzyme more hydrophilic and provided increased stability in aqueous solution.

Precision is essential for efficient catalysis in an evolved Kemp eliminase.

PubMed

Blomberg, Rebecca; Kries, Hajo; Pinkas, Daniel M; Mittl, Peer R E; Grütter, Markus G; Privett, Heidi K; Mayo, Stephen L; Hilvert, Donald

2013-11-21

Linus Pauling established the conceptual framework for understanding and mimicking enzymes more than six decades ago. The notion that enzymes selectively stabilize the rate-limiting transition state of the catalysed reaction relative to the bound ground state reduces the problem of design to one of molecular recognition. Nevertheless, past attempts to capitalize on this idea, for example by using transition state analogues to elicit antibodies with catalytic activities, have generally failed to deliver true enzymatic rates. The advent of computational design approaches, combined with directed evolution, has provided an opportunity to revisit this problem. Starting from a computationally designed catalyst for the Kemp elimination--a well-studied model system for proton transfer from carbon--we show that an artificial enzyme can be evolved that accelerates an elementary chemical reaction 6 × 10(8)-fold, approaching the exceptional efficiency of highly optimized natural enzymes such as triosephosphate isomerase. A 1.09 Å resolution crystal structure of the evolved enzyme indicates that familiar catalytic strategies such as shape complementarity and precisely placed catalytic groups can be successfully harnessed to afford such high rate accelerations, making us optimistic about the prospects of designing more sophisticated catalysts.

Enzyme stabilization via computationally guided protein stapling.

PubMed

Moore, Eric J; Zorine, Dmitri; Hansen, William A; Khare, Sagar D; Fasan, Rudi

2017-11-21

Thermostabilization represents a critical and often obligatory step toward enhancing the robustness of enzymes for organic synthesis and other applications. While directed evolution methods have provided valuable tools for this purpose, these protocols are laborious and time-consuming and typically require the accumulation of several mutations, potentially at the expense of catalytic function. Here, we report a minimally invasive strategy for enzyme stabilization that relies on the installation of genetically encoded, nonreducible covalent staples in a target protein scaffold using computational design. This methodology enables the rapid development of myoglobin-based cyclopropanation biocatalysts featuring dramatically enhanced thermostability (Δ T m = +18.0 °C and Δ T 50 = +16.0 °C) as well as increased stability against chemical denaturation [Δ C m (GndHCl) = 0.53 M], without altering their catalytic efficiency and stereoselectivity properties. In addition, the stabilized variants offer superior performance and selectivity compared with the parent enzyme in the presence of a high concentration of organic cosolvents, enabling the more efficient cyclopropanation of a water-insoluble substrate. This work introduces and validates an approach for protein stabilization which should be applicable to a variety of other proteins and enzymes.

The limits to biocatalysis: pushing the envelope.

PubMed

Sheldon, Roger A; Brady, Dean

2018-06-12

In the period 1985 to 1995 applications of biocatalysis, driven by the need for more sustainable manufacture of chemicals and catalytic, (enantio)selective methods for the synthesis of pharmaceutical intermediates, largely involved the available hydrolases. This was followed, in the next two decades, by revolutionary developments in protein engineering and directed evolution for the optimisation of enzyme function and performance that totally changed the biocatalysis landscape. In the same period, metabolic engineering and synthetic biology revolutionised the use of whole cell biocatalysis in the synthesis of commodity chemicals by fermentation. In particular, developments in the enzymatic enantioselective synthesis of chiral alcohols and amines are highlighted. Progress in enzyme immobilisation facilitated applications under harsh industrial conditions, such as in organic solvents. The emergence of biocatalytic or chemoenzymatic cascade processes, often with co-immobilised enzymes, has enabled telescoping of multi-step processes. Discovering and inventing new biocatalytic processes, based on (meta)genomic sequencing, evolving enzyme promiscuity, chemomimetic biocatalysis, artificial metalloenzymes, and the introduction of non-canonical amino acids into proteins, are pushing back the limits of biocatalysis function. Finally, the integral role of biocatalysis in developing a biobased carbon-neutral economy is discussed.

Enzymatic analysis of a thermostabilized mutant of an Escherichia coli hygromycin B phosphotransferase.

PubMed

Nakamura, Akira; Takakura, Yasuaki; Sugimoto, Naohisa; Takaya, Naoki; Shiraki, Kentaro; Hoshino, Takayuki

2008-09-01

An Escherichia coli hygromycin B phosphotransferase (HPH) and its thermostabilized mutant protein, HPH5, containing five amino acid substitutions, D20G, A118V, S225P, Q226L, and T246A (Nakamura et al., J. Biosci. Bioeng., 100, 158-163 (2005)), obtained by an in vivo directed evolution procedure in Thermus thermophilus, were produced and purified from E. coli recombinants, and enzymatic comparisons were performed. The optimum temperatures for enzyme activity were 50 and 55 degrees C for HPH and HPH5 respectively, but the thermal stability of the enzyme activity and the temperature for protein denaturation of HPH5 increased, from 36 and 37.2 degrees C of HPH to 53 and 58.8 degrees C respectively. Specific activities and steady-state kinetics measured at 25 degrees C showed only slight differences between the two enzymes. From these results we concluded that HPH5 was thermostabilized at the protein level, and that the mutations introduced did not affect its enzyme activity, at least under the assay conditions.

Convergent evolution of chromatin modification by structurally distinct enzymes: comparative enzymology of histone H3 Lys²⁷ methylation by human polycomb repressive complex 2 and vSET.

PubMed

Swalm, Brooke M; Hallenbeck, Kenneth K; Majer, Christina R; Jin, Lei; Scott, Margaret Porter; Moyer, Mikel P; Copeland, Robert A; Wigle, Tim J

2013-07-15

H3K27 (histone H3 Lys27) methylation is an important epigenetic modification that regulates gene transcription. In humans, EZH (enhancer of zeste homologue) 1 and EZH2 are the only enzymes capable of catalysing methylation of H3K27. There is great interest in understanding structure-function relationships for EZH2, as genetic alterations in this enzyme are thought to play a causal role in a number of human cancers. EZH2 is challenging to study because it is only active in the context of the multi-subunit PRC2 (polycomb repressive complex 2). vSET is a viral lysine methyltransferase that represents the smallest protein unit capable of catalysing H3K27 methylation. The crystal structure of this minimal catalytic protein has been solved and researchers have suggested that vSET might prove useful as an EZH2 surrogate for the development of active site-directed inhibitors. To test this proposition, we conducted comparative enzymatic analysis of human EZH2 and vSET and report that, although both enzymes share similar preferences for methylation of H3K27, they diverge in terms of their permissiveness for catalysing methylation of alternative histone lysine sites, their relative preferences for utilization of multimeric macromolecular substrates, their active site primary sequences and, most importantly, their sensitivity to inhibition by drug-like small molecules. The cumulative data led us to suggest that EZH2 and vSET have very distinct active site structures, despite the commonality of the reaction catalysed by the two enzymes. Hence, the EZH2 and vSET pair of enzymes represent an example of convergent evolution in which distinct structural solutions have developed to solve a common catalytic need.

Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase.

PubMed Central

Lodola, A; Shore, J D; Parker, D M; Holbrook, J

1978-01-01

1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows 'all-of-the-sites' behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate dehydrogenase and this, and the structural results of others, can be explained if the two enzymes are a product of divergent evolution. PMID:217361

Emergence and Evolution

PubMed Central

Bullwinkle, Tammy J.

2013-01-01

The aminoacyl-tRNA synthetases (aaRSs) are essential components of the protein synthesis machinery responsible for defining the genetic code by pairing the correct amino acids to their cognate tRNAs. The aaRSs are an ancient enzyme family believed to have origins that may predate the last common ancestor and as such they provide insights into the evolution and development of the extant genetic code. Although the aaRSs have long been viewed as a highly conserved group of enzymes, findings within the last couple of decades have started to demonstrate how diverse and versatile these enzymes really are. Beyond their central role in translation, aaRSs and their numerous homologs have evolved a wide array of alternative functions both inside and outside translation. Current understanding of the emergence of the aaRSs, and their subsequent evolution into a functionally diverse enzyme family, are discussed in this chapter. PMID:23478877

Rapid bursts and slow declines: on the possible evolutionary trajectories of enzymes

PubMed Central

Newton, Matilda S.; Arcus, Vickery L.; Patrick, Wayne M.

2015-01-01

The evolution of enzymes is often viewed as following a smooth and steady trajectory, from barely functional primordial catalysts to the highly active and specific enzymes that we observe today. In this review, we summarize experimental data that suggest a different reality. Modern examples, such as the emergence of enzymes that hydrolyse human-made pesticides, demonstrate that evolution can be extraordinarily rapid. Experiments to infer and resurrect ancient sequences suggest that some of the first organisms present on the Earth are likely to have possessed highly active enzymes. Reconciling these observations, we argue that rapid bursts of strong selection for increased catalytic efficiency are interspersed with much longer periods in which the catalytic power of an enzyme erodes, through neutral drift and selection for other properties such as cellular energy efficiency or regulation. Thus, many enzymes may have already passed their catalytic peaks. PMID:25926697

Detection of biological threats. A challenge for directed molecular evolution.

PubMed

Petrenko, Valery A; Sorokulova, Iryna B

2004-08-01

The probe technique originated from early attempts of Anton van Leeuwenhoek to contrast microorganisms under the microscope using plant juices, successful staining of tubercle bacilli with synthetic dyes by Paul Ehrlich and discovery of a stain for differentiation of gram-positive and gram-negative bacteria by Hans Christian Gram. The technique relies on the principle that pathogens have unique structural features, which can be recognized by specifically labeled organic molecules. A hundred years of extensive screening efforts led to discovery of a limited assortment of organic probes that are used for identification and differentiation of bacteria. A new challenge--continuous monitoring of biological threats--requires long lasting molecular probes capable of tight specific binding of pathogens in unfavorable conditions. To respond to the challenge, probe technology is being revolutionized by utilizing methods of combinatorial chemistry, phage display and directed molecular evolution. This review describes how molecular evolution methods are applied for development of peptide, antibody and phage probes, and summarizes the author's own data on development of landscape phage probes against Salmonella typhimurium. The performance of the probes in detection of Salmonella is illustrated by a precipitation test, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS) and fluorescent, optical and electron microscopy.

Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering.

PubMed

Xie, Wenping; Lv, Xiaomei; Ye, Lidan; Zhou, Pingping; Yu, Hongwei

2015-07-01

Improved supply of farnesyl diphosphate (FPP) is often considered as a typical strategy for engineering Saccharomyces cerevisiae towards efficient terpenoid production. However, in the engineered strains with enhanced precursor supply, the production of the target metabolite is often impeded by insufficient capacity of the heterologous terpenoid pathways, which limits further conversion of FPP. Here, we tried to assemble an unimpeded biosynthesis pathway by combining directed evolution and metabolic engineering in S. cerevisiae for lycopene-overproduction. First, the catalytic ability of phytoene syntheses from different sources was investigated based on lycopene accumulation. Particularly, the lycopene cyclase function of the bifunctional enzyme CrtYB from Xanthophyllomyces dendrorhous was inactivated by deletion of functional domain and directed evolution to obtain mutants with solely phytoene synthase function. Coexpression of the resulting CrtYB11M mutant along with the CrtE and CrtI genes from X. dendrorhous, and the tHMG1 gene from S. cerevisiae led to production of 4.47 mg/g DCW (Dry cell weight) of lycopene and 25.66 mg/g DCW of the by-product squalene. To further increase the FPP competitiveness of the lycopene synthesis pathway, we tried to enhance the catalytic performance of CrtE by directed evolution and created a series of pathway variants by varying the copy number of Crt genes. Finally, fed-batch fermentation was conducted for the diploid strain YXWPD-14 resulting in accumulation of 1.61 g/L (24.41 mg/g DCW) of lycopene, meanwhile, the by-production of squalene was reduced to below 1 mg/g DCW. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein

PubMed Central

Tan, Wei-Hung; Cheng, Shu-Chun; Liu, Yu-Tung; Wu, Cheng-Guo; Lin, Min-Han; Chen, Chiao-Che; Lin, Chao-Hsiung; Chou, Chi-Yuan

2016-01-01

Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We’ve also identified four historical mutations that are able to produce a “GST-like” S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution. PMID:27499004

Early evolution of efficient enzymes and genome organization

PubMed Central

2012-01-01

Background Cellular life with complex metabolism probably evolved during the reign of RNA, when it served as both information carrier and enzyme. Jensen proposed that enzymes of primordial cells possessed broad specificities: they were generalist. When and under what conditions could primordial metabolism run by generalist enzymes evolve to contemporary-type metabolism run by specific enzymes? Results Here we show by numerical simulation of an enzyme-catalyzed reaction chain that specialist enzymes spread after the invention of the chromosome because protocells harbouring unlinked genes maintain largely non-specific enzymes to reduce their assortment load. When genes are linked on chromosomes, high enzyme specificity evolves because it increases biomass production, also by reducing taxation by side reactions. Conclusion The constitution of the genetic system has a profound influence on the limits of metabolic efficiency. The major evolutionary transition to chromosomes is thus proven to be a prerequisite for a complex metabolism. Furthermore, the appearance of specific enzymes opens the door for the evolution of their regulation. Reviewers This article was reviewed by Sándor Pongor, Gáspár Jékely, and Rob Knight. PMID:23114029

Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Opportunitistic Enzymes, Catalytic Promiscuity and the Evolution of Chemodiversity in Nature (2010 JGI User Meeting)

ScienceCinema

Noel, Joseph

2018-04-26

Joseph Noel from the Salk Institute on "Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Enzymes, Catalytic Promiscuity and the Evolution of Chemodiversity in Nature" on March 26, 2010 at the 5th Annual DOE JGI User Meeting.

Tracing the Repertoire of Promiscuous Enzymes along the Metabolic Pathways in Archaeal Organisms.

PubMed

Martínez-Núñez, Mario Alberto; Rodríguez-Escamilla, Zuemy; Rodríguez-Vázquez, Katya; Pérez-Rueda, Ernesto

2017-07-13

The metabolic pathways that carry out the biochemical transformations sustaining life depend on the efficiency of their associated enzymes. In recent years, it has become clear that promiscuous enzymes have played an important role in the function and evolution of metabolism. In this work we analyze the repertoire of promiscuous enzymes in 89 non-redundant genomes of the Archaea cellular domain. Promiscuous enzymes are defined as those proteins with two or more different Enzyme Commission (E.C.) numbers, according the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. From this analysis, it was found that the fraction of promiscuous enzymes is lower in Archaea than in Bacteria. A greater diversity of superfamily domains is associated with promiscuous enzymes compared to specialized enzymes, both in Archaea and Bacteria, and there is an enrichment of substrate promiscuity rather than catalytic promiscuity in the archaeal enzymes. Finally, the presence of promiscuous enzymes in the metabolic pathways was found to be heterogeneously distributed at the domain level and in the phyla that make up the Archaea. These analyses increase our understanding of promiscuous enzymes and provide additional clues to the evolution of metabolism in Archaea.

Conflict RNA modification, host-parasite co-evolution, and the origins of DNA and DNA-binding proteins1.

PubMed

McLaughlin, Paul J; Keegan, Liam P

2014-08-01

Nearly 150 different enzymatically modified forms of the four canonical residues in RNA have been identified. For instance, enzymes of the ADAR (adenosine deaminase acting on RNA) family convert adenosine residues into inosine in cellular dsRNAs. Recent findings show that DNA endonuclease V enzymes have undergone an evolutionary transition from cleaving 3' to deoxyinosine in DNA and ssDNA to cleaving 3' to inosine in dsRNA and ssRNA in humans. Recent work on dsRNA-binding domains of ADARs and other proteins also shows that a degree of sequence specificity is achieved by direct readout in the minor groove. However, the level of sequence specificity observed is much less than that of DNA major groove-binding helix-turn-helix proteins. We suggest that the evolution of DNA-binding proteins following the RNA to DNA genome transition represents the major advantage that DNA genomes have over RNA genomes. We propose that a hypothetical RNA modification, a RRAR (ribose reductase acting on genomic dsRNA) produced the first stretches of DNA in RNA genomes. We discuss why this is the most satisfactory explanation for the origin of DNA. The evolution of this RNA modification and later steps to DNA genomes are likely to have been driven by cellular genome co-evolution with viruses and intragenomic parasites. RNA modifications continue to be involved in host-virus conflicts; in vertebrates, edited cellular dsRNAs with inosine-uracil base pairs appear to be recognized as self RNA and to suppress activation of innate immune sensors that detect viral dsRNA.

On-Demand Production of Flow-Reactor Cartridges by 3D Printing of Thermostable Enzymes.

PubMed

Maier, Manfred; Radtke, Carsten P; Hubbuch, Jürgen; Niemeyer, Christof M; Rabe, Kersten S

2018-05-04

The compartmentalization of chemical reactions is an essential principle of life that provides a major source of innovation for the development of novel approaches in biocatalysis. To implement spatially controlled biotransformations, rapid manufacturing methods are needed for the production of biocatalysts that can be applied in flow systems. Whereas three-dimensional (3D) printing techniques offer high-throughput manufacturing capability, they are usually not compatible with the delicate nature of enzymes, which call for physiological processing parameters. We herein demonstrate the utility of thermostable enzymes in the generation of biocatalytic agarose-based inks for a simple temperature-controlled 3D printing process. As examples we utilized an esterase and an alcohol dehydrogenase from thermophilic organisms as well as a decarboxylase that was thermostabilized by directed protein evolution. We used the resulting 3D-printed parts for a continuous, two-step sequential biotransformation in a fluidic setup. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Compartmentalization - A Prerequisite for Maintaining and Changing an Identity.

PubMed

Rottmann, Philipp; Ward, Thomas; Panke, Sven

2016-01-01

The chemical manipulation of DNA is much more convenient than the manipulation of the bioproducts, such as enzymes, that it encodes. The optimization of bioproducts requires cycles of diversification of DNA followed by read-out of the information into the bioproduct. Maintaining the link between the information - the genotype - and the properties of the bioproduct - the phenotype - through some form of compartmentalization is therefore an essential aspect in directed evolution. While the ideal compartment is a biological cell, many projects involving more radical changes in the bioproduct, such as the introduction of novel cofactors, may not be suitable for expression of the information in cells, and alternative in vitro methods have to be applied. Consequently, the possibility to produce simple and advanced micro compartments at high rates and to combine them with the ability to translate the information into proteins represents a unique opportunity to explore demanding enzyme engineering projects that require the evaluation of at least hundreds of thousands of enzyme variants over multiple generations.

Prospecting Metagenomic Enzyme Subfamily Genes for DNA Family Shuffling by a Novel PCR-based Approach*

PubMed Central

Wang, Qiuyan; Wu, Huili; Wang, Anming; Du, Pengfei; Pei, Xiaolin; Li, Haifeng; Yin, Xiaopu; Huang, Lifeng; Xiong, Xiaolong

2010-01-01

DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64–99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering. PMID:20962349

The Role of Distant Mutations and Allosteric Regulation on LovD Active Site Dynamics

PubMed Central

Jiménez-Osés, Gonzalo; Osuna, Sílvia; Gao, Xue; Sawaya, Michael R.; Gilson, Lynne; Collier, Steven J.; Huisman, Gjalt W.; Yeates, Todd O.; Tang, Yi; Houk, K. N.

2014-01-01

Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester, and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is the first non-patent report of the enzyme currently used for the manufacture of simvastatin, as well as the intermediate evolved variants. Crystal structures and microsecond molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations dramatically altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF. PMID:24727900

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

PubMed

Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt

2002-08-16

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

Evolution of neurotransmitter receptor systems.

PubMed

Venter, J C; di Porzio, U; Robinson, D A; Shreeve, S M; Lai, J; Kerlavage, A R; Fracek, S P; Lentes, K U; Fraser, C M

1988-01-01

The presence of hormones, neurotransmitters, their receptors and biosynthetic and degradative enzymes is clearly not only associated with the present and the recent past but with the past several hundred million years. Evidence is mounting which indicates substantial conservation of protein structure and function of these receptors and enzymes over these tremendous periods of time. These findings indicate that the evolution and development of the nervous system was not dependent upon the formation of new or better transmitter substances, receptor proteins, transducers and effector proteins but involved better utilization of these highly developed elements in creating advanced and refined circuitry. This is not a new concept; it is one that is now substantiated by increasingly sophisticated studies. In a 1953 article discussing chemical aspects of evolution (Danielli, 1953) Danielli quotes Medawar, "... endocrine evolution is not an evolution of hormones but an evolution of the uses to which they are put; an evolution not, to put it crudely, of chemical formulae but of reactivities, reaction patterns and tissue competences." To also quote Danielli, "In terms of comparative biochemistry, one must ask to what extent the evolution of these reactivities, reaction patterns and competences is conditional upon the evolution of methods of synthesis of new proteins, etc., and to what extent the proteins, etc., are always within the synthetic competence of an organism. In the latter case evolution is the history of changing uses of molecules, and not of changing synthetic abilities." (Danielli, 1953). Figure 4 outlines a phylogenetic tree together with an indication of where evidence exists for both the enzymes that determine the biosynthesis and metabolism of the cholinergic and adrenergic transmitters and their specific cholinergic and adrenergic receptors. This figure illustrates a number of important points. For example, the evidence appears to show that the transmitters and their associated enzymes existed for a substantial period before their respective receptor proteins. While the transmitters and enzymes appear to exist in single cellular organisms, there is no solid evidence for the presence of adrenergic or cholinergic receptors until multicellular organisms where the receptors appear to be clearly associated with specific cellular and neuronal communication (Fig. 4). One can only speculate as to the possible role for acetylcholine and the catecholamine in single cell organisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

PubMed

Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a β-lactamase fold: X-ray crystallographic analysis of directly evolved 6-aminohexanoate-dimer hydrolase

PubMed Central

Ohki, Taku; Shibata, Naoki; Higuchi, Yoshiki; Kawashima, Yasuyuki; Takeo, Masahiro; Kato, Dai-ichiro; Negoro, Seiji

2009-01-01

Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a β-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (Km = 15 mM) and turnover (kcat = 3.1 s−1) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the Ald—COOH and Tyr370, a hydrogen-bonding network from Ser187 to , and interaction between and Gln27-Oɛ derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with (Kawashima et al., FEBS J 2009; 276:2547–2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a β-lactamase fold. PMID:19521995

Expanding the Scope of Site-Specific Recombinases for Genetic and Metabolic Engineering

PubMed Central

Gaj, Thomas; Sirk, Shannon J.; Barbas, Carlos F.

2014-01-01

Site-specific recombinases are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, site-specific recombination systems have empowered researchers with unprecedented control over diverse biological functions, enabling countless insights into cellular structure and function. The rigid target specificities of many sites-specific recombinases, however, have limited their adoption in fields that require highly flexible recognition abilities. As a result, intense effort has been directed toward altering the properties of site-specific recombination systems by protein engineering. Here, we review key developments in the rational design and directed molecular evolution of site-specific recombinases, highlighting the numerous applications of these enzymes across diverse fields of study. PMID:23982993

Directed evolution improves the fibrinolytic activity of nattokinase from Bacillus natto.

PubMed

Yongjun, Cai; Wei, Bao; Shujun, Jiang; Meizhi, Weng; Yan, Jia; Yan, Yin; Zhongliang, Zheng; Goulin, Zou

2011-12-01

Nattokinase (subtilisin NAT, NK) is a relatively effective microbial fibrinolytic enzyme that has been identified and characterized from Bacillus natto. In the current report, DNA family shuffling was used to improve the fibrinolytic activity of nattokinase. Three homologous genes from B. natto AS 1.107, Bacillus amyloliquefaciens CICC 20164 and Bacillus licheniformis CICC 10092 were shuffled to generate a mutant library. A plate-based method was used to screen the mutant libraries for improved activity. After three rounds of DNA shuffling, one desirable mutant with 16 amino acid substitutions was obtained. The mutant enzyme was purified and characterized. The kinetic measurements showed that the catalytic efficiency of the mutant NK was approximately 2.3 times higher than that of the wild-type nattokinase. In addition, the molecular modeling analysis suggested that the mutations affect the enzymatic function by changing the surface conformation of the substrate-binding pocket. The current study shows that the evolution of nattokinase with improved fibrinolytic activity by DNA family shuffling is feasible and provides useful references to facilitate the application of nattokinase in thrombolytic therapy. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Models of Protocellular Structure, Function and Evolution

NASA Technical Reports Server (NTRS)

New, Michael H.; Pohorille, Andrew; Szostak, Jack W.; Keefe, Tony; Lanyi, Janos K.; DeVincenzi, Donald L. (Technical Monitor)

2001-01-01

In the absence of any record of protocells, the most direct way to test our understanding, of the origin of cellular life is to construct laboratory models that capture important features of protocellular systems. Such efforts are currently underway in a collaborative project between NASA-Ames, Harvard Medical School and University of California. They are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures. The centerpiece of this project is a method for the in vitro evolution of protein enzymes toward arbitrary catalytic targets. A similar approach has already been developed for nucleic acids in which a small number of functional molecules are selected from a large, random population of candidates. The selected molecules are next vastly multiplied using the polymerase chain reaction.

Rapid bursts and slow declines: on the possible evolutionary trajectories of enzymes.

PubMed

Newton, Matilda S; Arcus, Vickery L; Patrick, Wayne M

2015-06-06

The evolution of enzymes is often viewed as following a smooth and steady trajectory, from barely functional primordial catalysts to the highly active and specific enzymes that we observe today. In this review, we summarize experimental data that suggest a different reality. Modern examples, such as the emergence of enzymes that hydrolyse human-made pesticides, demonstrate that evolution can be extraordinarily rapid. Experiments to infer and resurrect ancient sequences suggest that some of the first organisms present on the Earth are likely to have possessed highly active enzymes. Reconciling these observations, we argue that rapid bursts of strong selection for increased catalytic efficiency are interspersed with much longer periods in which the catalytic power of an enzyme erodes, through neutral drift and selection for other properties such as cellular energy efficiency or regulation. Thus, many enzymes may have already passed their catalytic peaks. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

A 13-week research-based biochemistry laboratory curriculum.

PubMed

Lefurgy, Scott T; Mundorff, Emily C

2017-09-01

Here, we present a 13-week research-based biochemistry laboratory curriculum designed to provide the students with the experience of engaging in original research while introducing foundational biochemistry laboratory techniques. The laboratory experience has been developed around the directed evolution of an enzyme chosen by the instructor, with mutations designed by the students. Ideal enzymes for this curriculum are able to be structurally modeled, solubly expressed, and monitored for activity by UV/Vis spectroscopy, and an example curriculum for haloalkane dehalogenase is given. Unique to this curriculum is a successful implementation of saturation mutagenesis and high-throughput screening of enzyme function, along with bioinformatics analysis, homology modeling, structural analysis, protein expression and purification, polyacrylamide gel electrophoresis, UV/Vis spectroscopy, and enzyme kinetics. Each of these techniques is carried out using a novel student-designed mutant library or enzyme variant unique to the lab team and, importantly, not described previously in the literature. Use of a well-established set of protocols promotes student data quality. Publication may result from the original student-generated hypotheses and data, either from the class as a whole or individual students that continue their independent projects upon course completion. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):437-448, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Biocatalysis in organic chemistry and biotechnology: past, present, and future.

PubMed

Reetz, Manfred T

2013-08-28

Enzymes as catalysts in synthetic organic chemistry gained importance in the latter half of the 20th century, but nevertheless suffered from two major limitations. First, many enzymes were not accessible in large enough quantities for practical applications. The advent of recombinant DNA technology changed this dramatically in the late 1970s. Second, many enzymes showed a narrow substrate scope, often poor stereo- and/or regioselectivity and/or insufficient stability under operating conditions. With the development of directed evolution beginning in the 1990s and continuing to the present day, all of these problems can be addressed and generally solved. The present Perspective focuses on these and other developments which have popularized enzymes as part of the toolkit of synthetic organic chemists and biotechnologists. Included is a discussion of the scope and limitation of cascade reactions using enzyme mixtures in vitro and of metabolic engineering of pathways in cells as factories for the production of simple compounds such as biofuels and complex natural products. Future trends and problems are also highlighted, as is the discussion concerning biocatalysis versus nonbiological catalysis in synthetic organic chemistry. This Perspective does not constitute a comprehensive review, and therefore the author apologizes to those researchers whose work is not specifically treated here.

Back-reactions, short-circuits, leaks and other energy wasteful reactions in biological electron transfer: redox tuning to survive life in O(2).

PubMed

Rutherford, A William; Osyczka, Artur; Rappaport, Fabrice

2012-03-09

The energy-converting redox enzymes perform productive reactions efficiently despite the involvement of high energy intermediates in their catalytic cycles. This is achieved by kinetic control: with forward reactions being faster than competing, energy-wasteful reactions. This requires appropriate cofactor spacing, driving forces and reorganizational energies. These features evolved in ancestral enzymes in a low O(2) environment. When O(2) appeared, energy-converting enzymes had to deal with its troublesome chemistry. Various protective mechanisms duly evolved that are not directly related to the enzymes' principal redox roles. These protective mechanisms involve fine-tuning of reduction potentials, switching of pathways and the use of short circuits, back-reactions and side-paths, all of which compromise efficiency. This energetic loss is worth it since it minimises damage from reactive derivatives of O(2) and thus gives the organism a better chance of survival. We examine photosynthetic reaction centres, bc(1) and b(6)f complexes from this view point. In particular, the evolution of the heterodimeric PSI from its homodimeric ancestors is explained as providing a protective back-reaction pathway. This "sacrifice-of-efficiency-for-protection" concept should be generally applicable to bioenergetic enzymes in aerobic environments. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

Life-history evolution and the microevolution of intermediary metabolism: activities of lipid-metabolizing enzymes in life-history morphs of a wing-dimorphic cricket.

PubMed

Zera, Anthony J; Zhao, Zhangwu

2003-03-01

Although a considerable amount of information is available on the ecology, genetics, and physiology of life-history traits, much more limited data are available on the biochemical and genetic correlates of life-history variation within species. Specific activities of five enzymes of lipid biosynthesis and two enzymes of amino acid catabolism were compared among lines selected for flight-capable (LW[f]) versus flightless (SW) morphs of the cricket Gryllus firmus. These morphs, which exist in natural populations, differ genetically in ovarian growth (100-400% higher in SW) and aspects of flight capability including the size of wings and flight muscles, and the concentration of triglyceride flight fuel (40% greater in LW[f]). Consistently higher activity of each enzyme in LW(f) versus SW-selected lines, and strong co-segregation between morph and enzyme activity, demonstrated genetically based co-variance between wing morph and enzyme activity. Developmental profiles of enzyme activities strongly paralleled profiles of triglyceride accumulation during adulthood and previous measures of in vivo lipid biosynthesis. These data strongly imply that genetically based elevation in activities of lipogenic enzymes, and enzymes controlling the conversion of amino acids into lipids, is an important cause underlying the elevated accumulation of triglyceride in the LW(f) morph, a key biochemical component of the trade-off between elevated early fecundity and flight capability. Global changes in lipid and amino-acid metabolism appear to have resulted from microevolutionary alteration of regulators of metabolism. Finally, strong genotype x environment (diet) interactions were observed for most enzyme activities. Future progress in understanding the functional causes of life-history evolution requires a more detailed synthesis of the fields of life-history evolution and metabolic biochemistry. Wing polymorphism is a powerful experimental model in such integrative studies.

Iron-Sulfur Cluster Engineering Provides Insight into the Evolution of Substrate Specificity among Sulfonucleotide Reductases

PubMed Central

Bhave, Devayani P.; Hong, Jiyoung A.; Keller, Rebecca L.; Krebs, Carsten; Carroll, Kate S.

2011-01-01

Assimilatory sulfate reduction supplies prototrophic organisms with reduced sulfur that is required for the biosynthesis of all sulfur-containing metabolites, including cysteine and methionine. The reduction of sulfate requires its activation via an ATP-dependent activation to form adenosine-5′-phosphosulfate (APS). Depending on the species, APS can be reduced directly to sulfite by APS reductase (APR) or undergo a second phosphorylation to yield 3′-phosphoadenosine-5′-phosphosulfate (PAPS), the substrate for PAPS reductase (PAPR). These essential enzymes have no human homolog, rendering them attractive targets for the development of novel antibacterial drugs. APR and PAPR share sequence and structure homology as well as a common catalytic mechanism, but the enzymes are distinguished by two features, namely, the amino acid sequence of the phosphate-binding loop (P-loop) and an iron-sulfur cofactor in APRs. Based on the crystal structures of APR and PAPR, two P-loop residues are proposed to determine substrate specificity; however, this hypothesis has not been tested. In contrast to this prevailing view, we report here that the P-loop motif has a modest effect on substrate discrimination. Instead, by means of metalloprotein engineering, spectroscopic and kinetic analyses, we demonstrate that the iron-sulfur cluster cofactor enhances APS reduction by nearly 1000-fold, thereby playing a pivotal role in substrate specificity and catalysis. These findings offer new insights into the evolution of this enzyme family, and extend the known functions of protein-bound iron-sulfur clusters. PMID:22023093

A Computational Tale of Two Enzymes: Glycerol Dehydration With or Without B12.

PubMed

Kovačević, Borislav; Barić, Danijela; Babic, Darko; Bilić, Luka; Hanževački, Marko; Sandala, Gregory M; Radom, Leo; Smith, David M

2018-06-12

We present a series of QM/MM calculations aimed at understanding the mechanism of the biological dehydration of glycerol. Strikingly and unusually, this process is catalyzed by two different radical enzymes, one of which is a coenzyme-B 12 - dependent enzyme and the other which is a coenzyme-B 12 - independent enzyme. We show that glycerol dehydration in the presence of the coenzyme-B 12 -dependent enzyme proceeds via a 1,2-OH shift, which benefits from a significant catalytic reduction in the barrier. In contrast, the same reaction in the presence of the coenzyme-B 12 -independent enzyme is unlikely to involve the 1,2-OH shift; instead, a strong preference for direct loss of water from a radical intermediate is indicated. We show that this preference and, ultimately the evolution of such enzymes, is strongly linked with the reactivities of the species responsible for abstracting a hydrogen atom from the substrate. It appears that the hydrogen re-abstraction step involving the product-related radical is fundamental to the mechanistic preference. The unconventional 1,2-OH shift seems to be required to generate a product-related radical of sufficient reactivity to cleave the relatively inactive C-H bond arising from the B 12 cofactor. In the absence of B 12 , it is the relatively weak S-H bond of a cysteine residue that must be homolyzed. Such a transformation is much less demanding and its inclusion apparently enables a simpler overall dehydration mechanism.

Something Old, Something New: Conserved Enzymes and the Evolution of Novelty in Plant Specialized Metabolism1

PubMed Central

Moghe, Gaurav D.; Last, Robert L.

2015-01-01

Plants produce hundreds of thousands of small molecules known as specialized metabolites, many of which are of economic and ecological importance. This remarkable variety is a consequence of the diversity and rapid evolution of specialized metabolic pathways. These novel biosynthetic pathways originate via gene duplication or by functional divergence of existing genes, and they subsequently evolve through selection and/or drift. Studies over the past two decades revealed that diverse specialized metabolic pathways have resulted from the incorporation of primary metabolic enzymes. We discuss examples of enzyme recruitment from primary metabolism and the variety of paths taken by duplicated primary metabolic enzymes toward integration into specialized metabolism. These examples provide insight into processes by which plant specialized metabolic pathways evolve and suggest approaches to discover enzymes of previously uncharacterized metabolic networks. PMID:26276843

Structural intermediates and directionality of the swiveling motion of Pyruvate Phosphate Dikinase

NASA Astrophysics Data System (ADS)

Minges, Alexander; Ciupka, Daniel; Winkler, Christian; Höppner, Astrid; Gohlke, Holger; Groth, Georg

2017-03-01

Pyruvate phosphate dikinase (PPDK) is a vital enzyme in cellular energy metabolism catalyzing the ATP- and Pi-dependent formation of phosphoenolpyruvate from pyruvate in C4 -plants, but the reverse reaction forming ATP in bacteria and protozoa. The multi-domain enzyme is considered an efficient molecular machine that performs one of the largest single domain movements in proteins. However, a comprehensive understanding of the proposed swiveling domain motion has been limited by not knowing structural intermediates or molecular dynamics of the catalytic process. Here, we present crystal structures of PPDKs from Flaveria, a model genus for studying the evolution of C4 -enzymes from phylogenetic ancestors. These structures resolve yet unknown conformational intermediates and provide the first detailed view on the large conformational transitions of the protein in the catalytic cycle. Independently performed unrestrained MD simulations and configurational free energy calculations also identified these intermediates. In all, our experimental and computational data reveal strict coupling of the CD swiveling motion to the conformational state of the NBD. Moreover, structural asymmetries and nucleotide binding states in the PPDK dimer support an alternate binding change mechanism for this intriguing bioenergetic enzyme.

Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.

PubMed

Kaltenbach, Miriam; Emond, Stephane; Hollfelder, Florian; Tokuriki, Nobuhiko

2016-10-01

The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and the emergence of generalist enzymes.

METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS

DOE Office of Scientific and Technical Information (OSTI.GOV)

John J. Kilbane II

The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project was focused on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate deaminase. The objective of the final phase of the project will bemore » to develop derivative C-N bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments resulted in the isolation of microbial cultures that utilize aromatic amides as sole nitrogen sources, several amidase genes were cloned and were included in directed evolution experiments to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. During the second year of the project (October, 2003-September, 2004) enrichment culture experiments succeeded in isolating a mixed bacterial culture that can utilize 2-aminobiphenyl as a sole nitrogen source, directed evolution experiments were focused on the aniline dioxygenase enzyme that is capable of deaminating aniline, and expression vectors were constructed to enable the expression of genes encoding C-N bond cleaving enzymes in Rhodococcus hosts. The construction of a new metabolic pathway to selectively remove nitrogen from carbazole and other molecules typically found in petroleum should lead to the development of a process to improve oil refinery efficiency by reducing the poisoning, by nitrogen, of catalysts used in the hydrotreating and catalytic cracking of petroleum. Aromatic compounds such as carbazole are representative of the difficult-to-treat organonitrogen compounds most commonly encountered in petroleum. There are two C-N bonds in carbazole and the construction of a metabolic pathway for the removal of nitrogen from carbazole will require enzymes capable cleaving both C-N bonds. A multi-component enzyme, carbazole dioxygenase, which can selectively cleave the first C-N bond has been identified and the genes that encode this enzyme have been cloned, sequenced, and are being expressed in Rhodococcus erythropolis, a bacterial culture that tolerates exposure to petroleum. An enzyme capable of selectively cleaving the second C-N bond in carbazole has not yet been identified, but enrichment culture experiments have recently succeeded in isolating a bacterial culture that is a likely candidate and may possess a suitable enzyme. Research in the near future will verify if a suitable enzyme for the cleavage of the second C-N bond in carbazole has indeed been found, then the genes encoding a suitable enzyme will be identified, cloned, and sequenced. Ultimately genes encoding enzymes for selective cleavage of both C-N bonds in carbazole will be assembled into a new metabolic pathway and the ability of the resulting bacterial culture to remove nitrogen from petroleum will be determined.« less

A route from darkness to light: emergence and evolution of luciferase activity in AMP-CoA-ligases inferred from a mealworm luciferase-like enzyme.

PubMed

Viviani, V R; Prado, R A; Neves, D R; Kato, D; Barbosa, J A

2013-06-11

The origin of luciferases and of bioluminescence is enigmatic. In beetles, luciferases seem to have evolved from AMP-CoA-ligases. How the new oxygenase luminogenic function originated from AMP-ligases leading to luciferases is one of the most challenging mysteries of bioluminescence. Comparison of the cloned luciferase-like enzyme from the nonluminescent Zophobas morio mealworm and beetle luciferases showed that the oxygenase activity may have emerged as a stereoselective oxidative drift with d-luciferin, a substrate that cannot be easily thioesterified to CoA as in the case of the l-isomer. While the overall kcat displayed by beetle luciferases is orders of magnitude greater than that of the luciferase-like enzyme, the respective oxidation rates and quantum yields of bioluminescence are roughly similar, suggesting that the rate constant of the AMP-ligase activity exerted on the new d-luciferin substrate in beetle protoluciferases was the main enzymatic property that suffered optimization during the evolution of luciferases. The luciferase-like enzyme and luciferases boost the rate of luciferyl-adenylate chemiluminescent oxidation by factors of 10(6) and 10(7), respectively, as compared to the substrate spontaneous oxidation in buffer. A similar enhancement of luciferyl-adenylate chemiluminescence is provided by nucleophilic aprotic solvents, implying that the peptide bonds in the luciferin binding site of beetle luciferase could provide a similar catalytically favorable environment. These data suggest that the luciferase-like enzyme and other similar AMP-ligases are potential alternative oxygenases. Site-directed mutagenesis studies of the luciferase-like enzyme and the red light-producing luciferase of Phrixotrix hirtus railroadworm confirm here a critical role for T/S345 in luciferase function. Mutations such as I327T/S in the luciferase-like enzyme, which simultaneously increases luciferase activity and promotes blue shifts in the emission spectrum, could have been critical for evolving functional bioluminescence from red-emitting protoluciferases. Through the combination of I327T/S mutations and N-terminal fusion, the luminescence activity of this enzyme was increased to visible levels, with the development of a totally new orange-emitting luciferase. These results open the possibility of engineering luciferase activity in a set of AMP-CoA-ligases.

Computational Protein Engineering: Bridging the Gap between Rational Design and Laboratory Evolution

PubMed Central

Barrozo, Alexandre; Borstnar, Rok; Marloie, Gaël; Kamerlin, Shina Caroline Lynn

2012-01-01

Enzymes are tremendously proficient catalysts, which can be used as extracellular catalysts for a whole host of processes, from chemical synthesis to the generation of novel biofuels. For them to be more amenable to the needs of biotechnology, however, it is often necessary to be able to manipulate their physico-chemical properties in an efficient and streamlined manner, and, ideally, to be able to train them to catalyze completely new reactions. Recent years have seen an explosion of interest in different approaches to achieve this, both in the laboratory, and in silico. There remains, however, a gap between current approaches to computational enzyme design, which have primarily focused on the early stages of the design process, and laboratory evolution, which is an extremely powerful tool for enzyme redesign, but will always be limited by the vastness of sequence space combined with the low frequency for desirable mutations. This review discusses different approaches towards computational enzyme design and demonstrates how combining newly developed screening approaches that can rapidly predict potential mutation “hotspots” with approaches that can quantitatively and reliably dissect the catalytic step can bridge the gap that currently exists between computational enzyme design and laboratory evolution studies. PMID:23202907

Plasticity and evolution of (+)-3-carene synthase and (-)-sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance.

PubMed

Roach, Christopher R; Hall, Dawn E; Zerbe, Philipp; Bohlmann, Jörg

2014-08-22

The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (-)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3-carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (-)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene, whereas phenylalanine promotes (-)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Evolution of catalytic RNA in the laboratory

NASA Technical Reports Server (NTRS)

Joyce, Gerald F.

1992-01-01

We are interested in the biochemistry of existing RNA enzymes and in the development of RNA enzymes with novel catalytic function. The focal point of our research program has been the design and operation of a laboratory system for the controlled evolution of catalytic RNA. This system serves as working model of RNA-based life and can be used to explore the catalytic potential of RNA. Evolution requires the integration of three chemical processes: amplification, mutation, and selection. Amplification results in additional copies of the genetic material. Mutation operates at the level of genotype to introduce variability, this variability in turn being expressed as a range of phenotypes. Selection operates at the level of phenotype to reduce variability by excluding those individuals that do not conform to the prevailing fitness criteria. These three processes must be linked so that only the selected individuals are amplified, subject to mutational error, to produce a progeny distribution of mutant individuals. We devised techniques for the amplification, mutation, and selection of catalytic RNA, all of which can be performed rapidly in vitro within a single reaction vessel. We integrated these techniques in such a way that they can be performed iteratively and routinely. This allowed us to conduct evolution experiments in response to artificially-imposed selection constraints. Our objective was to develop novel RNA enzymes by altering the selection constraints in a controlled manner. In this way we were able to expand the catalytic repertoire of RNA. Our long-range objective is to develop an RNA enzyme with RNA replicase activity. If such an enzyme had the ability to produce additional copies of itself, then RNA evolution would operate autonomously and the origin of life will have been realized in the laboratory.

Molecular Phylogeny of Heme Peroxidases

NASA Astrophysics Data System (ADS)

Zámocký, Marcel; Obinger, Christian

All currently available gene sequences of heme peroxidases can be phylogenetically divided in two superfamilies and three families. In this chapter, the phylogenetics and genomic distribution of each group are presented. Within the peroxidase-cyclooxygenase superfamily, the main evolutionary direction developed peroxidatic heme proteins involved in the innate immune defense system and in biosynthesis of (iodinated) hormones. The peroxidase-catalase superfamily is widely spread mainly among bacteria, fungi, and plants, and particularly in Class I led to the evolution of bifunctional catalase-peroxidases. Its numerous fungal representatives of Class II are involved in carbon recycling via lignin degradation, whereas Class III secretory peroxidases from algae and plants are included in various forms of secondary metabolism. The family of di-heme peroxidases are predominantly bacteria-inducible enzymes; however, a few corresponding genes were also detected in archaeal genomes. Four subfamilies of dyp-type peroxidases capable of degradation of various xenobiotics are abundant mainly among bacteria and fungi. Heme-haloperoxidase genes are widely spread among sac and club fungi, but corresponding genes were recently found also among oomycetes. All described families herein represent heme peroxidases of broad diversity in structure and function. Our accumulating knowledge about the evolution of various enzymatic functions and physiological roles can be exploited in future directed evolution approaches for engineering peroxidase genes de novo for various demands.

Multifunctional enzymes from reduced genomes - model proteins for simple primordial metabolism?

PubMed

Seelig, Burckhard

2017-08-01

Billions of years of evolution have yielded today's complex metabolic networks driven by efficient and highly specialized enzymes. In contrast, the metabolism of the earliest cellular life forms was likely much simpler with only a few enzymes of comparatively low activity. It has been speculated that these early enzymes had low specificities and in turn were able to perform multiple functions. In this issue of Molecular Microbiology, Ferla et al. describe examples of enzymes that catalyze chemically distinct reactions while using the same active site. Most importantly, the authors demonstrated that the comparatively weak activities of these multifunctional enzymes are each physiologically relevant. These findings contrast with simply promiscuous enzyme activities, which have been described numerous times but are not physiologically relevant. Ferla et al. elegantly combined initial bioinformatics searches for enzyme candidates with sound kinetic measurements, evolutionary considerations and even structural discussions. The phenomenon of multifunctionality appears to be a mechanism for bacteria with reduced genomes to compensate for their lack of certain enzymes. In the broader context of evolution, these organisms could be considered living model systems to study features of long-extinct early cellular life. © 2017 John Wiley & Sons Ltd.

RNA-Cleaving DNA Enzymes with Altered Regio- or Enantioselectivity

NASA Technical Reports Server (NTRS)

Ordoukhanian, Phillip; Joyce, Gerald F.

2002-01-01

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5' - phosphodiester following a wibonucleotide or a 3',5' -phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5' -phosphodiester exhibits a k(sub cat) of approx. 0.01/ min and catalytic efficiency, k(sub cat)/k(sub m) of approx. 10(exp 5)/ M min. The enzyme that cleaves an L-ribonudeotide is about 10-fold slower and has a catalytic efficiency of approx. 4 x 10(exp 5)/ M min. Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 C. In a comparison of each enzyme s activity with either its corresponding substrate that contains an unnatural ribonudeotide or a substrate that instead contains a standard ribonucleotide, the 2',5' -phosphodiester-deaving DNA enzyme exhibited a regioselectivity of 6000- fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 50-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.

Escherichia coli arabinose isomerase and Staphylococcus aureus tagatose-6-phosphate isomerase: which is a better template for directed evolution of non-natural substrate isomerization?

PubMed

Kim, Hye Jung; Uhm, Tae Guk; Kim, Seong Bo; Kim, Pil

2010-06-01

Metallic and non-metallic isomerases can be used to produce commercially important monosaccharides. To determine which category of isomerase is more suitable as a template for directed evolution to improve enzymes for galactose isomerization, L-arabinose isomerase from Escherichia coli (ECAI; E.C. 5.3.1.4) and tagatose-6-phosphate isomerase from Staphylococcus aureus (SATI; E.C. 5.3.1.26) were chosen as models of a metallic and non-metallic isomerase, respectively. Random mutations were introduced into the genes encoding ECAI and SATI at the same rate, resulting in the generation of 515 mutants of each isomerase. The isomerization activity of each of the mutants toward a non-natural substrate (galactose) was then measured. With an average mutation rate of 0.2 mutations/kb, 47.5% of the mutated ECAIs showed an increase in activity compared with wild-type ECAI, and the remaining 52.5% showed a decrease in activity. Among the mutated SATIs, 58.6% showed an increase in activity, whereas 41.4% showed a decrease in activity. Mutant clones showing a significant change in relative activity were sequenced and specific increases in activity were measured. The maximum increase in activity achieved by mutation of ECAI was 130%, and that for SATI was 190%. Based on these results, the characteristics of the different isomerases are discussed in terms of their usefulness for directed evolution of non-natural substrate isomerization.

Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution.

PubMed

Shivange, Amol V; Roccatano, Danilo; Schwaneberg, Ulrich

2016-01-01

Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE,  an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The "best" combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes.

Expanding the scope of site-specific recombinases for genetic and metabolic engineering.

PubMed

Gaj, Thomas; Sirk, Shannon J; Barbas, Carlos F

2014-01-01

Site-specific recombinases are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, site-specific recombination systems have empowered researchers with unprecedented control over diverse biological functions, enabling countless insights into cellular structure and function. The rigid target specificities of many sites-specific recombinases, however, have limited their adoption in fields that require highly flexible recognition abilities. As a result, intense effort has been directed toward altering the properties of site-specific recombination systems by protein engineering. Here, we review key developments in the rational design and directed molecular evolution of site-specific recombinases, highlighting the numerous applications of these enzymes across diverse fields of study. © 2013 Wiley Periodicals, Inc.

Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability.

PubMed

Axarli, Irine; Muleta, Abdi W; Chronopoulou, Evangelia G; Papageorgiou, Anastassios C; Labrou, Nikolaos E

2017-01-01

Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher k cat /K m and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Šand 2.3Šresolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

PubMed

Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

2014-11-25

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

A Thermostable β-Glucuronidase Obtained by Directed Evolution as a Reporter Gene in Transgenic Plants

PubMed Central

Xiong, Ai-Sheng; Peng, Ri-He; Zhuang, Jing; Chen, Jian-Min; Zhang, Bin; Zhang, Jian; Yao, Quan-Hong

2011-01-01

A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter. PMID:22096498

Engineering Cellulases for Biorefinery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2010-06-27

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Thermostable Cellulases: Why & How?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2010-04-19

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

PubMed Central

Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M.; Wong, Margaret; Kiessling, Laura L.; Steitz, Thomas A.; O’Donoghue, Patrick; Söll, Dieter

2014-01-01

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. PMID:25385624

Engineering 7β-Hydroxysteroid Dehydrogenase for Enhanced Ursodeoxycholic Acid Production by Multiobjective Directed Evolution.

PubMed

Zheng, Ming-Min; Chen, Ke-Cai; Wang, Ru-Feng; Li, Hao; Li, Chun-Xiu; Xu, Jian-He

2017-02-15

Ursodeoxycholic acid (UDCA) is the main active ingredient of natural bear bile powder with multiple pharmacological functions. 7β-Hydroxysteroid dehydrogenase (HSDH) is a key biocatalyst for the synthesis of UDCA. However, all the 7β-HSDHs reported commonly suffer from poor activity and thermostability, resulting in limited productivity of UDCA. In this study, a multiobjective directed evolution (MODE) strategy was proposed and applied to improve the activity, thermostability, and pH optimum of a 7β-HSDH. The best variant (V 3-1 ) showed a specific activity 5.5-fold higher than and a half-life 3-fold longer than those of the wild type. In addition, the pH optimum of the variant was shifted to a weakly alkaline value. In the cascade reaction, the productivity of UDCA with V 3-1 increased to 942 g L -1 day -1 , in contrast to 141 g L -1 day -1 with the wild type. Therefore, this study provides a useful strategy for improving the catalytic efficiency of a key enzyme that significantly facilitated the bioproduction of UDCA.

Design Strategy of Multi-electron Transfer Catalysts Based on a Bioinformatic Analysis of Oxygen Evolution and Reduction Enzymes.

PubMed

Ooka, Hideshi; Hashimoto, Kazuhito; Nakamura, Ryuhei

2018-05-14

Understanding the design strategy of photosynthetic and respiratory enzymes is important to develop efficient artificial catalysts for oxygen evolution and reduction reactions. Here, based on a bioinformatic analysis of cyanobacterial oxygen evolution and reduction enzymes (photosystem II: PS II and cytochrome c oxidase: COX, respectively), the gene encoding the catalytic D1 subunit of PS II was found to be expressed individually across 38 phylogenetically diverse strains, which is in contrast to the operon structure of the genes encoding major COX subunits. Selective synthesis of the D1 subunit minimizes the repair cost of PS II, which allows compensation for its instability by lowering the turnover number required to generate a net positive energy yield. The different bioenergetics observed between PS II and COX suggest that in addition to the catalytic activity rationalized by the Sabatier principle, stability factors have also provided a major influence on the design strategy of biological multi-electron transfer enzymes. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative genomics and evolution of eukaryotic phospholipidbiosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lykidis, Athanasios

2006-12-01

Phospholipid biosynthetic enzymes produce diverse molecular structures and are often present in multiple forms encoded by different genes. This work utilizes comparative genomics and phylogenetics for exploring the distribution, structure and evolution of phospholipid biosynthetic genes and pathways in 26 eukaryotic genomes. Although the basic structure of the pathways was formed early in eukaryotic evolution, the emerging picture indicates that individual enzyme families followed unique evolutionary courses. For example, choline and ethanolamine kinases and cytidylyltransferases emerged in ancestral eukaryotes, whereas, multiple forms of the corresponding phosphatidyltransferases evolved mainly in a lineage specific manner. Furthermore, several unicellular eukaryotes maintain bacterial-type enzymesmore » and reactions for the synthesis of phosphatidylglycerol and cardiolipin. Also, base-exchange phosphatidylserine synthases are widespread and ancestral enzymes. The multiplicity of phospholipid biosynthetic enzymes has been largely generated by gene expansion in a lineage specific manner. Thus, these observations suggest that phospholipid biosynthesis has been an actively evolving system. Finally, comparative genomic analysis indicates the existence of novel phosphatidyltransferases and provides a candidate for the uncharacterized eukaryotic phosphatidylglycerol phosphate phosphatase.« less

Detoxification of organophosphate nerve agents by bacterial phosphotriesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghanem, Eman; Raushel, Frank M.

2005-09-01

Organophosphates have been widely used as insecticides and chemical warfare agents. The health risks associated with these agents have necessitated the need for better detoxification and bioremediation tools. Bacterial enzymes capable of hydrolyzing the lethal organophosphate nerve agents are of special interest. Phosphotriesterase (PTE) isolated from the soil bacteria Pseudomonas diminuta displays a significant rate enhancement and substrate promiscuity for the hydrolysis of organophosphate triesters. Directed evolution and rational redesign of the active site of PTE have led to the identification of new variants with enhanced catalytic efficiency and stereoselectivity toward the hydrolysis of organophosphate neurotoxins. PTE has been utilizedmore » to protect against organophosphate poisoning in vivo. Biotechnological applications of PTE for detection and decontamination of insecticides and chemical warfare agents are developing into useful tools. In this review, the catalytic properties and potential applications of this remarkable enzyme are discussed.« less

Comparative Genomics Provide Insights into Evolution of Trichoderma Nutrition Style

PubMed Central

Xie, Bin-Bin; Qin, Qi-Long; Shi, Mei; Chen, Lei-Lei; Shu, Yan-Li; Luo, Yan; Wang, Xiao-Wei; Rong, Jin-Cheng; Gong, Zhi-Ting; Li, Dan; Sun, Cai-Yun; Liu, Gui-Ming; Dong, Xiao-Wei; Pang, Xiu-Hua; Huang, Feng; Liu, Weifeng; Chen, Xiu-Lan; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Song, Xiao-Yan

2014-01-01

Saprotrophy on plant biomass is a recently developed nutrition strategy for Trichoderma. However, the physiology and evolution of this new nutrition strategy is still elusive. We report the deep sequencing and analysis of the genome of Trichoderma longibrachiatum, an efficient cellulase producer. The 31.7-Mb genome, smallest among the sequenced Trichoderma species, encodes fewer nutrition-related genes than saprotrophic T. reesei (Tr), including glycoside hydrolases and nonribosomal peptide synthetase–polyketide synthase. Homology and phylogenetic analyses suggest that a large number of nutrition-related genes, including GH18 chitinases, β-1,3/1,6-glucanases, cellulolytic enzymes, and hemicellulolytic enzymes, were lost in the common ancestor of T. longibrachiatum (Tl) and Tr. dN/dS (ω) calculation indicates that all the nutrition-related genes analyzed are under purifying selection. Cellulolytic enzymes, the key enzymes for saprotrophy on plant biomass, are under stronger purifying selection pressure in Tl and Tr than in mycoparasitic species, suggesting that development of the nutrition strategy of saprotrophy on plant biomass has increased the selection pressure. In addition, aspartic proteases, serine proteases, and metalloproteases are subject to stronger purifying selection pressure in Tl and Tr, suggesting that these enzymes may also play important roles in the nutrition. This study provides insights into the physiology and evolution of the nutrition strategy of Trichoderma. PMID:24482532

Comparative genomics provide insights into evolution of trichoderma nutrition style.

PubMed

Xie, Bin-Bin; Qin, Qi-Long; Shi, Mei; Chen, Lei-Lei; Shu, Yan-Li; Luo, Yan; Wang, Xiao-Wei; Rong, Jin-Cheng; Gong, Zhi-Ting; Li, Dan; Sun, Cai-Yun; Liu, Gui-Ming; Dong, Xiao-Wei; Pang, Xiu-Hua; Huang, Feng; Liu, Weifeng; Chen, Xiu-Lan; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Song, Xiao-Yan

2014-02-01

Saprotrophy on plant biomass is a recently developed nutrition strategy for Trichoderma. However, the physiology and evolution of this new nutrition strategy is still elusive. We report the deep sequencing and analysis of the genome of Trichoderma longibrachiatum, an efficient cellulase producer. The 31.7-Mb genome, smallest among the sequenced Trichoderma species, encodes fewer nutrition-related genes than saprotrophic T. reesei (Tr), including glycoside hydrolases and nonribosomal peptide synthetase-polyketide synthase. Homology and phylogenetic analyses suggest that a large number of nutrition-related genes, including GH18 chitinases, β-1,3/1,6-glucanases, cellulolytic enzymes, and hemicellulolytic enzymes, were lost in the common ancestor of T. longibrachiatum (Tl) and Tr. dN/dS (ω) calculation indicates that all the nutrition-related genes analyzed are under purifying selection. Cellulolytic enzymes, the key enzymes for saprotrophy on plant biomass, are under stronger purifying selection pressure in Tl and Tr than in mycoparasitic species, suggesting that development of the nutrition strategy of saprotrophy on plant biomass has increased the selection pressure. In addition, aspartic proteases, serine proteases, and metalloproteases are subject to stronger purifying selection pressure in Tl and Tr, suggesting that these enzymes may also play important roles in the nutrition. This study provides insights into the physiology and evolution of the nutrition strategy of Trichoderma.

The Cytophaga hutchinsonii ChTPSP: First characterized bifunctional TPS-TPP protein as putative ancestor of all eukaryotic trehalose biosynthesis proteins.

PubMed

Avonce, Nelson; Wuyts, Jan; Verschooten, Katrien; Vandesteene, Lies; Van Dijck, Patrick

2010-02-01

The most widely distributed pathway to synthesize trehalose in nature consists of two consecutive enzymatic reactions with a trehalose-6-P (T6P)-synthase (TPS) enzyme, producing the intermediate T6P, and a T6P-phosphatase (TPP) enzyme, which dephosphorylates T6P to produce trehalose and inorganic phosphate. In plants, these enzymes are called Class I and Class II proteins, respectively, with some Class I proteins being active enzymes. The Class II proteins possess both TPS and TPP consensus regions but appear to have lost enzymatic activity during evolution. Plants also contain an extra group of enzymes of small protein size, of which some members have been characterized as functional TPPs. These Class III proteins have less sequence similarity with the Class I and Class II proteins. Here, we characterize for the first time, by using biochemical analysis and yeast growth complementation assays, the existence of a natural TPS-TPP bifunctional enzyme found in the bacterial species Cytophaga hutchinsonii. Through phylogenetic analysis, we show that prokaryotic genes such as ChTPSP might be the ancestor of the eukaryotic trehalose biosynthesis genes. Second, we show that plants have recruited during evolution, possibly by horizontal transfer from bacteria such as Rhodoferax ferrireducens, a new type of small protein, encoding TPP activity, which have been named Class III proteins. RfTPP has very high TPP activity upon expression in yeast. Finally, we demonstrate that TPS gene duplication, the recruitment of the Class III enzymes, and recruitment of an N-terminal regulatory element, which regulates the Class I enzyme activity in higher plants, were initiated very early in eukaryan evolution as the three classes of trehalose biosynthesis genes are already present in the alga Ostreococcus tauri.

Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1.

PubMed

Wu, Tung-Yun; Chen, Chang-Ting; Liu, Jessica Tse-Jin; Bogorad, Igor W; Damoiseaux, Robert; Liao, James C

2016-06-01

Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

'Enzyme Test Bench': A biochemical application of the multi-rate modeling

NASA Astrophysics Data System (ADS)

Rachinskiy, K.; Schultze, H.; Boy, M.; Büchs, J.

2008-11-01

In the expanding field of 'white biotechnology' enzymes are frequently applied to catalyze the biochemical reaction from a resource material to a valuable product. Evolutionary designed to catalyze the metabolism in any life form, they selectively accelerate complex reactions under physiological conditions. Modern techniques, such as directed evolution, have been developed to satisfy the increasing demand on enzymes. Applying these techniques together with rational protein design, we aim at improving of enzymes' activity, selectivity and stability. To tap the full potential of these techniques, it is essential to combine them with adequate screening methods. Nowadays a great number of high throughput colorimetric and fluorescent enzyme assays are applied to measure the initial enzyme activity with high throughput. However, the prediction of enzyme long term stability within short experiments is still a challenge. A new high throughput technique for enzyme characterization with specific attention to the long term stability, called 'Enzyme Test Bench', is presented. The concept of the Enzyme Test Bench consists of short term enzyme tests conducted under partly extreme conditions to predict the enzyme long term stability under moderate conditions. The technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles in sequential experiments by optimum non-linear experimental design, the long term deactivation effects can be purposefully accelerated and detected within hours. During the experiment the enzyme activity is measured online to estimate the model parameters from the obtained data. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The results of the characterization, based on micro liter format experiments of hours, are in good agreement with the results of long term experiments in 1L format. Thus, the new technique allows for both: the enzyme screening with regard to the long term stability and the choice of the optimal process temperature. The presented article gives a successful example for the application of multi-rate modeling, experimental design and parameter estimation within biochemical engineering. At the same time, it shows the limitations of the methods at the state of the art and addresses the current problems to the applied mathematics community.

Regioselective alkane hydroxylation with a mutant AlkB enzyme

DOEpatents

Koch, Daniel J.; Arnold, Frances H.

2012-11-13

AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

Site-directed saturation mutagenesis of polyhydroxylalkanoate synthase for efficient microbial production of poly[(R)-2-hydroxybutyrate].

PubMed

Hori, Chiaki; Oishi, Kenta; Matsumoto, Ken'ichiro; Taguchi, Seiichi; Ooi, Toshihiko

2018-06-01

In our previous study, artificial polyhydroxyalkanoate (PHA) poly[(R)-2-hydroxybutyrate] [P(2HB)] was successfully biosynthesized from racemic 2HB in recombinant Escherichia coli using an engineered PHA synthase, PhaC1 Ps (S325T/Q481K). Although P(2HB) has promising material properties, the low level of polymer production was a drawback. In this study, we performed directed evolution of PhaC1 Ps towards enhanced P(2HB) accumulation in E. coli by site-directed dual saturation mutagenesis at the positions 477 and 481, which was known for their potential in enhancing natural PHA accumulation. By using a screening on agar plates with Nile red, eight colonies were isolated which produced a greater amount of P(2HB) compared to a colony expressing the parent enzyme PhaC1 Ps (S325T/Q481K). Among them, the cells expressing PhaC1 Ps (S325T/S477R/Q481G) [ST/SR/QG] accumulated polymer at the highest level (up to 2.9-fold). As seen in PhaC1 Ps (ST/SR/QG), glycine and basic amino acid residues (K or R) were frequently found at the two positions of the select mutated enzymes. The enzymatic activity of PhaC1 Ps (ST/SR/QG) toward 2HB-CoA was approximately 3-fold higher than that of the parent enzyme. Additionally, expression levels of the select mutated enzymes were lower than the parent. These results indicated that PhaC1 Ps mutagenesis at the positions 477 and 481 increased specific activity toward 2HB-CoA and it could result in the enhanced production of P(2HB). Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Characteristic Variations and Similarities in Biochemical, Molecular, and Functional Properties of Glyoxalases across Prokaryotes and Eukaryotes.

PubMed

Kaur, Charanpreet; Sharma, Shweta; Hasan, Mohammad Rokebul; Pareek, Ashwani; Singla-Pareek, Sneh L; Sopory, Sudhir K

2017-03-30

The glyoxalase system is the ubiquitous pathway for the detoxification of methylglyoxal (MG) in the biological systems. It comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), which act sequentially to convert MG into d-lactate, thereby helping living systems get rid of this otherwise cytotoxic byproduct of metabolism. In addition, a glutathione-independent GLYIII enzyme activity also exists in the biological systems that can directly convert MG to d-lactate. Humans and Escherichia coli possess a single copy of GLYI (encoding either the Ni- or Zn-dependent form) and GLYII genes, which through MG detoxification provide protection against various pathological and disease conditions. By contrast, the plant genome possesses multiple GLYI and GLYII genes with a role in abiotic stress tolerance. Plants possess both Ni 2+ - and Zn 2+ -dependent forms of GLYI, and studies on plant glyoxalases reveal the various unique features of these enzymes distinguishing them from prokaryotic and other eukaryotic glyoxalases. Through this review, we provide an overview of the plant glyoxalase family along with a comparative analysis of glyoxalases across various species, highlighting similarities as well as differences in the biochemical, molecular, and physiological properties of these enzymes. We believe that the evolution of multiple glyoxalases isoforms in plants is an important component of their robust defense strategies.

Surveying the expanding prokaryotic Rubisco multiverse.

PubMed

Liu, Di; Ramya, Ramaswamy Chettiyan Seetharaman; Mueller-Cajar, Oliver

2017-09-01

The universal, but catalytically modest, CO2-fixing enzyme Rubisco is currently experiencing intense interest by researchers aiming to enhance crop photosynthesis. These efforts are mostly focused on the highly conserved hexadecameric enzyme found in land plants. In comparison, prokaryotic organisms harbor a far greater diversity in Rubisco forms. Recent work towards improving our appreciation of microbial Rubisco properties and harnessing their potential is surveyed. New structural models are providing informative glimpses into catalytic subtleties and diverse oligomeric states. Ongoing characterization is informing us about the conservation of constraints, such as sugar phosphate inhibition and the associated dependence on Rubisco activase helper proteins. Prokaryotic Rubiscos operate under a far wider range of metabolic contexts than the photosynthetic function of higher plant enzymes. Relaxed selection pressures may have resulted in the exploration of a larger volume of sequence space than permitted in organisms performing oxygenic photosynthesis. To tap into the potential of microbial Rubiscos, in vivo selection systems are being used to discover functional metagenomic Rubiscos. Various directed evolution systems to optimize their function have been developed. It is anticipated that this approach will provide access to biotechnologically valuable enzymes that cannot be encountered in the higher plant Rubisco space. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus

PubMed Central

Wang, Yupeng; Khan, Iram F.; Boissel, Sandrine; Jarjour, Jordan; Pangallo, Joseph; Thyme, Summer; Baker, David; Scharenberg, Andrew M.; Rawlings, David J.

2014-01-01

LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20–22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)—a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering. PMID:24682825

The evolution of recombinant thrombolytics: Current status and future directions

PubMed Central

Adivitiya; Khasa, Yogender Pal

2017-01-01

ABSTRACT Cardiovascular disorders are on the rise worldwide due to alcohol abuse, obesity, hypertension, raised blood lipids, diabetes and age-related risks. The use of classical antiplatelet and anticoagulant therapies combined with surgical intervention helped to clear blood clots during the inceptive years. However, the discovery of streptokinase and urokinase ushered the way of using these enzymes as thrombolytic agents to degrade the fibrin network with an issue of systemic hemorrhage. The development of second generation plasminogen activators like anistreplase and tissue plasminogen activator partially controlled this problem. The third generation molecules, majorly t-PA variants, showed desirable properties of improved stability, safety and efficacy with enhanced fibrin specificity. Plasmin variants are produced as direct fibrinolytic agents as a futuristic approach with targeted delivery of these drugs using liposome technlogy. The novel molecules from microbial, plant and animal origin present the future of direct thrombolytics due to their safety and ease of administration. PMID:27696935

On the levels of enzymatic substrate specificity: Implications for the early evolution of metabolic pathways

NASA Technical Reports Server (NTRS)

Lazcano, A.; Diaz-Villagomez, E.; Mills, T.; Oro, J.

1995-01-01

The most frequently invoked explanation for the origin of metabolic pathways is the retrograde evolution hypothesis. In contrast, according to the so-called 'patchwork' theory, metabolism evolved by the recruitment of relatively inefficient small enzymes of broad specificity that could react with a wide range of chemically related substrates. In this paper it is argued that both sequence comparisons and experimental results on enzyme substrate specificity support the patchwork assembly theory. The available evidence supports previous suggestions that gene duplication events followed by a gradual neoDarwinian accumulation of mutations and other minute genetic changes lead to the narrowing and modification of enzyme function in at least some primordial metabolic pathways.

New lives for old: evolution of pseudoenzyme function illustrated by iRhoms.

PubMed

Adrain, Colin; Freeman, Matthew

2012-07-11

Large-scale sequencing of genomes has revealed that most enzyme families include inactive homologues. These pseudoenzymes are often well conserved, implying a selective pressure to retain them during evolution, and therefore that they have significant function. Mechanistic insights and evolutionary lessons are now emerging from the study of a broad range of such 'dead' enzymes. The recently discovered iRhoms - inactive homologues of rhomboid proteases - have joined derlins and other members of the rhomboid-like clan in regulating the fate of proteins as they pass through the secretory pathway. There is a strong case that dead enzymes, which have been rather overlooked, may be a rich source of biological regulators.

The tubulins of animals, plants, fungi and protists implications for metazoan evolution

NASA Astrophysics Data System (ADS)

Little, Melvyn; Ludueña, Richard F.; Morejohn, Louis C.; Asnes, Clara; Hoffman, Eugene

1984-03-01

α-Tubulin subunits from trout (S. gairdneri) sperm tails, sea urchin (S. purpuratus) cilia, protistan alga (C. elongatum) flagella and rose (Paul's Scarlet) cytoplasm have been characterized by limited proteolytic cleavage with the enzymeStaphylococcus aureus protease and electrophoresis of the digestion products on SDS-PAGE. The resulting patterns corresponded to either of two major types representative of animal and non-animal α-tubulins, respectively. A total of 28 α-tubulins have now been characterized by this method. They are classified in this paper according to the type of cleavage pattern generated by the enzymeS. aureus protease. The implications of these results for metazoan evolution are discussed.

On origin and evolution of carbonic anhydrase isozymes: A phylogenetic analysis from whole-enzyme to active site.

PubMed

Banerjee, Srijoni; Deshpande, Parag A

2016-04-01

Genetic evolution of carbonic anhydrase enzyme provides an interesting instance of functional similarity in spite of structural diversity of the members of a given family of enzymes. Phylogenetic analysis of α-, β- and γ-carbonic anhydrase was carried out to determine the evolutionary relationships among various members of the family with the enzyme marking its presence in a wide range of cellular and chromosomal locations. The presence of more than one class of enzymes in a particular organism was revealed by phylogenetic time tree. The evolutionary relationships among the members of animal, plant and microbial kingdom were developed. The study revises a long-established notion of kingdom-specificity of the different classes of carbonic anhydrases and provides a new version of the presence of multiple classes of carbonic anhydrases in a single organism and the presence of a given class of carbonic anhydrase across different kingdoms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Combinatorial evolution of site- and enantioselective catalysts for polyene epoxidation

NASA Astrophysics Data System (ADS)

Lichtor, Phillip A.; Miller, Scott J.

2012-12-01

Selectivity in the catalytic functionalization of complex molecules is a major challenge in chemical synthesis. The problem is magnified when there are several possible stereochemical outcomes and when similar functional groups occur repeatedly within the same molecule. Selective polyene oxidation provides an archetypical example of this challenge. Historically, enzymatic catalysis has provided the only precedents. Although non-enzymatic catalysts that meet some of these challenges became known, a comprehensive solution has remained elusive. Here, we describe low molecular weight peptide-based catalysts, discovered through a combinatorial synthesis and screening protocol, that exhibit site- and enantioselective oxidation of certain positions of various isoprenols. This diversity-based approach, which exhibits features reminiscent of the directed evolution of enzymes, delivers catalysts that compare favourably to the state-of-the-art for the asymmetric oxidation of these compounds. Moreover, the approach culminated in catalysts that exhibit alternative-site selectivity in comparison to oxidation catalysts previously described.

Gain-of-function mutations in beet DODA2 identify key residues for betalain pigment evolution.

PubMed

Bean, Alexander; Sunnadeniya, Rasika; Akhavan, Neda; Campbell, Annabelle; Brown, Matthew; Lloyd, Alan

2018-05-13

The key enzymatic step in betalain biosynthesis involves conversion of l-3,4-dihydroxyphenylalanine (l-DOPA) to betalamic acid. One class of enzymes capable of this is 3,4-dihydroxyphenylalanine 4,5-dioxygenase (DODA). In betalain-producing species, multiple paralogs of this gene are maintained. This study demonstrates which paralogs function in the betalain pathway and determines the residue changes required to evolve a betalain-nonfunctional DODA into a betalain-functional DODA. Functionalities of two pairs of DODAs were tested by expression in beets, Arabidopsis and yeast, and gene silencing was performed by virus-induced gene silencing. Site-directed mutagenesis identified amino acid residues essential for betalamic acid production. Beta vulgaris and Mirabilis jalapa both possess a DODA1 lineage that functions in the betalain pathway and at least one other lineage, DODA2, that does not. Site-directed mutagenesis resulted in betalain biosynthesis by a previously nonfunctional DODA, revealing key residues required for evolution of the betalain pathway. Divergent functionality of DODA paralogs, one clade involved in betalain biosynthesis but others not, is present in various Caryophyllales species. A minimum of seven amino acid residue changes conferred betalain enzymatic activity to a betalain-nonfunctional DODA paralog, providing insight into the evolution of the betalain pigment pathway in plants. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Genetic control of enzyme formation. Final technical report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mills, S. E.

1978-07-26

Research progress is reported on work on tryptophan biosynthesis in Euglena gracilis and higher plants. The experimental data provide an outline of the general evolution of the pathway. Structural analyses of the pathway proteins by quantitative immunochemical methods have been completed; this was done with the anthranilate synthase-1 phosphoribosyl transferase complex in Escherichia coli. An examination of the evolution, in the Enterobacteriaceae, of the enzyme activities anthranilate synthase and anthranilate-5-1 phosphoribosyl-1-pyrophosphate phosphoribosyltransferase has been begun. (ACR)

Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes.

PubMed

Huang, Ruiqi; O'Donnell, Andrew J; Barboline, Jessica J; Barkman, Todd J

2016-09-20

Convergent evolution is a process that has occurred throughout the tree of life, but the historical genetic and biochemical context promoting the repeated independent origins of a trait is rarely understood. The well-known stimulant caffeine, and its xanthine alkaloid precursors, has evolved multiple times in flowering plant history for various roles in plant defense and pollination. We have shown that convergent caffeine production, surprisingly, has evolved by two previously unknown biochemical pathways in chocolate, citrus, and guaraná plants using either caffeine synthase- or xanthine methyltransferase-like enzymes. However, the pathway and enzyme lineage used by any given plant species is not predictable from phylogenetic relatedness alone. Ancestral sequence resurrection reveals that this convergence was facilitated by co-option of genes maintained over 100 million y for alternative biochemical roles. The ancient enzymes of the Citrus lineage were exapted for reactions currently used for various steps of caffeine biosynthesis and required very few mutations to acquire modern-day enzymatic characteristics, allowing for the evolution of a complete pathway. Future studies aimed at manipulating caffeine content of plants will require the use of different approaches given the metabolic and genetic diversity revealed by this study.

Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes

PubMed Central

Huang, Ruiqi; O’Donnell, Andrew J.; Barboline, Jessica J.; Barkman, Todd J.

2016-01-01

Convergent evolution is a process that has occurred throughout the tree of life, but the historical genetic and biochemical context promoting the repeated independent origins of a trait is rarely understood. The well-known stimulant caffeine, and its xanthine alkaloid precursors, has evolved multiple times in flowering plant history for various roles in plant defense and pollination. We have shown that convergent caffeine production, surprisingly, has evolved by two previously unknown biochemical pathways in chocolate, citrus, and guaraná plants using either caffeine synthase- or xanthine methyltransferase-like enzymes. However, the pathway and enzyme lineage used by any given plant species is not predictable from phylogenetic relatedness alone. Ancestral sequence resurrection reveals that this convergence was facilitated by co-option of genes maintained over 100 million y for alternative biochemical roles. The ancient enzymes of the Citrus lineage were exapted for reactions currently used for various steps of caffeine biosynthesis and required very few mutations to acquire modern-day enzymatic characteristics, allowing for the evolution of a complete pathway. Future studies aimed at manipulating caffeine content of plants will require the use of different approaches given the metabolic and genetic diversity revealed by this study. PMID:27638206

Intron-loss evolution of hatching enzyme genes in Teleostei

PubMed Central

2010-01-01

Background Hatching enzyme, belonging to the astacin metallo-protease family, digests egg envelope at embryo hatching. Orthologous genes of the enzyme are found in all vertebrate genomes. Recently, we found that exon-intron structures of the genes were conserved among tetrapods, while the genes of teleosts frequently lost their introns. Occurrence of such intron losses in teleostean hatching enzyme genes is an uncommon evolutionary event, as most eukaryotic genes are generally known to be interrupted by introns and the intron insertion sites are conserved from species to species. Here, we report on extensive studies of the exon-intron structures of teleostean hatching enzyme genes for insight into how and why introns were lost during evolution. Results We investigated the evolutionary pathway of intron-losses in hatching enzyme genes of 27 species of Teleostei. Hatching enzyme genes of basal teleosts are of only one type, which conserves the 9-exon-8-intron structure of an assumed ancestor. On the other hand, otocephalans and euteleosts possess two types of hatching enzyme genes, suggesting a gene duplication event in the common ancestor of otocephalans and euteleosts. The duplicated genes were classified into two clades, clades I and II, based on phylogenetic analysis. In otocephalans and euteleosts, clade I genes developed a phylogeny-specific structure, such as an 8-exon-7-intron, 5-exon-4-intron, 4-exon-3-intron or intron-less structure. In contrast to the clade I genes, the structures of clade II genes were relatively stable in their configuration, and were similar to that of the ancestral genes. Expression analyses revealed that hatching enzyme genes were high-expression genes, when compared to that of housekeeping genes. When expression levels were compared between clade I and II genes, clade I genes tends to be expressed more highly than clade II genes. Conclusions Hatching enzyme genes evolved to lose their introns, and the intron-loss events occurred at the specific points of teleostean phylogeny. We propose that the high-expression hatching enzyme genes frequently lost their introns during the evolution of teleosts, while the low-expression genes maintained the exon-intron structure of the ancestral gene. PMID:20796321

The RNA-world and co-evolution hypothesis and the origin of life: Implications, research strategies and perspectives

NASA Technical Reports Server (NTRS)

Lahav, Noam

1993-01-01

The applicability of the RNA-world and co-evolution hypothesis to the study of the very first stages of the origin of life is discussed. The discussion focuses on the basic differences between the two hypotheses and their implications, with regard to the reconstruction methodology, ribosome emergence, balance between ribozymes and protein enzymes, and their major difficultites. Additional complexities of the two hypotheses, such as membranes and the energy source of the first reactions, are not treated in the present work. A central element in the proposed experimental strategies is the study of the catalytic activites of very small peptides and RNA-like oligomers, according to existing, as well as to yet-to-be-invented scenarios of the two hypothesis under consideration. It is suggested that the novel directed molecular evolution technology, and molecular computational modeling, can be applied to this research. This strategy is assumed to be essential for the suggested goal of future studies of the origin of life, namely, the establishment of a `Primordial Darwinian entity'.

Identification and Functional Characterization of Monofunctional ent-Copalyl Diphosphate and ent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms1[W][OA

PubMed Central

Keeling, Christopher I.; Dullat, Harpreet K.; Yuen, Mack; Ralph, Steven G.; Jancsik, Sharon; Bohlmann, Jörg

2010-01-01

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms. PMID:20044448

Segment swapping aided the evolution of enzyme function: The case of uroporphyrinogen III synthase.

PubMed

Szilágyi, András; Györffy, Dániel; Závodszky, Péter

2017-01-01

In an earlier study, we showed that two-domain segment-swapped proteins can evolve by domain swapping and fusion, resulting in a protein with two linkers connecting its domains. We proposed that a potential evolutionary advantage of this topology may be the restriction of interdomain motions, which may facilitate domain closure by a hinge-like movement, crucial for the function of many enzymes. Here, we test this hypothesis computationally on uroporphyrinogen III synthase, a two-domain segment-swapped enzyme essential in porphyrin metabolism. To compare the interdomain flexibility between the wild-type, segment-swapped enzyme (having two interdomain linkers) and circular permutants of the same enzyme having only one interdomain linker, we performed geometric and molecular dynamics simulations for these species in their ligand-free and ligand-bound forms. We find that in the ligand-free form, interdomain motions in the wild-type enzyme are significantly more restricted than they would be with only one interdomain linker, while the flexibility difference is negligible in the ligand-bound form. We also estimated the entropy costs of ligand binding associated with the interdomain motions, and find that the change in domain connectivity due to segment swapping results in a reduction of this entropy cost, corresponding to ∼20% of the total ligand binding free energy. In addition, the restriction of interdomain motions may also help the functional domain-closure motion required for catalysis. This suggests that the evolution of the segment-swapped topology facilitated the evolution of enzyme function for this protein by influencing its dynamic properties. Proteins 2016; 85:46-53. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Evolution of allosteric regulation in chorismate mutases from early plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kroll, Kourtney; Holland, Cynthia K.; Starks, Courtney M.

Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plantmore » chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 Å X-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes.« less

The evolution of energy-transducing systems. Studies with an extremely halophilic archaebacterium

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga

1992-01-01

The F-type ATPases are found in remarkably similar versions in the energy-transducing membranes of eubacteria, chloroplasts, and mitochondria. Thus, it is likely that they have originated early in the evolution of life, which is consistent with their function as key enzymes of cellular metabolism. The archaebacteria are a group of microorganisms which, as shown by molecular sequencing and biochemical data, have diverged early from the main line of prokaryotic evolution. From studies of members of all three major groups of archaebacteria - the halophiles, methanogens, and thermoacidophiles - it emerged that they possess a membrane ATPase which differs from the F-ATPases. The goal of this project was a comparison of the ATPase from the halophilic archaebacterium Halobacterium saccharovorum with the well-characterized F-type ATPases on the molecular level. Amino acid sequences of critical regions of the enzyme were to be determined, as well as immunoreactions of single subunits in the search for common epitopes. The results were expected to allow a decision about the nature of archaebacterial ATPases, their classification as one of the known or, alternatively, novel enzyme complexes, and possibly deduction of events during the early evolution of energy-transducing systems.

A novel helper phage for HaloTag-mediated co-display of enzyme and substrate on phage.

PubMed

Delespaul, Wouter; Peeters, Yves; Herdewijn, Piet; Robben, Johan

2015-05-01

Phage display is an established technique for the molecular evolution of peptides and proteins. For the selection of enzymes based on catalytic activity however, simultaneous coupling of an enzyme and its substrate to the phage surface is required. To facilitate this process of co-display, we developed a new helper phage displaying HaloTag, a modified haloalkane dehalogenase that binds specifically and covalently to functionalized haloalkane ligands. The display of functional HaloTag was demonstrated by capture on streptavidin-coated magnetic beads, after coupling a biotinylated haloalkane ligand, or after on-phage extension of a DNA oligonucleotide primer with a biotinylated nucleotide by phi29 DNA polymerase. We also achieved co-display of HaloTag and phi29 DNA polymerase, thereby opening perspectives for the molecular evolution of this enzyme (and others) towards new substrate specificities. Copyright © 2015 Elsevier Inc. All rights reserved.

Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution.

PubMed

Goedegebuur, Frits; Dankmeyer, Lydia; Gualfetti, Peter; Karkehabadi, Saeid; Hansson, Henrik; Jana, Suvamay; Huynh, Vicky; Kelemen, Bradley R; Kruithof, Paulien; Larenas, Edmund A; Teunissen, Pauline J M; Ståhlberg, Jerry; Payne, Christina M; Mitchinson, Colin; Sandgren, Mats

2017-10-20

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in T m and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Temporal variations in the gene expression levels of cyanobacterial anti-oxidant enzymes through geological history: implications for biological evolution during the Great Oxidation Event

NASA Astrophysics Data System (ADS)

Harada, M.; Furukawa, R.; Yokobori, S. I.; Tajika, E.; Yamagishi, A.

2016-12-01

A significant rise in atmospheric O2 levels during the GOE (Great Oxidation Event), ca. 2.45-2.0 Ga, must have caused a great stress to biosphere, enforcing life to adapt to oxic conditions. Cyanobacteria, oxygenic photosynthetic bacteria that had been responsible for the GOE, are at the same time one of the organisms that would have been greatly affected by the rise of O2 level in the surface environments. Knowledge on the evolution of cyanobacteria is not only important to elucidate the cause of the GOE, but also helps us to better understand the adaptive evolution of life in response to the GOE. Here we performed phylogenetic analysis of an anti-oxidant enzyme Fe-SOD (iron superoxide dismutase) of cyanobacteria, to assess the adaptive evolution of life under the GOE. The rise of O2 level must have increased the level of toxic reactive oxygen species in cyanobacterial cells, thus forced them to change activities or the gene expression levels of Fe-SOD. In the present study, we focus on the change in the gene expression levels of the enzyme, which can be estimated from the promoter sequences of the gene. Promoters are DNA sequences found upstream of protein encoding regions, where RNA polymerase binds and initiates transcription. "Strong" promoters that efficiently interact with RNA polymerase induce high rates of transcription, leading to high levels of gene expression. Thus, from the temporal changes in the promoter sequences, we can estimate the variations in the gene expression levels during the geological time. Promoter sequences of Fe-SOD at each ancestral node of cyanobacteria were predicted from phylogenetic analysis, and the ancestral promoter sequences were compared to the promoters of known highly expressed genes. The similarity was low at the time of the emergence of cyanobacteria; however, increased at the branching nodes diverged 2.4 billon years ago. This roughly coincided with the onset of the GOE, implying that the transition from low to high gene expression levels of Fe-SOD occurred in response to the GOE. We propose that this is the first direct evidence of the evolution of cyanobacteria related to the rise of O2, and that the methodologies of ancestral promoter analysis used in this study can be a novel tools to reveal the biological adaptation to such a significant geologic event.

A phylogenetic analysis of normal modes evolution in enzymes and its relationship to enzyme function

PubMed Central

Lai, Jason; Jin, Jing; Kubelka, Jan; Liberles, David A.

2012-01-01

Since the dynamic nature of protein structures is essential for enzymatic function, it is expected that the functional evolution can be inferred from the changes in the protein dynamics. However, dynamics can also diverge neutrally with sequence substitution between enzymes without changes of function. In this study, a phylogenetic approach is implemented to explore the relationship between enzyme dynamics and function through evolutionary history. Protein dynamics are described by normal mode analysis based on a simplified harmonic potential force field applied to the reduced Cα representation of the protein structure while enzymatic function is described by Enzyme Commission (EC) numbers. Similarity of the binding pocket dynamics at each branch of the protein family’s phylogeny was analyzed in two ways: 1) explicitly by quantifying the normal mode overlap calculated for the reconstructed ancestral proteins at each end and 2) implicitly using a diffusion model to obtain the reconstructed lineage-specific changes in the normal modes. Both explicit and implicit ancestral reconstruction identified generally faster rates of change in dynamics compared with the expected change from neutral evolution at the branches of potential functional divergences for the alpha-amylase, D-isomer specific 2-hydroxyacid dehydrogenase, and copper-containing amine oxidase protein families. Normal modes analysis added additional information over just comparing the RMSD of static structures. However, the branch-specific changes were not statistically significant compared to background function-independent neutral rates of change of dynamic properties and blind application of the analysis would not enable prediction of changes in enzyme specificity. PMID:22651983

A phylogenetic analysis of normal modes evolution in enzymes and its relationship to enzyme function.

PubMed

Lai, Jason; Jin, Jing; Kubelka, Jan; Liberles, David A

2012-09-21

Since the dynamic nature of protein structures is essential for enzymatic function, it is expected that functional evolution can be inferred from the changes in protein dynamics. However, dynamics can also diverge neutrally with sequence substitution between enzymes without changes of function. In this study, a phylogenetic approach is implemented to explore the relationship between enzyme dynamics and function through evolutionary history. Protein dynamics are described by normal mode analysis based on a simplified harmonic potential force field applied to the reduced C(α) representation of the protein structure while enzymatic function is described by Enzyme Commission numbers. Similarity of the binding pocket dynamics at each branch of the protein family's phylogeny was analyzed in two ways: (1) explicitly by quantifying the normal mode overlap calculated for the reconstructed ancestral proteins at each end and (2) implicitly using a diffusion model to obtain the reconstructed lineage-specific changes in the normal modes. Both explicit and implicit ancestral reconstruction identified generally faster rates of change in dynamics compared with the expected change from neutral evolution at the branches of potential functional divergences for the α-amylase, D-isomer-specific 2-hydroxyacid dehydrogenase, and copper-containing amine oxidase protein families. Normal mode analysis added additional information over just comparing the RMSD of static structures. However, the branch-specific changes were not statistically significant compared to background function-independent neutral rates of change of dynamic properties and blind application of the analysis would not enable prediction of changes in enzyme specificity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Pumping ions: rapid parallel evolution of ionic regulation following habitat invasions.

PubMed

Lee, Carol Eunmi; Kiergaard, Michael; Gelembiuk, Gregory William; Eads, Brian Donovan; Posavi, Marijan

2011-08-01

Marine to freshwater colonizations constitute among the most dramatic evolutionary transitions in the history of life. This study examined evolution of ionic regulation following saline-to-freshwater transitions in an invasive species. In recent years, the copepod Eurytemora affinis has invaded freshwater habitats multiple times independently. We found parallel evolutionary shifts in ion-motive enzyme activity (V-type H(+) ATPase, Na(+) /K(+) -ATPase) across independent invasions and in replicate laboratory selection experiments. Freshwater populations exhibited increased V-type H(+) ATPase activity in fresh water (0 PSU) and declines at higher salinity (15 PSU) relative to saline populations. This shift represented marked evolutionary increases in plasticity. In contrast, freshwater populations displayed reduced Na(+) /K(+) -ATPase activity across all salinities. Most notably, modifying salinity alone during laboratory selection experiments recapitulated the evolutionary shifts in V-type H(+) ATPase activity observed in nature. Maternal and embryonic acclimation could not account for the observed shifts in enzyme activity. V-type H(+) ATPase function has been hypothesized to be critical for freshwater and terrestrial adaptations, but evolution of this enzyme function had not been previously demonstrated in the context of habitat transitions. Moreover, the speed of these evolutionary shifts was remarkable, within a few generations in the laboratory and a few decades in the wild. © 2011 The Author(s). Evolution© 2011 The Society for the Study of Evolution.

Evolution of functional specialization and division of labor.

PubMed

Rueffler, Claus; Hermisson, Joachim; Wagner, Günter P

2012-02-07

Division of labor among functionally specialized modules occurs at all levels of biological organization in both animals and plants. Well-known examples include the evolution of specialized enzymes after gene duplication, the evolution of specialized cell types, limb diversification in arthropods, and the evolution of specialized colony members in many taxa of marine invertebrates and social insects. Here, we identify conditions favoring the evolution of division of labor by means of a general mathematical model. Our starting point is the assumption that modules contribute to two different biological tasks and that the potential of modules to contribute to these tasks is traded off. Our results are phrased in terms of properties of performance functions that map the phenotype of modules to measures of performance. We show that division of labor is favored by three factors: positional effects that predispose modules for one of the tasks, accelerating performance functions, and synergistic interactions between modules. If modules can be lost or damaged, selection for robustness can counteract selection for functional specialization. To illustrate our theory we apply it to the evolution of specialized enzymes coded by duplicated genes.

Thermoadaptation of a mesophilic hygromycin B phosphotransferase by directed evolution in hyperthermophilic Archaea: selection of a stable genetic marker for DNA transfer into Sulfolobus solfataricus.

PubMed

Cannio, R; Contursi, P; Rossi, M; Bartolucci, S

2001-06-01

A mutated version of the hygromycin B phosphotransferase (hph(mut)) gene from Escherichia coli, isolated by directed evolution at 75 degrees C in transformants of a thermophilic strain of Sulfolobus solfataricus, was characterized with respect to its genetic stability in both the original mesophilic and the new thermophilic hosts. This gene was demonstrated to be able to express the hygromycin B resistance phenotype and to be steadily maintained and propagated also in other, more thermophilic strains of S. solfataricus, i.e., up to 82 degrees C. Furthermore, it may be transferred to S. solfataricus cells by cotransformation with pKMSD48, another extrachromosomal element derived from the virus SSV1 of Sulfolobus shibatae, without any loss of stability and without affecting the replication and infectivity of this viral DNA. The hph(mut) and the wild-type gene products were expressed at higher levels in E. coli and purified by specific affinity chromatography on immobilized hygromycin B. Comparative characterization revealed that the mutant enzyme had acquired significant thermoresistance and displayed higher thermal activity with augmented catalytic efficiency.

Thermostable Cellulases: Why & How?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2010-03-24

These are a set of slides from the conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for eachmore » cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Highly Efficient Thermostable DSM Cellulases: Why & How?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Manoj

2011-04-26

These are the slides from this presentation. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase componentmore » enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

DOE PAGES

Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.; ...

2014-12-31

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

PubMed

Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

2016-04-04

Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Genetically programmed chiral organoborane synthesis

NASA Astrophysics Data System (ADS)

Kan, S. B. Jennifer; Huang, Xiongyi; Gumulya, Yosephine; Chen, Kai; Arnold, Frances H.

2017-12-01

Recent advances in enzyme engineering and design have expanded nature’s catalytic repertoire to functions that are new to biology. However, only a subset of these engineered enzymes can function in living systems. Finding enzymatic pathways that form chemical bonds that are not found in biology is particularly difficult in the cellular environment, as this depends on the discovery not only of new enzyme activities, but also of reagents that are both sufficiently reactive for the desired transformation and stable in vivo. Here we report the discovery, evolution and generalization of a fully genetically encoded platform for producing chiral organoboranes in bacteria. Escherichia coli cells harbouring wild-type cytochrome c from Rhodothermus marinus (Rma cyt c) were found to form carbon-boron bonds in the presence of borane-Lewis base complexes, through carbene insertion into boron-hydrogen bonds. Directed evolution of Rma cyt c in the bacterial catalyst provided access to 16 novel chiral organoboranes. The catalyst is suitable for gram-scale biosynthesis, providing up to 15,300 turnovers, a turnover frequency of 6,100 h-1, a 99:1 enantiomeric ratio and 100% chemoselectivity. The enantiopreference of the biocatalyst could also be tuned to provide either enantiomer of the organoborane products. Evolved in the context of whole-cell catalysts, the proteins were more active in the whole-cell system than in purified forms. This study establishes a DNA-encoded and readily engineered bacterial platform for borylation; engineering can be accomplished at a pace that rivals the development of chemical synthetic methods, with the ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than those of known chiral catalysts for the same class of transformation. This tunable method for manipulating boron in cells could expand the scope of boron chemistry in living systems.

Functional Analogy in Human Metabolism: Enzymes with Different Biological Roles or Functional Redundancy?

PubMed Central

Piergiorge, Rafael Mina; de Miranda, Antonio Basílio; Catanho, Marcos

2017-01-01

Abstract Since enzymes catalyze almost all chemical reactions that occur in living organisms, it is crucial that genes encoding such activities are correctly identified and functionally characterized. Several studies suggest that the fraction of enzymatic activities in which multiple events of independent origin have taken place during evolution is substantial. However, this topic is still poorly explored, and a comprehensive investigation of the occurrence, distribution, and implications of these events has not been done so far. Fundamental questions, such as how analogous enzymes originate, why so many events of independent origin have apparently occurred during evolution, and what are the reasons for the coexistence in the same organism of distinct enzymatic forms catalyzing the same reaction, remain unanswered. Also, several isofunctional enzymes are still not recognized as nonhomologous, even with substantial evidence indicating different evolutionary histories. In this work, we begin to investigate the biological significance of the cooccurrence of nonhomologous isofunctional enzymes in human metabolism, characterizing functional analogous enzymes identified in metabolic pathways annotated in the human genome. Our hypothesis is that the coexistence of multiple enzymatic forms might not be interpreted as functional redundancy. Instead, these enzymatic forms may be implicated in distinct (and probably relevant) biological roles. PMID:28854631

Bio-inspired co-catalysts bonded to a silicon photocathode for solar hydrogen evolution

NASA Astrophysics Data System (ADS)

Hou, Yidong; Abrams, Billie L.; Vesborg, Peter C. K.; Bjørketun, Mårten E.; Herbst, Konrad; Bech, Lone; Seger, Brian; Pedersen, Thomas; Hansen, Ole; Rossmeisl, Jan; Dahl, Søren; Nørskov, Jens K.; Chorkendorff, Ib

2011-10-01

The production of fuels directly or indirectly from sunlight represents one of the major challenges to the development of a sustainable energy system. Hydrogen is the simplest fuel to produce and while platinum and other noble metals are efficient catalysts for photoelectrochemical hydrogen evolution, earth-abundant alternatives are needed for largescale use. We show that bio-inspired molecular clusters based on molybdenum sulfides and tungsten sulfides mimic nature's enzymes for hydrogen evolution, molybdenum sulfides evolve hydrogen at a slightly higher overpotential than platinum when deposited on various supports. It will be demonstrated how this overpotential can be eliminated by depositing the same type of hydrogen evolution catalyst on p-type Si which can harvest the red part of the solar spectrum. Such a system could constitute the cathode part of a tandem dream device where the red part of the spectrum is utilized for hydrogen evolution while the blue part is reserved for the more difficult oxygen evolution. The samples have been illuminated with a simulated red part of the solar spectrum i.e. long wavelength (" > 620 nm) part of simulated AM 1.5G radiation. The current densities at the reversible potential match the requirement of a photoelectrochemical hydrogen production system with a solar-to-hydrogen efficiency in excess of 10%. The experimental observations are supported by DFT calculations of the Mo3S4 cluster adsorbed on the hydrogen-terminated silicon surface providing insights into the nature of the active site.

Net light-induced oxygen evolution in photosystem I deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Wang, Qing Jun; Singh, Abhay; Li, Hong; Nedbal, Ladislav; Sherman, Louis A; Govindjee; Whitmarsh, John

2012-05-01

Oxygenic photosynthesis in cyanobacteria, algae, and plants requires photosystem II (PSII) to extract electrons from H(2)O and depends on photosystem I (PSI) to reduce NADP(+). Here we demonstrate that mixotrophically-grown mutants of the cyanobacterium Synechocystis sp. PCC 6803 that lack PSI (ΔPSI) are capable of net light-induced O(2) evolution in vivo. The net light-induced O(2) evolution requires glucose and can be sustained for more than 30 min. Utilizing electron transport inhibitors and chlorophyll a fluorescence measurements, we show that in these mutants PSII is the source of the light-induced O(2) evolution, and that the plastoquinone pool is reduced by PSII and subsequently oxidized by an unidentified electron acceptor that does not involve the plastoquinol oxidase site of the cytochrome b(6)f complex. Moreover, both O(2) evolution and chlorophyll a fluorescence kinetics of the ΔPSI mutants are highly sensitive to KCN, indicating the involvement of a KCN-sensitive enzyme(s). Experiments using (14)C-labeled bicarbonate show that the ΔPSI mutants assimilate more CO(2) in the light compared to the dark. However, the rate of the light-minus-dark CO(2) assimilation accounts for just over half of the net light-induced O(2) evolution rate, indicating the involvement of unidentified terminal electron acceptors. Based on these results we suggest that O(2) evolution in ΔPSI cells can be sustained by an alternative electron transport pathway that results in CO(2) assimilation and that includes PSII, the platoquinone pool, and a KCN-sensitive enzyme. Copyright © 2012 Elsevier B.V. All rights reserved.

Carbohydrates as efficient catalysts for the hydration of α-amino nitriles.

PubMed

Chitale, Sampada; Derasp, Joshua S; Hussain, Bashir; Tanveer, Kashif; Beauchemin, André M

2016-11-01

Directed hydration of α-amino nitriles was achieved under mild conditions using simple carbohydrates as catalysts exploiting temporary intramolecularity. A broadly applicable procedure using both formaldehyde and NaOH as catalysts efficiently hydrated a variety of primary and secondary susbtrates, and allowed the hydration of enantiopure substrates to proceed without racemization. This work also provides a rare comparison of the catalytic activity of carbohydrates, and shows that the simple aldehydes at the basis of chemical evolution are efficient organocatalysts mimicking the function of hydratase enzymes. Optimal catalytic efficiency was observed with destabilized aldehydes, and with difficult substrates only simple carbohydrates such as formaldehyde and glycolaldehyde proved reliable.

Playing RNase P evolution: swapping the RNA catalyst for a protein reveals functional uniformity of highly divergent enzyme forms.

PubMed

Weber, Christoph; Hartig, Andreas; Hartmann, Roland K; Rossmanith, Walter

2014-08-01

The RNase P family is a diverse group of endonucleases responsible for the removal of 5' extensions from tRNA precursors. The diversity of enzyme forms finds its extremes in the eukaryal nucleus where RNA-based catalysis by complex ribonucleoproteins in some organisms contrasts with single-polypeptide enzymes in others. Such structural contrast suggests associated functional differences, and the complexity of the ribonucleoprotein was indeed proposed to broaden the enzyme's functionality beyond tRNA processing. To explore functional overlap and differences between most divergent forms of RNase P, we replaced the nuclear RNase P of Saccharomyces cerevisiae, a 10-subunit ribonucleoprotein, with Arabidopsis thaliana PRORP3, a single monomeric protein. Surprisingly, the RNase P-swapped yeast strains were viable, displayed essentially unimpaired growth under a wide variety of conditions, and, in a certain genetic background, their fitness even slightly exceeded that of the wild type. The molecular analysis of the RNase P-swapped strains showed a minor disturbance in tRNA metabolism, but did not point to any RNase P substrates or functions beyond that. Altogether, these results indicate the full functional exchangeability of the highly dissimilar enzymes. Our study thereby establishes the RNase P family, with its combination of structural diversity and functional uniformity, as an extreme case of convergent evolution. It moreover suggests that the apparently gratuitous complexity of some RNase P forms is the result of constructive neutral evolution rather than reflecting increased functional versatility.

Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5).

PubMed

Aspeborg, Henrik; Coutinho, Pedro M; Wang, Yang; Brumer, Harry; Henrissat, Bernard

2012-09-20

The large Glycoside Hydrolase family 5 (GH5) groups together a wide range of enzymes acting on β-linked oligo- and polysaccharides, and glycoconjugates from a large spectrum of organisms. The long and complex evolution of this family of enzymes and its broad sequence diversity limits functional prediction. With the objective of improving the differentiation of enzyme specificities in a knowledge-based context, and to obtain new evolutionary insights, we present here a new, robust subfamily classification of family GH5. About 80% of the current sequences were assigned into 51 subfamilies in a global analysis of all publicly available GH5 sequences and associated biochemical data. Examination of subfamilies with catalytically-active members revealed that one third are monospecific (containing a single enzyme activity), although new functions may be discovered with biochemical characterization in the future. Furthermore, twenty subfamilies presently have no characterization whatsoever and many others have only limited structural and biochemical data. Mapping of functional knowledge onto the GH5 phylogenetic tree revealed that the sequence space of this historical and industrially important family is far from well dispersed, highlighting targets in need of further study. The analysis also uncovered a number of GH5 proteins which have lost their catalytic machinery, indicating evolution towards novel functions. Overall, the subfamily division of GH5 provides an actively curated resource for large-scale protein sequence annotation for glycogenomics; the subfamily assignments are openly accessible via the Carbohydrate-Active Enzyme database at http://www.cazy.org/GH5.html.

Characteristic Variations and Similarities in Biochemical, Molecular, and Functional Properties of Glyoxalases across Prokaryotes and Eukaryotes

PubMed Central

Kaur, Charanpreet; Sharma, Shweta; Hasan, Mohammad Rokebul; Pareek, Ashwani; Singla-Pareek, Sneh L.; Sopory, Sudhir K.

2017-01-01

The glyoxalase system is the ubiquitous pathway for the detoxification of methylglyoxal (MG) in the biological systems. It comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), which act sequentially to convert MG into d-lactate, thereby helping living systems get rid of this otherwise cytotoxic byproduct of metabolism. In addition, a glutathione-independent GLYIII enzyme activity also exists in the biological systems that can directly convert MG to d-lactate. Humans and Escherichia coli possess a single copy of GLYI (encoding either the Ni- or Zn-dependent form) and GLYII genes, which through MG detoxification provide protection against various pathological and disease conditions. By contrast, the plant genome possesses multiple GLYI and GLYII genes with a role in abiotic stress tolerance. Plants possess both Ni2+- and Zn2+-dependent forms of GLYI, and studies on plant glyoxalases reveal the various unique features of these enzymes distinguishing them from prokaryotic and other eukaryotic glyoxalases. Through this review, we provide an overview of the plant glyoxalase family along with a comparative analysis of glyoxalases across various species, highlighting similarities as well as differences in the biochemical, molecular, and physiological properties of these enzymes. We believe that the evolution of multiple glyoxalases isoforms in plants is an important component of their robust defense strategies. PMID:28358304

Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

PubMed

Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

2016-03-01

Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.

Functional metagenomic selection of RubisCOs from uncultivated bacteria

USGS Publications Warehouse

Varaljay, Vanessa A; Satagopan, Sriram; North, Justin A.; Witteveen, Briana; Dourado, Manuella N.; Anantharaman, Karthik; Arbing, Mark A.; McCann, Shelley; Oremland, Ronald S.; Banfield, Jillian F.; Wrighton, Kelly C.; Tabita, F. Robert

2016-01-01

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2-dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of theGallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possessCO2/O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2-fixing enzymes not previously characterized.

White biotechnology: State of the art strategies for the development of biocatalysts for biorefining.

PubMed

Heux, S; Meynial-Salles, I; O'Donohue, M J; Dumon, C

2015-12-01

White biotechnology is a term that is now often used to describe the implementation of biotechnology in the industrial sphere. Biocatalysts (enzymes and microorganisms) are the key tools of white biotechnology, which is considered to be one of the key technological drivers for the growing bioeconomy. Biocatalysts are already present in sectors such as the chemical and agro-food industries, and are used to manufacture products as diverse as antibiotics, paper pulp, bread or advanced polymers. This review proposes an original and global overview of highly complementary fields of biotechnology at both enzyme and microorganism level. A certain number of state of the art approaches that are now being used to improve the industrial fitness of biocatalysts particularly focused on the biorefinery sector are presented. The first part deals with the technologies that underpin the development of industrial biocatalysts, notably the discovery of new enzymes and enzyme improvement using directed evolution techniques. The second part describes the toolbox available by the cell engineer to shape the metabolism of microorganisms. And finally the last part focuses on the 'omic' technologies that are vital for understanding and guide microbial engineering toward more efficient microbial biocatalysts. Altogether, these techniques and strategies will undoubtedly help to achieve the challenging task of developing consolidated bioprocessing (i.e. CBP) readily available for industrial purpose. Copyright © 2015 Elsevier Inc. All rights reserved.

Simultaneous use of in silico design and a correlated mutation network as a tool to efficiently guide enzyme engineering.

PubMed

Nobili, Alberto; Tao, Yifeng; Pavlidis, Ioannis V; van den Bergh, Tom; Joosten, Henk-Jan; Tan, Tianwei; Bornscheuer, Uwe T

2015-03-23

In order to improve the efficiency of directed evolution experiments, in silico multiple-substrate clustering was combined with an analysis of the variability of natural enzymes within a protein superfamily. This was applied to a Pseudomonas fluorescens esterase (PFE I) targeting the enantioselective hydrolysis of 3-phenylbutyric acid esters. Data reported in the literature for nine substrates were used for the clustering meta-analysis of the docking conformations in wild-type PFE I, and this highlighted a tryptophan residue (W28) as an interesting target. Exploration of the most frequently, naturally occurring amino acids at this position suggested that the reduced flexibility observed in the case of the W28F variant leads to enhancement of the enantioselectivity. This mutant was subsequently combined with mutations identified in a library based on analysis of a correlated mutation network. By interrogation of <80 variants a mutant with 15-fold improved enantioselectivity was found. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Emerging engineering principles for yield improvement in microbial cell design.

PubMed

Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo

2012-01-01

Metabolic Engineering has undertaken a rapid transformation in the last ten years making real progress towards the production of a wide range of molecules and fine chemicals using a designed cellular host. However, the maximization of product yields through pathway optimization is a constant and central challenge of this field. Traditional methods used to improve the production of target compounds from engineered biosynthetic pathways in non-native hosts include: codon usage optimization, elimination of the accumulation of toxic intermediates or byproducts, enhanced production of rate-limiting enzymes, selection of appropriate promoter and ribosome binding sites, application of directed evolution of enzymes, and chassis re-circuit. Overall, these approaches tend to be specific for each engineering project rather than a systematic practice based on a more generalizable strategy. In this mini-review, we highlight some novel and extensive approaches and tools intended to address the improvement of a target product formation, founded in sophisticated principles such as dynamic control, pathway genes modularization, and flux modeling.

Emerging engineering principles for yield improvement in microbial cell design

PubMed Central

Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo

2012-01-01

Metabolic Engineering has undertaken a rapid transformation in the last ten years making real progress towards the production of a wide range of molecules and fine chemicals using a designed cellular host. However, the maximization of product yields through pathway optimization is a constant and central challenge of this field. Traditional methods used to improve the production of target compounds from engineered biosynthetic pathways in non-native hosts include: codon usage optimization, elimination of the accumulation of toxic intermediates or byproducts, enhanced production of rate-limiting enzymes, selection of appropriate promoter and ribosome binding sites, application of directed evolution of enzymes, and chassis re-circuit. Overall, these approaches tend to be specific for each engineering project rather than a systematic practice based on a more generalizable strategy. In this mini-review, we highlight some novel and extensive approaches and tools intended to address the improvement of a target product formation, founded in sophisticated principles such as dynamic control, pathway genes modularization, and flux modeling. PMID:24688676

Protein conformational disorder and enzyme catalysis.

PubMed

Schulenburg, Cindy; Hilvert, Donald

2013-01-01

Though lacking a well-defined three-dimensional structure, intrinsically unstructured proteins are ubiquitous in nature. These molecules play crucial roles in many cellular processes, especially signaling and regulation. Surprisingly, even enzyme catalysis can tolerate substantial disorder. This observation contravenes conventional wisdom but is relevant to an understanding of how protein dynamics modulates enzyme function. This chapter reviews properties and characteristics of disordered proteins, emphasizing examples of enzymes that lack defined structures, and considers implications of structural disorder for catalytic efficiency and evolution.

Molecular evolution of multiple-level control of heme biosynthesis pathway in animal kingdom.

PubMed

Tzou, Wen-Shyong; Chu, Ying; Lin, Tzung-Yi; Hu, Chin-Hwa; Pai, Tun-Wen; Liu, Hsin-Fu; Lin, Han-Jia; Cases, Ildeofonso; Rojas, Ana; Sanchez, Mayka; You, Zong-Ye; Hsu, Ming-Wei

2014-01-01

Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5' untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway.

2-Oxoacid Metabolism in Methanogenic CoM and CoB Biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Graham, David E

Coenzyme M (CoM) and coenzyme B (CoB) are essential for methane production by the euryarchaea that employ this specialized anaerobic metabolism. Two pathways are known to produce CoM, 2-mercaptoethanesulfonate, and both converge on the 2-oxoacid sulfopyruvate. These cells have recruited the rich biochemistry of amino acid and 2-oxoacid metabolizing enzymes to produce a compound that resembles oxaloacetate, but with a more stable and acidic sulfonate group. 7-Mercaptoheptanoylthreonine phosphate, CoB, likewise owes its carbon backbone to a 2-oxoacid. Three enzymes recruited from leucine biosynthesis have evolved to catalyze the elongation of 2-oxoglutarate to 2-oxosuberate in CoB biosynthesis. This chapter describes themore » enzymology, synthesis and analytical techniques used to study 2-oxoacid metabolism in these pathways. Protein structure and mechanistic information from enzymes provides insight into the evolution of new enzymatic activity, and the evolution of substrate specificity from promiscuous enzyme scaffolds.« less

Massive Thermal Acceleration of the Emergence of Primordial Chemistry, the Incidence of Spontaneous Mutation, and the Evolution of Enzymes*

PubMed Central

Wolfenden, Richard

2014-01-01

Kelvin considered it unlikely that sufficient time had elapsed on the earth for life to have reached its present level of complexity. In the warm surroundings in which life first appeared, however, elevated temperatures would have reduced the kinetic barriers to reaction. Recent experiments disclose the profound extent to which very slow reactions are accelerated by elevated temperatures, collapsing the time that would have been required for early events in primordial chemistry before the advent of enzymes. If a primitive enzyme, like model catalysts and most modern enzymes, accelerated a reaction by lowering its enthalpy of activation, then the rate enhancement that it produced would have increased automatically as the environment cooled, quite apart from any improvements in catalytic activity that arose from mutation and natural selection. The chemical events responsible for spontaneous mutation are also highly sensitive to temperature, furnishing an independent mechanism for accelerating evolution. PMID:25210030

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

PubMed Central

Antoniali, Giulia; Lirussi, Lisa; Poletto, Mattia

2014-01-01

Abstract Significance: An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. Recent Advances: After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. Critical Issues: A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. Future Directions: A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world. Antioxid. Redox Signal. 20, 621–639. PMID:23879289

Direct enzymatic bioelectrocatalysis: differentiating between myth and reality

PubMed Central

2017-01-01

Enzymatic bioelectrocatalysis is being increasingly exploited to better understand oxidoreductase enzymes, to develop minimalistic yet specific biosensor platforms, and to develop alternative energy conversion devices and bioelectrosynthetic devices for the production of energy and/or important chemical commodities. In some cases, these enzymes are able to electronically communicate with an appropriately designed electrode surface without the requirement of an electron mediator to shuttle electrons between the enzyme and electrode. This phenomenon has been termed direct electron transfer or direct bioelectrocatalysis. While many thorough studies have extensively investigated this fascinating feat, it is sometimes difficult to differentiate desirable enzymatic bioelectrocatalysis from electrocatalysis deriving from inactivated enzyme that may have also released its catalytic cofactor. This article will review direct bioelectrocatalysis of several oxidoreductases, with an emphasis on experiments that provide support for direct bioelectrocatalysis versus denatured enzyme or dissociated cofactor. Finally, this review will conclude with a series of proposed control experiments that could be adopted to discern successful direct electronic communication of an enzyme from its denatured counterpart. PMID:28637918

Human Origins: Problems in the Interpretation of New Evidence. Third Edition. AAAS Study Guides on Contemporary Problems.

ERIC Educational Resources Information Center

Almquist, Alan J.; Cronin, John E.

This Chautauqua-type short course in human evolution is divided into two parts: The Biochemical Evidence for Human Evolution, and the Fossil Evidence for Human Evolution. The first part covers the comparison of macromolecular differences between species. This includes comparison of DNA base-ratios and amino acid substitution in enzymes and other…

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

PubMed Central

2014-01-01

Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme. PMID:24934472

Evolutionary analysis of the TPP-dependent enzyme family.

PubMed

Costelloe, Seán J; Ward, John M; Dalby, Paul A

2008-01-01

The evolutionary relationships of the thiamine pyrophosphate (TPP)-dependent family of enzymes was investigated by generation of a neighbor joining phylogenetic tree using sequences from the conserved pyrophosphate (PP) and pyrimidine (Pyr) binding domains of 17 TPP-dependent enzymes. This represents the most comprehensive analysis of TPP-dependent enzyme evolution to date. The phylogeny was shown to be robust by comparison with maximum likelihood trees generated for each individual enzyme and also broadly confirms the evolutionary history proposed recently from structural comparisons alone (Duggleby 2006). The phylogeny is most parsimonious with the TPP enzymes having arisen from a homotetramer which subsequently diverged into an alpha(2)beta(2) heterotetramer. The relationship between the PP- and Pyr-domains and the recruitment of additional protein domains was examined using the transketolase C-terminal (TKC)-domain as an example. This domain has been recruited by several members of the family and yet forms no part of the active site and has unknown function. Removal of the TKC-domain was found to increase activity toward beta-hydroxypyruvate and glycolaldehyde. Further truncations of the Pyr-domain yielded several variants with retained activity. This suggests that the influence of TKC-domain recruitment on the evolution of the mechanism and specificity of transketolase (TK) has been minor, and that the smallest functioning unit of TK comprises the PP- and Pyr-domains, whose evolutionary histories extend to all TPP-dependent enzymes.

FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

PubMed

Bednar, David; Beerens, Koen; Sebestova, Eva; Bendl, Jaroslav; Khare, Sagar; Chaloupkova, Radka; Prokop, Zbynek; Brezovsky, Jan; Baker, David; Damborsky, Jiri

2015-11-01

There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

DNA's Encounter with Ultraviolet Light: An Instinct for Self-Preservation?

PubMed

Barlev, Adam; Sen, Dipankar

2018-02-20

Photochemical modification is the major class of environmental damage suffered by DNA, the genetic material of all free-living organisms. Photolyases are enzymes that carry out direct photochemical repair (photoreactivation) of covalent pyrimidine dimers formed in DNA from exposure to ultraviolet light. The discovery of catalytic RNAs in the 1980s led to the "RNA world hypothesis", which posits that early in evolution RNA or a similar polymer served both genetic and catalytic functions. Intrigued by the RNA world hypothesis, we set out to test whether a catalytic RNA (or a surrogate, a catalytic DNA) with photolyase activity could be contemplated. In vitro selection from a random-sequence DNA pool yielded two DNA enzymes (DNAzymes): Sero1C, which requires serotonin as an obligate cofactor, and UV1C, which is cofactor-independent and optimally uses light of 300-310 nm wavelength to repair cyclobutane thymine dimers within a gapped DNA substrate. Both Sero1C and UV1C show multiple turnover kinetics, and UV1C repairs its substrate with a quantum yield of ∼0.05, on the same order as the quantum yields of certain classes of photolyase enzymes. Intensive study of UV1C has revealed that its catalytic core consists of a guanine quadruplex (G-quadruplex) positioned proximally to the bound substrate's thymine dimer. We hypothesize that electron transfer from photoexcited guanines within UV1C's G-quadruplex is responsible for substrate photoreactivation, analogous to electron transfer to pyrimidine dimers within a DNA substrate from photoexcited flavin cofactors located within natural photolyase enzymes. Though the analogy to evolution is necessarily limited, a comparison of the properties of UV1C and Sero1C, which arose out of the same in vitro selection experiment, reveals that although the two DNAzymes comparably accelerate the rate of thymine dimer repair, Sero1C has a substantially broader substrate repertoire, as it can repair many more kinds of pyrimidine dimers than UV1C. Therefore, the co-opting of an amino acid-like cofactor by a nucleic acid enzyme in this case contributes functional versatility rather than a greater rate enhancement. In recent work on UV1C, we have succeeded in shifting its action spectrum from the UVB into the blue region of the spectrum and determined that although it catalyzes both repair and de novo formation of thymine dimers, UV1C is primarily a catalyst for thymine dimer repair. Our work on photolyase DNAzymes has stimulated broader questions about whether analogous, purely nucleotide-based photoreactivation also occurs in double-helical DNA, the dominant form of DNA in living cells. Recently, a number of different groups have reported that this kind of repair is indeed operational in DNA duplexes, i.e., that there exist nucleotide sequences that actively protect, by way of photoreactivation (rather than by simply preventing their formation), pyrimidine dimers located proximal to them. Nucleotide-based photoreactivation thus appears to be a salient, if unanticipated, property of DNA and RNA. The phenomenon also offers pointers in the direction of how in primordial evolution-in an RNA world-early nucleic acids may have protected themselves from structural and functional damage wrought by ultraviolet light.

Molecular evolution accompanying functional divergence of duplicated genes along the plant starch biosynthesis pathway

PubMed Central

2014-01-01

Background Starch is the main source of carbon storage in the Archaeplastida. The starch biosynthesis pathway (sbp) emerged from cytosolic glycogen metabolism shortly after plastid endosymbiosis and was redirected to the plastid stroma during the green lineage divergence. The SBP is a complex network of genes, most of which are members of large multigene families. While some gene duplications occurred in the Archaeplastida ancestor, most were generated during the sbp redirection process, and the remaining few paralogs were generated through compartmentalization or tissue specialization during the evolution of the land plants. In the present study, we tested models of duplicated gene evolution in order to understand the evolutionary forces that have led to the development of SBP in angiosperms. We combined phylogenetic analyses and tests on the rates of evolution along branches emerging from major duplication events in six gene families encoding sbp enzymes. Results We found evidence of positive selection along branches following cytosolic or plastidial specialization in two starch phosphorylases and identified numerous residues that exhibited changes in volume, polarity or charge. Starch synthases, branching and debranching enzymes functional specializations were also accompanied by accelerated evolution. However, none of the sites targeted by selection corresponded to known functional domains, catalytic or regulatory. Interestingly, among the 13 duplications tested, 7 exhibited evidence of positive selection in both branches emerging from the duplication, 2 in only one branch, and 4 in none of the branches. Conclusions The majority of duplications were followed by accelerated evolution targeting specific residues along both branches. This pattern was consistent with the optimization of the two sub-functions originally fulfilled by the ancestral gene before duplication. Our results thereby provide strong support to the so-called “Escape from Adaptive Conflict” (EAC) model. Because none of the residues targeted by selection occurred in characterized functional domains, we propose that enzyme specialization has occurred through subtle changes in affinity, activity or interaction with other enzymes in complex formation, while the basic function defined by the catalytic domain has been maintained. PMID:24884572

The protein cofactor allows the sequence of an RNase P ribozyme to diversify by maintaining the catalytically active structure of the enzyme.

PubMed Central

Kim, J J; Kilani, A F; Zhan, X; Altman, S; Liu, F

1997-01-01

To study the effect proteins have on the catalysis and evolution of RNA enzymes, we simulated evolution of RNase P catalytic M1 RNA in vitro, in the presence and absence of its C5 protein cofactor. In the presence of C5, functional M1 sequence variants (not catalytically active in the absence of C5) were selected in addition to those identical to M1. C5 maintains the catalytically active structure of the variants and allows for an enhanced spectrum of M1 molecules to function in the context of a ribonucleoprotein (RNP) complex. The generation of an RNP enzyme, requiring both RNA and protein components, from a catalytically active RNA molecule has implications for how modern RNP complexes evolved from ancestral RNAs. PMID:9174096

Inside the trap: gland morphologies, digestive enzymes, and the evolution of plant carnivory in the Caryophyllales⋆

PubMed Central

Renner, Tanya; Specht, Chelsea D

2013-01-01

The digestion of prey by carnivorous plants is determined in part by suites of enzymes that are associated with morphologically and anatomically diverse trapping mechanisms. Chitinases represent a group of enzymes known to be integral to effective plant carnivory. In non-carnivorous plants, chitinases commonly act as pathogenesis-related proteins, which are either induced in response to insect herbivory and fungal elicitors, or constitutively expressed in tissues vulnerable to attack. In the Caryophyllales carnivorous plant lineage, multiple classes of chitinases are likely involved in both pathogenic response and digestion of prey items. We review what is currently known about trap morphologies, provide an examination of the diversity, roles, and evolution of chitinases, and examine how herbivore and pathogen defense mechanisms may have been coopted for plant carnivory in the Caryophyllales. PMID:23830995

Direct Electron Transfer of Enzymes in a Biologically Assembled Conductive Nanomesh Enzyme Platform.

PubMed

Lee, Seung-Woo; Lee, Ki-Young; Song, Yong-Won; Choi, Won Kook; Chang, Joonyeon; Yi, Hyunjung

2016-02-24

Nondestructive assembly of a nanostructured enzyme platform is developed in combination of the specific biomolecular attraction and electrostatic coupling for highly efficient direct electron transfer (DET) of enzymes with unprecedented applicability and versatility. The biologically assembled conductive nanomesh enzyme platform enables DET-based flexible integrated biosensors and DET of eight different enzyme with various catalytic activities. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evolutionary history of versatile-lipases from Agaricales through reconstruction of ancestral structures.

PubMed

Barriuso, Jorge; Martínez, María Jesús

2017-01-03

Fungal "Versatile carboxylic ester hydrolases" are enzymes with great biotechnological interest. Here we carried out a bioinformatic screening to find these proteins in genomes from Agaricales, by means of searching for conserved motifs, sequence and phylogenetic analysis, and three-dimensional modeling. Moreover, we reconstructed the molecular evolution of these enzymes along the time by inferring and analyzing the sequence of ancestral intermediate forms. The properties of the ancestral candidates are discussed on the basis of their three-dimensional structural models, the hydrophobicity of the lid, and the substrate binding intramolecular tunnel, revealing all of them featured properties of these enzymes. The evolutionary history of the putative lipases revealed an increase on the length and hydrophobicity of the lid region, as well as in the size of the substrate binding pocket, during evolution time. These facts suggest the enzymes' specialization towards certain substrates and their subsequent loss of promiscuity. These results bring to light the presence of different pools of lipases in fungi with different habitats and life styles. Despite the consistency of the data gathered from reconstruction of ancestral sequences, the heterologous expression of some of these candidates would be essential to corroborate enzymes' activities.

Enzyme Sequestration as a Tuning Point in Controlling Response Dynamics of Signalling Networks

PubMed Central

Ollivier, Julien F.; Soyer, Orkun S.

2016-01-01

Signalling networks result from combinatorial interactions among many enzymes and scaffolding proteins. These complex systems generate response dynamics that are often essential for correct decision-making in cells. Uncovering biochemical design principles that underpin such response dynamics is a prerequisite to understand evolved signalling networks and to design synthetic ones. Here, we use in silico evolution to explore the possible biochemical design space for signalling networks displaying ultrasensitive and adaptive response dynamics. By running evolutionary simulations mimicking different biochemical scenarios, we find that enzyme sequestration emerges as a key mechanism for enabling such dynamics. Inspired by these findings, and to test the role of sequestration, we design a generic, minimalist model of a signalling cycle, featuring two enzymes and a single scaffolding protein. We show that this simple system is capable of displaying both ultrasensitive and adaptive response dynamics. Furthermore, we find that tuning the concentration or kinetics of the sequestering protein can shift system dynamics between these two response types. These empirical results suggest that enzyme sequestration through scaffolding proteins is exploited by evolution to generate diverse response dynamics in signalling networks and could provide an engineering point in synthetic biology applications. PMID:27163612

Carbon and hydrogen metabolism of green algae in light and dark

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1990-01-01

After adaptation to a hydrogen metabolism, Chlamydomonas reinhardtii can photoanaerobically metabolize acetate with the evolution of H{sub 2} and CO{sub 2}. An enzyme profile of the chloroplastic, cytoplasmic, and mitochondrial fractions were obtained with a cellular fractionation procedure that incorporated cell wall removal by autolysine, digestion of the plasmalemma with digitonin and fractionation by differential centrifugation on a Percoll step gradient. The sequence of events leading to the photo-evolution of H{sub 2} from acetate includes the conversion of acetate into succinate via the extraplastidic glyoxylate cycle, the oxidation of succinate to fumarate by chloroplastic succinic dehydrogenase and the oxidation ofmore » malate to oxaloacetate in the chloroplast by NAD dependent malate dehydrogenase. The level of potential activity of the enzymes was sufficient to accommodate the observed rate of gas evolution. The isolated darkened chloroplast evolves aerobically CO{sub 2} from glucose indicating a chloroplastic respiratory pathway. Evolution of CO{sub 2} is blocked by mitochondrial inhibitors.« less

Biomimicry as a basis for drug discovery.

PubMed

Kolb, V M

1998-01-01

Selected works are discussed which clearly demonstrate that mimicking various aspects of the process by which natural products evolved is becoming a powerful tool in contemporary drug discovery. Natural products are an established and rich source of drugs. The term "natural product" is often used synonymously with "secondary metabolite." Knowledge of genetics and molecular evolution helps us understand how biosynthesis of many classes of secondary metabolites evolved. One proposed hypothesis is termed "inventive evolution." It invokes duplication of genes, and mutation of the gene copies, among other genetic events. The modified duplicate genes, per se or in conjunction with other genetic events, may give rise to new enzymes, which, in turn, may generate new products, some of which may be selected for. Steps of the inventive evolution can be mimicked in several ways for purpose of drug discovery. For example, libraries of chemical compounds of any imaginable structure may be produced by combinatorial synthesis. Out of these libraries new active compounds can be selected. In another example, genetic system can be manipulated to produce modified natural products ("unnatural natural products"), from which new drugs can be selected. In some instances, similar natural products turn up in species that are not direct descendants of each other. This is presumably due to a horizontal gene transfer. The mechanism of this inter-species gene transfer can be mimicked in therapeutic gene delivery. Mimicking specifics or principles of chemical evolution including experimental and test-tube evolution also provides leads for new drug discovery.

Cognitive enhancers (nootropics). Part 2: drugs interacting with enzymes. Update 2014.

PubMed

Froestl, Wolfgang; Muhs, Andreas; Pfeifer, Andrea

2014-01-01

Scientists working in the field of Alzheimer's disease and, in particular, cognitive enhancers are very productive. The review on Drugs interacting with Enzymes was accepted in August 2012. However, this field is very dynamic. New potential targets for the treatment of Alzheimer's disease were identified. This update describes drugs interacting with 60 enzymes versus 43 enzymes in the first paper. Some compounds progressed in their development, while many others were discontinued. The present review covers the evolution of research in this field through April 2014.

A Promiscuous De Novo Retro-Aldolase Catalyzes Asymmetric Michael Additions via Schiff Base Intermediates.

PubMed

Garrabou, Xavier; Beck, Tobias; Hilvert, Donald

2015-05-04

Recent advances in computational design have enabled the development of primitive enzymes for a range of mechanistically distinct reactions. Here we show that the rudimentary active sites of these catalysts can give rise to useful chemical promiscuity. Specifically, RA95.5-8, designed and evolved as a retro-aldolase, also promotes asymmetric Michael additions of carbanions to unsaturated ketones with high rates and selectivities. The reactions proceed by amine catalysis, as indicated by mutagenesis and X-ray data. The inherent flexibility and tunability of this catalyst should make it a versatile platform for further optimization and/or mechanistic diversification by directed evolution. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Redox chemistry of molybdenum in natural waters and its involvement in biological evolution

PubMed Central

Wang, Deli

2012-01-01

The transition element molybdenum (Mo) possesses diverse valances (+II to +VI), and is involved in forming cofactors in more than 60 enzymes in biology. Redox switching of the element in these enzymes catalyzes a series of metabolic reactions in both prokaryotes and eukaryotes, and the element therefore plays a fundamental role in the global carbon, nitrogen, and sulfur cycling. In the present oxygenated waters, oxidized Mo(VI) predominates thermodynamically, whilst reduced Mo species are mainly confined within specific niches including cytoplasm. Only recently has the reduced Mo(V) been separated from Mo(VI) in sulfidic mats and even in some reducing waters. Given the presence of reduced Mo(V) in contemporary anaerobic habitats, it seems that reduced Mo species were present in the ancient reducing ocean (probably under both ferruginous and sulfidic conditions), prompting the involvement of Mo in enzymes including nitrogenase and nitrate reductase. During the global transition to oxic conditions, reduced Mo species were constrained to specific anaerobic habitats, and efficient uptake systems of oxidized Mo(VI) became a selective advantage for current prokaryotic and eukaryotic cells. Some prokaryotes are still able to directly utilize reduced Mo if any exists in ambient environments. In total, this mini-review describes the redox chemistry and biogeochemistry of Mo over the Earth’s history. PMID:23267355

FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.

2014-01-01

Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were isolated. All mutants selected against one antibiotic were cross-resistant to the other two antibiotics. Mutations were not detected in fabF, but the resistant strains harbored missense mutations in fabH. The altered amino acids clustered in and around the FabH active-site tunnel. The mutant FabH proteins were catalytically compromised based on the low activities of the purified enzymes, a fatty acid-dependent growth phenotype, and elevated expression of the fabHF operon in the mutant strains. Independent manipulation of fabF and fabH expression levels showed that the FabH/FabF activity ratio was a major determinant of antibiotic sensitivity. Missense mutations that reduce FabH activity are sufficient to confer resistance to multiple antibiotics that bind to the FabF acyl-enzyme intermediate in S. aureus. PMID:25403676

Models of Protocellular Structure, Function and Evolution

NASA Technical Reports Server (NTRS)

New, Michael H.; Pohorille, Andrew; Szostak, Jack W.; Keefe, Tony; Lanyi, Janos K.

2001-01-01

In the absence of any record of protocells, the most direct way to test our understanding of the origin of cellular life is to construct laboratory models that capture important features of protocellular systems. Such efforts are currently underway in a collaborative project between NASA-Ames, Harvard Medical School and University of California. They are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures. The centerpiece of this project is a method for the in vitro evolution of protein enzymes toward arbitrary catalytic targets. A similar approach has already been developed for nucleic acids in which a small number of functional molecules are selected from a large, random population of candidates. The selected molecules are next vastly multiplied using the polymerase chain reaction. A mutagenic approach, in which the sequences of selected molecules are randomly altered, can yield further improvements in performance or alterations of specificities. Unfortunately, the catalytic potential of nucleic acids is rather limited. Proteins are more catalytically capable but cannot be directly amplified. In the new technique, this problem is circumvented by covalently linking each protein of the initial, diverse, pool to the RNA sequence that codes for it. Then, selection is performed on the proteins, but the nucleic acids are replicated. Additional information is contained in the original extended abstract.

Identification of key residues modulating the stereoselectivity of nitrile hydratase toward rac-mandelonitrile by semi-rational engineering.

PubMed

Cheng, Zhongyi; Peplowski, Lukasz; Cui, Wenjing; Xia, Yuanyuan; Liu, Zhongmei; Zhang, Jialei; Kobayashi, Michihiko; Zhou, Zhemin

2018-03-01

Optically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high-value chiral chemicals is often time-, money-, and resource-intensive; traditional semi-rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot-spot residues. In this study, through ligand-protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity toward rac-mandelonitrile (MAN). All the four selected residues had numerous collisions with rac-MAN. Five mutants significantly shifting stereoselectivity towards (S)-MAN were obtained from site-saturation mutagenesis, one of them, at position βPhe37, exhibiting efficient production of (S)-MAN with 96.8% ee p , was isolated and further analyzed. The increased pulling force observed during SMD simulation was found to be in good coincidence with the formation of hydrogen bonds between (R)-MAN and residue βHis37. (R)-MAN had to break these barriers to enter the active site of nitrile hydratase and S selectivity was thus improved. The results indicated that combining steered molecular dynamics simulation with a traditional semi-rational design significantly reduced the select range of hot-spot residues for the evolution of NHase stereoselectivity, which could serve as an alternative for the modulation of enzyme stereoselectivity. © 2017 Wiley Periodicals, Inc.

Assembling high activity phosphotriesterase composites using hybrid nanoparticle peptide-DNA scaffolded architectures

NASA Astrophysics Data System (ADS)

Breger, Joyce C.; Buckhout-White, Susan; Walper, Scott A.; Oh, Eunkeu; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

2017-06-01

Nanoparticle (NP) display potentially offers a new way to both stabilize and, in many cases, enhance enzyme activity over that seen for native protein in solution. However, the large, globular and sometimes multimeric nature of many enzymes limits their ability to attach directly to the surface of NPs, especially when the latter are colloidally stabilized with bulky PEGylated ligands. Engineering extended protein linkers into the enzymes to achieve direct attachment through the PEG surface often detrimentally alters the enzymes catalytic ability. Here, we demonstrate an alternate, hybrid biomaterials-based approach to achieving directed enzyme assembly on PEGylated NPs. We self-assemble a unique architecture consisting of a central semiconductor quantum dot (QD) scaffold displaying controlled ratios of extended peptide-DNA linkers which penetrate through the PEG surface to directly couple enzymes to the QD surface. As a test case, we utilize phosphotriesterase (PTE), an enzyme of bio-defense interest due to its ability to hydrolyze organophosphate nerve agents. Moreover, this unique approach still allows PTE to maintain enhanced activity while also suggesting the ability of DNA to enhance enzyme activity in and of itself.

Crystal Structure of 4,6-α-Glucanotransferase Supports Diet-Driven Evolution of GH70 Enzymes from α-Amylases in Oral Bacteria.

PubMed

Bai, Yuxiang; Gangoiti, Joana; Dijkstra, Bauke W; Dijkhuizen, Lubbert; Pijning, Tjaard

2017-02-07

Food processing and refining has dramatically changed the human diet, but little is known about whether this affected the evolution of enzymes in human microbiota. We present evidence that glycoside hydrolase family 70 (GH70) glucansucrases from lactobacilli, synthesizing α-glucan-type extracellular polysaccharides from sucrose, likely evolved from GH13 starch-acting α-amylases, via GH70 4,6-α-glucanotransferases. The crystal structure of a 4,6-α-glucanotransferase explains the mode of action and unique product specificity of these enzymes. While the α-amylase substrate-binding scaffold is retained, active-site loops adapted to favor transglycosylation over hydrolysis; the structure also gives clues as to how 4,6-α-glucanotransferases may have evolved further toward sucrose utilization instead of starch. Further supported by genomic, phylogenetic, and in vivo studies, we propose that dietary changes involving starch (and starch derivatives) and sucrose intake were critical factors during the evolution of 4,6-α-GTs and glucansucrases from α-amylases, allowing oral bacteria to produce extracellular polymers that contribute to biofilm formation from different substrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterising Complex Enzyme Reaction Data

PubMed Central

Rahman, Syed Asad; Thornton, Janet M.

2016-01-01

The relationship between enzyme-catalysed reactions and the Enzyme Commission (EC) number, the widely accepted classification scheme used to characterise enzyme activity, is complex and with the rapid increase in our knowledge of the reactions catalysed by enzymes needs revisiting. We present a manual and computational analysis to investigate this complexity and found that almost one-third of all known EC numbers are linked to more than one reaction in the secondary reaction databases (e.g., KEGG). Although this complexity is often resolved by defining generic, alternative and partial reactions, we have also found individual EC numbers with more than one reaction catalysing different types of bond changes. This analysis adds a new dimension to our understanding of enzyme function and might be useful for the accurate annotation of the function of enzymes and to study the changes in enzyme function during evolution. PMID:26840640

A non-synonymous nucleotide substitution can account for one evolutionary route to sesquiterpene synthase activity in the TPS-b subgroup.

PubMed

Green, Sol; Baker, Edward N; Laing, William

2011-06-23

Plant sesquiterpene and hemiterpene synthases in the monoterpene synthase dominated TPS-b subgroup are thought to have evolved independently from a monoterpene synthase ancestor. A TPS-b sesquiterpene synthase from apple (MdAFS1), which predominantly produces α-farnesene, can also synthesize the monoterpene (E)-β-ocimene. The dual activity offered a functional link to an ancestral MdAFS1 enzyme and a rational basis for investigation of the evolution of TPS-b sesquiterpene enzymes. Protein modelling and mutagenesis analysis of the MdAFS1 active site identified a non-synonymous nucleotide substitution that could account for the requisite shift in substrate specificity necessary for the emergence of its sesquiterpene activity during the evolution of the TPS-b enzymes. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Human β-glucuronidase: structure, function, and application in enzyme replacement therapy.

PubMed

Naz, Huma; Islam, Asimul; Waheed, Abdul; Sly, William S; Ahmad, Faizan; Hassan, Imtaiyaz

2013-10-01

Lysosomal storage diseases occur due to incomplete metabolic degradation of macromolecules by various hydrolytic enzymes in the lysosome. Despite structural differences, most of the lysosomal enzymes share many common features including a lysosomal targeting motif and phosphotransferase recognition sites. β-Glucuronidase (GUSB) is an important lysosomal enzyme involved in the degradation of glucuronate-containing glycosaminoglycan. The deficiency of GUSB causes mucopolysaccharidosis type VII (MPSVII), leading to lysosomal storage in the brain. GUSB is a well-studied protein for its expression, sequence, structure, and function. The purpose of this review is to summarize our current understanding of sequence, structure, function, and evolution of GUSB and its lysosomal enzyme targeting. Enzyme replacement therapy reported for this protein is also discussed.

Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases.

PubMed

Sugrue, Elena; Carr, Paul D; Scott, Colin; Jackson, Colin J

2016-11-15

The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little about how enzymes can naturally evolve to include active sites with highly reactive and desolvated charges is known. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214 and Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a noncatalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214 and His215). To understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues increases the pK a of Glu241, allowing it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.

The evolution of respiratory O2/NO reductases: an out-of-the-phylogenetic-box perspective

PubMed Central

Ducluzeau, Anne-Lise; Schoepp-Cothenet, Barbara; van Lis, Robert; Baymann, Frauke; Russell, Michael J.; Nitschke, Wolfgang

2014-01-01

Complex life on our planet crucially depends on strong redox disequilibria afforded by the almost ubiquitous presence of highly oxidizing molecular oxygen. However, the history of O2-levels in the atmosphere is complex and prior to the Great Oxidation Event some 2.3 billion years ago, the amount of O2 in the biosphere is considered to have been extremely low as compared with present-day values. Therefore the evolutionary histories of life and of O2-levels are likely intricately intertwined. The obvious biological proxy for inferring the impact of changing O2-levels on life is the evolutionary history of the enzyme allowing organisms to tap into the redox power of molecular oxygen, i.e. the bioenergetic O2 reductases, alias the cytochrome and quinol oxidases. Consequently, molecular phylogenies reconstructed for this enzyme superfamily have been exploited over the last two decades in attempts to elucidate the interlocking between O2 levels in the environment and the evolution of respiratory bioenergetic processes. Although based on strictly identical datasets, these phylogenetic approaches have led to diametrically opposite scenarios with respect to the history of both the enzyme superfamily and molecular oxygen on the Earth. In an effort to overcome the deadlock of molecular phylogeny, we here review presently available structural, functional, palaeogeochemical and thermodynamic information pertinent to the evolution of the superfamily (which notably also encompasses the subfamily of nitric oxide reductases). The scenario which, in our eyes, most closely fits the ensemble of these non-phylogenetic data, sees the low O2-affinity SoxM- (or A-) type enzymes as the most recent evolutionary innovation and the high-affinity O2 reductases (SoxB or B and cbb3 or C) as arising independently from NO-reducing precursor enzymes. PMID:24968694

METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS

DOE Office of Scientific and Technical Information (OSTI.GOV)

John J. Kilbane III

The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project will focus on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate amidase. The objective of the final phase of the project will bemore » to develop derivative CN bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. The project is on schedule and no major difficulties have been encountered. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments have resulted in the isolation of promising cultures that may be capable of cleaving C-N bonds in aromatic amides, several amidase genes have been cloned and are currently undergoing directed evolution to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. Future research will address expression of these genes in Rhodococcus erythropolis. Enrichment culture experiments and directed evolution experiments continue to be a main focus of research activity and further work is required to obtain an appropriate amidase that will selectively cleave C-N bonds in aromatic substrates. Once an appropriate amidase gene is obtained it must be combined with genes encoding an enzyme capable of converting carbazole to 2'aminobiphenyl-2,3-diol: specifically carA genes. The carA genes from two sources have been cloned and are ready for construction of C-N bond cleavage pathway. The construction of a new metabolic pathway to selectively remove nitrogen from carbazole and other molecules typically found in petroleum should lead to the development of a process to improve oil refinery efficiency by reducing the poisoning, by nitrogen, of catalysts used in the hydrotreating and catalytic cracking of petroleum.« less

Models of the Protocellular Structures, Functions and Evolution

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; New, Michael; Keefe, Anthony; Szostak, Jack W.; Lanyi, Janos F.; DeVincenzi, Donald L. (Technical Monitor)

2000-01-01

In the absence of extinct or extant record of protocells, the most direct way to test our understanding of the origin of cellular life is to construct laboratory models that capture important features of protocellular systems. Such efforts are currently underway in a collaborative project between NASA-Ames, Harvard medical School and University of California. They are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures. The centerpiece of this project is a method for the in vitro evolution of protein enzymes toward arbitrary catalytic targets. A similar approach has already been developed for nucleic acids: First, a very large population of candidate molecules is generated using a random synthetic approach. Next, the small numbers of molecules that can accomplish the desired task are selected. These molecules are next vastly multiplied using the polymerase chain reaction. A mutagenic approach, in which the sequences of selected molecules are randomly altered, can yield further improvements in performance or alterations of specificities. Unfortunately, the catalytic potential of nucleic acids is rather limited. Proteins are more catalytically capable but cannot be directly amplified. In the new technique, this problem is circumvented by covalently linking each protein of the initial, diverse, pool to the RNA sequence that codes for it. Then, selection is performed on the proteins, but the nucleic acids are replicated. To date, we have obtained "a proof of concept" by evolving simple, novel proteins capable of selectively binding adenosine tri-phosphate (ATP). Our next goal is to create an enzyme that can phosphorylate amino acids and another to catalyze the formation of peptide bonds in the absence of nucleic acid templates. This latter reaction does not take place in contemporary cells. once developed, these enzymes will be encapsulated in liposomes so that they will function in a simulated cellular environment. To provide a continuous energy supply, usually needed to activate the substrates, an energy transduction complex which generates ATP from adenosine diphosphate, inorganic phosphate and light will be used. This system, consisting of two modern proteins, ATP synthase and bacteriorhodopsin, has already been built and shown to work efficiently. By coupling chemical synthesis to such a system, it will be possible to drive chemical reactions by light if only the substrates for these reactions are supplied.

The visualCMAT: A web-server to select and interpret correlated mutations/co-evolving residues in protein families.

PubMed

Suplatov, Dmitry; Sharapova, Yana; Timonina, Daria; Kopylov, Kirill; Švedas, Vytas

2018-04-01

The visualCMAT web-server was designed to assist experimental research in the fields of protein/enzyme biochemistry, protein engineering, and drug discovery by providing an intuitive and easy-to-use interface to the analysis of correlated mutations/co-evolving residues. Sequence and structural information describing homologous proteins are used to predict correlated substitutions by the Mutual information-based CMAT approach, classify them into spatially close co-evolving pairs, which either form a direct physical contact or interact with the same ligand (e.g. a substrate or a crystallographic water molecule), and long-range correlations, annotate and rank binding sites on the protein surface by the presence of statistically significant co-evolving positions. The results of the visualCMAT are organized for a convenient visual analysis and can be downloaded to a local computer as a content-rich all-in-one PyMol session file with multiple layers of annotation corresponding to bioinformatic, statistical and structural analyses of the predicted co-evolution, or further studied online using the built-in interactive analysis tools. The online interactivity is implemented in HTML5 and therefore neither plugins nor Java are required. The visualCMAT web-server is integrated with the Mustguseal web-server capable of constructing large structure-guided sequence alignments of protein families and superfamilies using all available information about their structures and sequences in public databases. The visualCMAT web-server can be used to understand the relationship between structure and function in proteins, implemented at selecting hotspots and compensatory mutations for rational design and directed evolution experiments to produce novel enzymes with improved properties, and employed at studying the mechanism of selective ligand's binding and allosteric communication between topologically independent sites in protein structures. The web-server is freely available at https://biokinet.belozersky.msu.ru/visualcmat and there are no login requirements.

Functional cooperation between exonucleases and endonucleases—basis for the evolution of restriction enzymes

PubMed Central

Raghavendra, Nidhanapathi K.; Rao, Desirazu N.

2003-01-01

Many types of restriction enzymes cleave DNA away from their recognition site. Using the type III restriction enzyme, EcoP15I, which cleaves DNA 25–27 bp away from its recognition site, we provide evidence to show that an intact recognition site on the cleaved DNA sequesters the restriction enzyme and decreases the effective concentration of the enzyme. EcoP15I restriction enzyme is shown here to perform only a single round of DNA cleavage. Significantly, we show that an exonuclease activity is essential for EcoP15I restriction enzyme to perform multiple rounds of DNA cleavage. This observation may hold true for all restriction enzymes cleaving DNA sufficiently far away from their recognition site. Our results highlight the importance of functional cooperation in the modulation of enzyme activity. Based on results presented here and other data on well-characterised restriction enzymes, a functional evolutionary hierarchy of restriction enzymes is discussed. PMID:12655005

Impact of enzyme replacement therapy on cardiac morphology and function and late enhancement in Fabry's cardiomyopathy.

PubMed

Beer, Meinrad; Weidemann, Frank; Breunig, Frank; Knoll, Anita; Koeppe, Sabrina; Machann, Wolfram; Hahn, Dietbert; Wanner, Christoph; Strotmann, Jörg; Sandstede, Jörn

2006-05-15

The present study evaluated the evolution of cardiac morphology, function, and late enhancement as a noninvasive marker of myocardial fibrosis, and their inter-relation during enzyme replacement therapy in patients with Fabry's disease using magnetic resonance imaging and color Doppler myocardial imaging. Late enhancement, which was present in up to 50% of patients, was associated with increased left ventricular mass, the failure of a significant regression of hypertrophy during enzyme replacement therapy, and worse segmental myocardial function. Late enhancement may predict the effect of enzyme replacement therapy on left ventricular mass and cardiac function.

The role of protein dynamics in the evolution of new enzyme function.

PubMed

Campbell, Eleanor; Kaltenbach, Miriam; Correy, Galen J; Carr, Paul D; Porebski, Benjamin T; Livingstone, Emma K; Afriat-Jurnou, Livnat; Buckle, Ashley M; Weik, Martin; Hollfelder, Florian; Tokuriki, Nobuhiko; Jackson, Colin J

2016-11-01

Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.

Directions for Optimization of Photosynthetic Carbon Fixation: RuBisCO's Efficiency May Not Be So Constrained After All.

PubMed

Cummins, Peter L; Kannappan, Babu; Gready, Jill E

2018-01-01

The ubiquitous enzyme Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) fixes atmospheric carbon dioxide within the Calvin-Benson cycle that is utilized by most photosynthetic organisms. Despite this central role, RuBisCO's efficiency surprisingly struggles, with both a very slow turnover rate to products and also impaired substrate specificity, features that have long been an enigma as it would be assumed that its efficiency was under strong evolutionary pressure. RuBisCO's substrate specificity is compromised as it catalyzes a side-fixation reaction with atmospheric oxygen; empirical kinetic results show a trend to tradeoff between relative specificity and low catalytic turnover rate. Although the dominant hypothesis has been that the active-site chemistry constrains the enzyme's evolution, a more recent study on RuBisCO stability and adaptability has implicated competing selection pressures. Elucidating these constraints is crucial for directing future research on improving photosynthesis, as the current literature casts doubt on the potential effectiveness of site-directed mutagenesis to improve RuBisCO's efficiency. Here we use regression analysis to quantify the relationships between kinetic parameters obtained from empirical data sets spanning a wide evolutionary range of RuBisCOs. Most significantly we found that the rate constant for dissociation of CO 2 from the enzyme complex was much higher than previous estimates and comparable with the corresponding catalytic rate constant. Observed trends between relative specificity and turnover rate can be expressed as the product of negative and positive correlation factors. This provides an explanation in simple kinetic terms of both the natural variation of relative specificity as well as that obtained by reported site-directed mutagenesis results. We demonstrate that the kinetic behaviour shows a lesser rather than more constrained RuBisCO, consistent with growing empirical evidence of higher variability in relative specificity. In summary our analysis supports an explanation for the origin of the tradeoff between specificity and turnover as due to competition between protein stability and activity, rather than constraints between rate constants imposed by the underlying chemistry. Our analysis suggests that simultaneous improvement in both specificity and turnover rate of RuBisCO is possible.

Visualizing and Clustering Protein Similarity Networks: Sequences, Structures, and Functions.

PubMed

Mai, Te-Lun; Hu, Geng-Ming; Chen, Chi-Ming

2016-07-01

Research in the recent decade has demonstrated the usefulness of protein network knowledge in furthering the study of molecular evolution of proteins, understanding the robustness of cells to perturbation, and annotating new protein functions. In this study, we aimed to provide a general clustering approach to visualize the sequence-structure-function relationship of protein networks, and investigate possible causes for inconsistency in the protein classifications based on sequences, structures, and functions. Such visualization of protein networks could facilitate our understanding of the overall relationship among proteins and help researchers comprehend various protein databases. As a demonstration, we clustered 1437 enzymes by their sequences and structures using the minimum span clustering (MSC) method. The general structure of this protein network was delineated at two clustering resolutions, and the second level MSC clustering was found to be highly similar to existing enzyme classifications. The clustering of these enzymes based on sequence, structure, and function information is consistent with each other. For proteases, the Jaccard's similarity coefficient is 0.86 between sequence and function classifications, 0.82 between sequence and structure classifications, and 0.78 between structure and function classifications. From our clustering results, we discussed possible examples of divergent evolution and convergent evolution of enzymes. Our clustering approach provides a panoramic view of the sequence-structure-function network of proteins, helps visualize the relation between related proteins intuitively, and is useful in predicting the structure and function of newly determined protein sequences.

Isolation of novel ribozymes that ligate AMP-activated RNA substrates

NASA Technical Reports Server (NTRS)

Hager, A. J.; Szostak, J. W.

1997-01-01

BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

Evolution of the ferric reductase domain (FRD) superfamily: modularity, functional diversification, and signature motifs.

PubMed

Zhang, Xuezhi; Krause, Karl-Heinz; Xenarios, Ioannis; Soldati, Thierry; Boeckmann, Brigitte

2013-01-01

A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria.

Possibilities for the evolution of the genetic code from a preceding form

NASA Technical Reports Server (NTRS)

Jukes, T. H.

1973-01-01

Analysis of the interaction between mRNA codons and tRNA anticodons suggests a model for the evolution of the genetic code. Modification of the nucleic acid following the anticodon is at present essential in both eukaryotes and prokaryotes to ensure fidelity of translation of codons starting with A, and the amino acids which could be coded for before the evolution of the modifying enzymes can be deduced.

Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

PubMed

Collins, Kathleen; Nilsen, Timothy W

2013-08-01

Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

Biochemical Evolution of Iron and Copper Proteins, Substances Vital to Life

ERIC Educational Resources Information Center

Frieden, Earl

1974-01-01

Summarizes studies in the area of biochemical evolution of iron, copper, and heme proteins to provide an historical outline. Included are lists of major kinds of proteins and enzymes and charts illustrating electron flow in a cytochrome electron transport system and interconversion of jerrous to ferric ion in iron metabolism. (CC)

The Evolution of Energy-Transducing Systems. Studies with an Extremely Halophilic Archaebacterium

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga

1997-01-01

The F-type ATPases are found in remarkably similar versions in the energy-transducing membranes of bacteria, chloroplasts and mitochondria (1). Thus, it is likely that they have originated early in the evolution of life, which is consistent with their function as key enzymes of cellular metabolism. The archaea (formerly called archaebacteria) are a group of microorganisms which, as shown by molecular sequencing and biochemical data, have diverged early from the main line of prokaryotic evolution (2). From studies of members of all three major groups of archaea, the halophiles, methanogens and thermoacidophiles, it emerged that they possess a membrane ATPase, which differs from the F-ATPases. The goal of this project was a comparison of the ATPase from the halophilic archaebacterium Halobacterium saccharovorum with the well-characterized F-type ATPases on the molecular level. The results were expected to allow a decision about the nature of archaebacterial ATPases, their classification as one of the known or, alternatively, novel enzyme complex, and possibly a deduction of events during the early evolution of energy-transducing systems.

DNA and RNA editing of retrotransposons accelerate mammalian genome evolution.

PubMed

Knisbacher, Binyamin A; Levanon, Erez Y

2015-04-01

Genome evolution is commonly viewed as a gradual process that is driven by random mutations that accumulate over time. However, DNA- and RNA-editing enzymes have been identified that can accelerate evolution by actively modifying the genomically encoded information. The apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBECs) are potent restriction factors that can inhibit retroelements by cytosine-to-uridine editing of retroelement DNA after reverse transcription. In some cases, a retroelement may successfully integrate into the genome despite being hypermutated. Such events introduce unique sequences into the genome and are thus a source of genomic innovation. adenosine deaminases that act on RNA (ADARs) catalyze adenosine-to-inosine editing in double-stranded RNA, commonly formed by oppositely oriented retroelements. The RNA editing confers plasticity to the transcriptome by generating many transcript variants from a single genomic locus. If the editing produces a beneficial variant, the genome may maintain the locus that produces the RNA-edited transcript for its novel function. Here, we discuss how these two powerful editing mechanisms, which both target inserted retroelements, facilitate expedited genome evolution. © 2015 New York Academy of Sciences.

Diazonium-functionalized horseradish peroxidase immobilized via addressable electrodeposition: direct electron transfer and electrochemical detection.

PubMed

Polsky, Ronen; Harper, Jason C; Dirk, Shawn M; Arango, Dulce C; Wheeler, David R; Brozik, Susan M

2007-01-16

A simple one-step procedure is introduced for the preparation of diazonium-enzyme adducts. The direct electrically addressable deposition of diazonium-modified enzymes is examined for electrochemical sensor applications. The deposition of diazonium-horseradish peroxidase leads to the direct electron transfer between the enzyme and electrode exhibiting a heterogeneous rate constant, ks, of 10.3 +/- 0.7 s-1 and a DeltaEp of 8 mV (v = 150 mV/s). The large ks and low DeltaEp are attributed to the intimate contact between enzyme and electrode attached by one to three phenyl molecules. Such an electrode shows high nonmediated catalytic activity toward H2O2 reduction. Future generations of arrayed electrochemical sensors and studies of direct electron transfer of enzymes can benefit from protein electrodes prepared by this method.

Positive selection on D-lactate dehydrogenases of Lactobacillus delbrueckii subspecies bulgaricus.

PubMed

Zhang, Jifeng; Gong, Guangyu; Wang, Xiao; Zhang, Hao; Tian, Weidong

2015-08-01

Lactobacillus delbrueckii has been widely used for yogurt fermentation. It has genes encoding both D- and L-type lactate dehydrogenases (LDHs) that catalyse the production of L(+) or D(-) stereoisomer of lactic acid. D-lactic acid is the primary lactate product by L. delbrueckii, yet it cannot be metabolised by human intestine. Since it has been domesticated for long time, an interesting question arises regarding to whether the selection pressure has affected the evolution of both L-LDH and D-LDH genes in the genome. To answer this question, in this study the authors first investigated the evolution of these two genes by constructing phylogenetic trees. They found that D-LDH-based phylogenetic tree could better represent the phylogenetic relationship in the acidophilus complex than L-LDH-based tree. They next investigated the evolutions of LDH genes of L. delbrueckii at amino acid level, and found that D-LDH gene in L. delbrueckii is positively selected, possibly a consequence of long-term domestication. They further identified four amino acids that are under positive selection. One of them, V261, is located at the centre of three catalytic active sites, indicating likely functional effects on the enzyme activity. The selection from the domestication process thus provides direction for future engineering of D-LDH.

Directed evolution and expression tuning of geraniol synthase for efficient geraniol production in Escherichia coli.

PubMed

Tashiro, Miki; Fujii, Akira; Kawai-Noma, Shigeko; Saito, Kyoichi; Umeno, Daisuke

2017-11-17

To achieve an efficient production of geraniol and its derivatives in Escherichia coli, we aimed to improve the activity of geraniol synthase (GES) through a single round of mutagenesis and screening for higher substrate consumption. We isolated GES variants that outperform their parent in geraniol production. The analysis of GES variants indicated that the expression level of GES was the bottleneck for geraniol synthesis. Over-expression of the mutant GES M53 with a 5'-untranslated sequence designed for high translational efficiency, along with the additional expression of mevalonate pathway enzymes, isopentenyl pyrophosphate isomerase, and geranyl pyrophosphate synthase, yielded 300 mg/L/12 h geraniol and its derivatives (>1000 mg/L/42 h in total) in a shaking flask.

Structural basis of reverse nucleotide polymerization

PubMed Central

Nakamura, Akiyoshi; Nemoto, Taiki; Heinemann, Ilka U.; Yamashita, Keitaro; Sonoda, Tomoyo; Komoda, Keisuke; Tanaka, Isao; Söll, Dieter; Yao, Min

2013-01-01

Nucleotide polymerization proceeds in the forward (5′-3′) direction. This tenet of the central dogma of molecular biology is found in diverse processes including transcription, reverse transcription, DNA replication, and even in lagging strand synthesis where reverse polymerization (3′-5′) would present a “simpler” solution. Interestingly, reverse (3′-5′) nucleotide addition is catalyzed by the tRNA maturation enzyme tRNAHis guanylyltransferase, a structural homolog of canonical forward polymerases. We present a Candida albicans tRNAHis guanylyltransferase-tRNAHis complex structure that reveals the structural basis of reverse polymerization. The directionality of nucleotide polymerization is determined by the orientation of approach of the nucleotide substrate. The tRNA substrate enters the enzyme’s active site from the opposite direction (180° flip) compared with similar nucleotide substrates of canonical 5′-3′ polymerases, and the finger domains are on opposing sides of the core palm domain. Structural, biochemical, and phylogenetic data indicate that reverse polymerization appeared early in evolution and resembles a mirror image of the forward process. PMID:24324136

Retrovolution: HIV-driven evolution of cellular genes and improvement of anticancer drug activation.

PubMed

Rossolillo, Paola; Winter, Flore; Simon-Loriere, Etienne; Gallois-Montbrun, Sarah; Negroni, Matteo

2012-08-01

In evolution strategies aimed at isolating molecules with new functions, screening for the desired phenotype is generally performed in vitro or in bacteria. When the final goal of the strategy is the modification of the human cell, the mutants selected with these preliminary screenings may fail to confer the desired phenotype, due to the complex networks that regulate gene expression in higher eukaryotes. We developed a system where, by mimicking successive infection cycles with HIV-1 derived vectors containing the gene target of the evolution in their genome, libraries of gene mutants are generated in the human cell, where they can be directly screened. As a proof of concept we created a library of mutants of the human deoxycytidine kinase (dCK) gene, involved in the activation of nucleoside analogues used in cancer treatment, with the aim of isolating a variant sensitizing cancer cells to the chemotherapy compound Gemcitabine, to be used in gene therapy for anti-cancer approaches or as a poorly immunogenic negative selection marker for cell transplantation approaches. We describe the isolation of a dCK mutant, G12, inducing a 300-fold sensitization to Gemcitabine in cells originally resistant to the prodrug (Messa 10K), an effect 60 times stronger than the one induced by the wt enzyme. The phenotype is observed in different tumour cell lines irrespective of the insertion site of the transgene and is due to a change in specificity of the mutated kinase in favour of the nucleoside analogue. The mutations characterizing G12 are distant from the active site of the enzyme and are unpredictable on a rational basis, fully validating the pragmatic approach followed. Besides the potential interest of the G12 dCK variant for therapeutic purposes, the methodology developed is of interest for a large panel of applications in biotechnology and basic research.

Thoughts on the diversity of convergent evolution of bioluminescence on earth

NASA Astrophysics Data System (ADS)

Waldenmaier, Hans E.; Oliveira, Anderson G.; Stevani, Cassius V.

2012-10-01

The widespread independent evolution of analogous bioluminescent systems is one of the most impressive and diverse examples of convergent evolution on earth. There are roughly 30 extant bioluminescent systems that have evolved independently on Earth, with each system likely having unique enzymes responsible for catalysing the bioluminescent reaction. Bioluminescence is a chemical reaction involving a luciferin molecule and a luciferase or photoprotein that results in the emission of light. Some independent systems utilize the same luciferin, such as the use of tetrapyrrolic compounds by krill and dinoflagellates, and the wide use of coelenterazine by marine organisms, while the enzymes involved are unique. One common thread among all the different bioluminescent systems is the requirement of molecular oxygen. Bioluminescence is found in most forms of life, especially marine organisms. Bioluminescence in known to benefit the organism by: attraction, repulsion, communication, camouflage, and illumination. The marine ecosystem is significantly affected by bioluminescence, the only light found in the pelagic zone and below is from bioluminescent organisms. Transgenic bioluminescent organisms have revolutionized molecular research, medicine and the biotechnology industry. The use of bioluminescence in studying molecular pathways and disease allows for non-invasive and real-time analysis. Bioluminescence-based assays have been developed for several analytes by coupling luminescence to many enzyme-catalysed reactions.

BIOSENSOR FOR DIRECT DETERMINATION OF ORGANOPHOSPHATE NERVE AGENTS. 1. POTENTIOMETRIC ENZYME ELECTRODE. (R823663)

EPA Science Inventory

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP)
nerve agents was developed. The basic element of this enzyme electrode was a pH electrode
modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking
OPH ...

Electrochemical Reduction of Carbon Dioxide to Methanol by Direct Injection of Electrons into Immobilized Enzymes on a Modified Electrode.

PubMed

Schlager, Stefanie; Dumitru, Liviu Mihai; Haberbauer, Marianne; Fuchsbauer, Anita; Neugebauer, Helmut; Hiemetsberger, Daniela; Wagner, Annika; Portenkirchner, Engelbert; Sariciftci, Niyazi Serdar

2016-03-21

We present results for direct bio-electrocatalytic reduction of CO2 to C1 products using electrodes with immobilized enzymes. Enzymatic reduction reactions are well known from biological systems where CO2 is selectively reduced to formate, formaldehyde, or methanol at room temperature and ambient pressure. In the past, the use of such enzymatic reductions for CO2 was limited due to the necessity of a sacrificial co-enzyme, such as nicotinamide adenine dinucleotide (NADH), to supply electrons and the hydrogen equivalent. The method reported here in this paper operates without the co-enzyme NADH by directly injecting electrons from electrodes into immobilized enzymes. We demonstrate the immobilization of formate, formaldehyde, and alcohol dehydrogenases on one-and-the-same electrode for direct CO2 reduction. Carbon felt is used as working electrode material. An alginate-silicate hybrid gel matrix is used for the immobilization of the enzymes on the electrode. Generation of methanol is observed for the six-electron reduction with Faradaic efficiencies of around 10%. This method of immobilization of enzymes on electrodes offers the opportunity for electrochemical application of enzymatic electrodes to many reactions in which a substitution of the expensive sacrificial co-enzyme NADH is desired. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel Random Mutagenesis Method for Directed Evolution.

PubMed

Feng, Hong; Wang, Hai-Yan; Zhao, Hong-Yan

2017-01-01

Directed evolution is a powerful strategy for gene mutagenesis, and has been used for protein engineering both in scientific research and in the biotechnology industry. The routine method for directed evolution was developed by Stemmer in 1994 (Stemmer, Proc Natl Acad Sci USA 91, 10747-10751, 1994; Stemmer, Nature 370, 389-391, 1994). Since then, various methods have been introduced, each of which has advantages and limitations depending upon the targeted genes and procedure. In this chapter, a novel alternative directed evolution method which combines mutagenesis PCR with dITP and fragmentation by endonuclease V is described. The kanamycin resistance gene is used as a reporter gene to verify the novel method for directed evolution. This method for directed evolution has been demonstrated to be efficient, reproducible, and easy to manipulate in practice.

Integrating molecular dynamics and co-evolutionary analysis for reliable target prediction and deregulation of the allosteric inhibition of aspartokinase for amino acid production.

PubMed

Chen, Zhen; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

2011-07-20

Deregulation of allosteric inhibition of enzymes is a challenge for strain engineering and has been achieved so far primarily by random mutation and trial-and-error. In this work, we used aspartokinase, an important allosteric enzyme for industrial amino acids production, to demonstrate a predictive approach that combines protein dynamics and evolution for a rational reengineering of enzyme allostery. Molecular dynamic simulation of aspartokinase III (AK3) from Escherichia coli and statistical coupling analysis of protein sequences of the aspartokinase family allowed to identify a cluster of residues which are correlated during protein motion and coupled during the evolution. This cluster of residues forms an interconnected network mediating the allosteric regulation, including most of the previously reported positions mutated in feedback insensitive AK3 mutants. Beyond these mutation positions, we have successfully constructed another twelve targeted mutations of AK3 desensitized toward lysine inhibition. Six threonine-insensitive mutants of aspartokinase I-homoserine dehydrogenase I (AK1-HD1) were also created based on the predictions. The proposed approach can be widely applied for the deregulation of other allosteric enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.

The structure of a zeta class glutathione S-transferase from Arabidopsis thaliana: characterisation of a GST with novel active-site architecture and a putative role in tyrosine catabolism.

PubMed

Thom, R; Dixon, D P; Edwards, R; Cole, D J; Lapthorn, A J

2001-05-18

The cis-trans isomerisation of maleylacetoacetate to fumarylacetoacetate is the penultimate step in the tyrosine/phenylalanine catabolic pathway and has recently been shown to be catalysed by glutathione S-transferase enzymes belonging to the zeta class. Given this primary metabolic role it is unsurprising that zeta class glutathione S-transferases are well conserved over a considerable period of evolution, being found in vertebrates, plants, insects and fungi. The structure of this glutathione S-transferase, cloned from Arabidopsis thaliana, has been solved by single isomorphous replacement with anomalous scattering and refined to a final crystallographic R-factor of 19.6% using data from 25.0 A to 1.65 A. The zeta class enzyme adopts the canonical glutathione S-transferase fold and forms a homodimer with each subunit consisting of 221 residues. In agreement with structures of glutathione S-transferases from the theta and phi classes, a serine residue (Ser17) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione. Site-directed mutagenesis of this residue confirms its importance in catalysis. In addition, the role of a highly conserved cysteine residue (Cys19) present in the active site of the zeta class glutathione S-transferase enzymes is discussed. Copyright 2001 Academic Press.

Structural Evolution of Differential Amino Acid Effector Regulation in Plant Chorismate Mutases*

PubMed Central

Westfall, Corey S.; Xu, Ang; Jez, Joseph M.

2014-01-01

Chorismate mutase converts chorismate into prephenate for aromatic amino acid biosynthesis. To understand the molecular basis of allosteric regulation in the plant chorismate mutases, we analyzed the three Arabidopsis thaliana chorismate mutase isoforms (AtCM1–3) and determined the x-ray crystal structures of AtCM1 in complex with phenylalanine and tyrosine. Functional analyses show a wider range of effector control in the Arabidopsis chorismate mutases than previously reported. AtCM1 is activated by tryptophan with phenylalanine and tyrosine acting as negative effectors; however, tryptophan, cysteine, and histidine activate AtCM3. AtCM2 is a nonallosteric form. The crystal structure of AtCM1 in complex with tyrosine and phenylalanine identifies differences in the effector sites of the allosterically regulated yeast enzyme and the other two Arabidopsis isoforms. Site-directed mutagenesis of residues in the effector site reveals key features leading to differential effector regulation in these enzymes. In AtCM1, mutations of Gly-213 abolish allosteric regulation, as observed in AtCM2. A second effector site position, Gly-149 in AtCM1 and Asp-132 in AtCM3, controls amino acid effector specificity in AtCM1 and AtCM3. Comparisons of chorismate mutases from multiple plants suggest that subtle differences in the effector site are conserved in different lineages and may lead to specialized regulation of this branch point enzyme. PMID:25160622

Similarity in Shape Dictates Signature Intrinsic Dynamics Despite No Functional Conservation in TIM Barrel Enzymes

PubMed Central

Tiwari, Sandhya P.; Reuter, Nathalie

2016-01-01

The conservation of the intrinsic dynamics of proteins emerges as we attempt to understand the relationship between sequence, structure and functional conservation. We characterise the conservation of such dynamics in a case where the structure is conserved but function differs greatly. The triosephosphate isomerase barrel fold (TBF), renowned for its 8 β-strand-α-helix repeats that close to form a barrel, is one of the most diverse and abundant folds found in known protein structures. Proteins with this fold have diverse enzymatic functions spanning five of six Enzyme Commission classes, and we have picked five different superfamily candidates for our analysis using elastic network models. We find that the overall shape is a large determinant in the similarity of the intrinsic dynamics, regardless of function. In particular, the β-barrel core is highly rigid, while the α-helices that flank the β-strands have greater relative mobility, allowing for the many possibilities for placement of catalytic residues. We find that these elements correlate with each other via the loops that link them, as opposed to being directly correlated. We are also able to analyse the types of motions encoded by the normal mode vectors of the α-helices. We suggest that the global conservation of the intrinsic dynamics in the TBF contributes greatly to its success as an enzymatic scaffold both through evolution and enzyme design. PMID:27015412

Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

USDA-ARS?s Scientific Manuscript database

Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

Evolution of nonspectral rhodopsin function at high altitudes.

PubMed

Castiglione, Gianni M; Hauser, Frances E; Liao, Brian S; Lujan, Nathan K; Van Nynatten, Alexander; Morrow, James M; Schott, Ryan K; Bhattacharyya, Nihar; Dungan, Sarah Z; Chang, Belinda S W

2017-07-11

High-altitude environments present a range of biochemical and physiological challenges for organisms through decreases in oxygen, pressure, and temperature relative to lowland habitats. Protein-level adaptations to hypoxic high-altitude conditions have been identified in multiple terrestrial endotherms; however, comparable adaptations in aquatic ectotherms, such as fishes, have not been as extensively characterized. In enzyme proteins, cold adaptation is attained through functional trade-offs between stability and activity, often mediated by substitutions outside the active site. Little is known whether signaling proteins [e.g., G protein-coupled receptors (GPCRs)] exhibit natural variation in response to cold temperatures. Rhodopsin (RH1), the temperature-sensitive visual pigment mediating dim-light vision, offers an opportunity to enhance our understanding of thermal adaptation in a model GPCR. Here, we investigate the evolution of rhodopsin function in an Andean mountain catfish system spanning a range of elevations. Using molecular evolutionary analyses and site-directed mutagenesis experiments, we provide evidence for cold adaptation in RH1. We find that unique amino acid substitutions occur at sites under positive selection in high-altitude catfishes, located at opposite ends of the RH1 intramolecular hydrogen-bonding network. Natural high-altitude variants introduced into these sites via mutagenesis have limited effects on spectral tuning, yet decrease the stability of dark-state and light-activated rhodopsin, accelerating the decay of ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates-properties of rhodopsin that mediate rod sensitivity and visual performance. Our results support a role for natural variation in enhancing the performance of GPCRs in response to cold temperatures.

ScaffoldSeq: Software for characterization of directed evolution populations.

PubMed

Woldring, Daniel R; Holec, Patrick V; Hackel, Benjamin J

2016-07-01

ScaffoldSeq is software designed for the numerous applications-including directed evolution analysis-in which a user generates a population of DNA sequences encoding for partially diverse proteins with related functions and would like to characterize the single site and pairwise amino acid frequencies across the population. A common scenario for enzyme maturation, antibody screening, and alternative scaffold engineering involves naïve and evolved populations that contain diversified regions, varying in both sequence and length, within a conserved framework. Analyzing the diversified regions of such populations is facilitated by high-throughput sequencing platforms; however, length variability within these regions (e.g., antibody CDRs) encumbers the alignment process. To overcome this challenge, the ScaffoldSeq algorithm takes advantage of conserved framework sequences to quickly identify diverse regions. Beyond this, unintended biases in sequence frequency are generated throughout the experimental workflow required to evolve and isolate clones of interest prior to DNA sequencing. ScaffoldSeq software uniquely handles this issue by providing tools to quantify and remove background sequences, cluster similar protein families, and dampen the impact of dominant clones. The software produces graphical and tabular summaries for each region of interest, allowing users to evaluate diversity in a site-specific manner as well as identify epistatic pairwise interactions. The code and detailed information are freely available at http://research.cems.umn.edu/hackel. Proteins 2016; 84:869-874. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Nickel centred H+ reduction catalysis in a model of [NiFe] Hydrogenase

PubMed Central

Brazzolotto, Deborah; Gennari, Marcello; Queyriaux, Nicolas; Simmons, Trevor R.; Pécaut, Jacques; Demeshko, Serhiy; Meyer, Franc; Orio, Maylis; Artero, Vincent; Duboc, Carole

2017-01-01

Hydrogen production through water splitting is one of the most promising solutions for the storage of renewable energy. [NiFe] hydrogenases are organometallic enzymes containing nickel and iron centers that catalyze hydrogen evolution with performances that rival those of platinum. These enzymes provide inspiration for the design of new molecular catalysts that do not require precious metals. However, all heterodinuclear NiFe models reported so far do not reproduce the Ni-centered reactivity found at the active site of [NiFe] hydrogenases. Here we report a structural and functional NiFe mimic that displays reactivity at the Ni site. This is shown by the detection of two catalytic intermediates that reproduce structural and electronic features of the Ni-L and Ni-R states of the enzyme during catalytic turnover. Under electrocatalytic conditions, this mimic displays high rates for H2 evolution (second order rate constant of 2.5 104 M-1s-1; turnover frequency of 225 s-1 at 10 mM H+ concentration) from mildly acidic solutions. PMID:27768098

Evolution of substrate specificity in a retained enzyme driven by gene loss

PubMed Central

Juárez-Vázquez, Ana Lilia; Edirisinghe, Janaka N; Verduzco-Castro, Ernesto A; Michalska, Karolina; Wu, Chenggang; Noda-García, Lianet; Babnigg, Gyorgy; Endres, Michael; Medina-Ruíz, Sofía; Santoyo-Flores, Julián; Carrillo-Tripp, Mauricio; Ton-That, Hung; Joachimiak, Andrzej; Henry, Christopher S; Barona-Gómez, Francisco

2017-01-01

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Our results show how gene loss can drive the evolution of substrate specificity from retained enzymes. DOI: http://dx.doi.org/10.7554/eLife.22679.001 PMID:28362260

Evolution of Substrate Specificity in A Retained Enzyme Driven by Gene Loss

DOE PAGES

Juarez-Vazquez, Ana L.; Edirisinghe, Janaka N.; Verduzco-Castro, Ernesto A.; ...

2017-03-31

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. Here, we apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We also observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to amore » monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. These results show how gene loss can drive the evolution of substrate specificity from retained enzymes.« less

Evolution of substrate specificity in a retained enzyme driven by gene loss

DOE PAGES

Juárez-Vázquez, Ana Lilia; Edirisinghe, Janaka N.; Verduzco-Castro, Ernesto A.; ...

2017-03-31

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence oftrpandhisgenes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficientmore » form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Finally, our results show how gene loss can drive the evolution of substrate specificity from retained enzymes.« less

Evolution of substrate specificity in a retained enzyme driven by gene loss

DOE Office of Scientific and Technical Information (OSTI.GOV)

Juárez-Vázquez, Ana Lilia; Edirisinghe, Janaka N.; Verduzco-Castro, Ernesto A.

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence oftrpandhisgenes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficientmore » form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Finally, our results show how gene loss can drive the evolution of substrate specificity from retained enzymes.« less

Evolution of Substrate Specificity in A Retained Enzyme Driven by Gene Loss

DOE Office of Scientific and Technical Information (OSTI.GOV)

Juarez-Vazquez, Ana L.; Edirisinghe, Janaka N.; Verduzco-Castro, Ernesto A.

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. Here, we apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We also observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to amore » monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. These results show how gene loss can drive the evolution of substrate specificity from retained enzymes.« less

Primary enzyme quantitation

DOEpatents

Saunders, G.C.

1982-03-04

The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

Krebs cycle metabolon formation: metabolite concentration gradient enhanced compartmentation of sequential enzymes.

PubMed

Wu, Fei; Pelster, Lindsey N; Minteer, Shelley D

2015-01-25

Dynamics of metabolon formation in mitochondria was probed by studying diffusional motion of two sequential Krebs cycle enzymes in a microfluidic channel. Enhanced directional co-diffusion of both enzymes against a substrate concentration gradient was observed in the presence of intermediate generation. This reveals a metabolite directed compartmentation of metabolic pathways.

Evolution of the Ferric Reductase Domain (FRD) Superfamily: Modularity, Functional Diversification, and Signature Motifs

PubMed Central

Zhang, Xuezhi; Krause, Karl-Heinz; Xenarios, Ioannis; Soldati, Thierry; Boeckmann, Brigitte

2013-01-01

A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria. PMID:23505460

Rethinking the evolution of eukaryotic metabolism: novel cellular partitioning of enzymes in stramenopiles links serine biosynthesis to glycolysis in mitochondria.

PubMed

Abrahamian, Melania; Kagda, Meenakshi; Ah-Fong, Audrey M V; Judelson, Howard S

2017-12-04

An important feature of eukaryotic evolution is metabolic compartmentalization, in which certain pathways are restricted to the cytosol or specific organelles. Glycolysis in eukaryotes is described as a cytosolic process. The universality of this canon has been challenged by recent genome data that suggest that some glycolytic enzymes made by stramenopiles bear mitochondrial targeting peptides. Mining of oomycete, diatom, and brown algal genomes indicates that stramenopiles encode two forms of enzymes for the second half of glycolysis, one with and the other without mitochondrial targeting peptides. The predicted mitochondrial targeting was confirmed by using fluorescent tags to localize phosphoglycerate kinase, phosphoglycerate mutase, and pyruvate kinase in Phytophthora infestans, the oomycete that causes potato blight. A genome-wide search for other enzymes with atypical mitochondrial locations identified phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase, which form a pathway for generating serine from the glycolytic intermediate 3-phosphoglycerate. Fluorescent tags confirmed the delivery of these serine biosynthetic enzymes to P. infestans mitochondria. A cytosolic form of this serine biosynthetic pathway, which occurs in most eukaryotes, is missing from oomycetes and most other stramenopiles. The glycolysis and serine metabolism pathways of oomycetes appear to be mosaics of enzymes with different ancestries. While some of the noncanonical oomycete mitochondrial enzymes have the closest affinity in phylogenetic analyses with proteins from other stramenopiles, others cluster with bacterial, plant, or animal proteins. The genes encoding the mitochondrial phosphoglycerate kinase and serine-forming enzymes are physically linked on oomycete chromosomes, which suggests a shared origin. Stramenopile metabolism appears to have been shaped through the acquisition of genes by descent and lateral or endosymbiotic gene transfer, along with the targeting of the proteins to locations that are novel compared to other eukaryotes. Colocalization of the glycolytic and serine biosynthesis enzymes in mitochondria is apparently necessary since they share a common intermediate. The results indicate that descriptions of metabolism in textbooks do not cover the full diversity of eukaryotic biology.

SlyD-dependent nickel delivery limits maturation of [NiFe]-hydrogenases in late-stationary phase Escherichia coli cells.

PubMed

Pinske, Constanze; Sargent, Frank; Sawers, R Gary

2015-04-01

Fermentatively growing Escherichia coli cells have three active [NiFe]-hydrogenases (Hyd), two of which, Hyd-1 and Hyd-2, contribute to H2 oxidation while Hyd-3 couples formate oxidation to H2 evolution. Biosynthesis of all Hyd involves the insertion of a Fe(CN)2CO group and a subsequent insertion of nickel ions through the HypA/HybF, HypB and SlyD proteins. With high nickel concentrations the presence of none of these proteins is required, but under normal growth conditions and during late stationary growth SlyD is important for hydrogenase activities. The slyD mutation reduced H2 production during exponential phase growth by about 50%. Assaying stationary phase grown cells for the coupling of Hyd activity to the respiratory chain or formate-dependent H2 evolution showed that SlyD is essential for both H2 evolution and H2 oxidation. Although introduction of plasmid-coded slyD resulted in an overall decrease of Hyd-2 polypeptides in slyD and hypA slyD mutants, processing and dye-reducing activity of the Hyd-2 enzyme was nevertheless restored. Similarly, introduction of the slyD plasmid restored only some H2 evolution in the slyD mutant while Hyd-3 polypeptides and dye-reducing activity were fully restored. Taken together, these results indicate an essential role for SlyD in the generation of the fully cofactor-equipped hydrogenase large subunits in the stationary phase where the level of each Hyd enzyme is finely tuned by SlyD for optimal enzyme activity.

Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

PubMed Central

Cozens, Christopher

2018-01-01

Abstract Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation. PMID:29409059

Functional cell-surface display of a lipase-specific chaperone.

PubMed

Wilhelm, Susanne; Rosenau, Frank; Becker, Stefan; Buest, Sebastian; Hausmann, Sascha; Kolmar, Harald; Jaeger, Karl-Erich

2007-01-02

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.

In vitro selection of functional nucleic acids

NASA Technical Reports Server (NTRS)

Wilson, D. S.; Szostak, J. W.

1999-01-01

In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes.

PubMed

Deponte, Marcel

2013-05-01

Glutathione-dependent catalysis is a metabolic adaptation to chemical challenges encountered by all life forms. In the course of evolution, nature optimized numerous mechanisms to use glutathione as the most versatile nucleophile for the conversion of a plethora of sulfur-, oxygen- or carbon-containing electrophilic substances. This comprehensive review summarizes fundamental principles of glutathione catalysis and compares the structures and mechanisms of glutathione-dependent enzymes, including glutathione reductase, glutaredoxins, glutathione peroxidases, peroxiredoxins, glyoxalases 1 and 2, glutathione transferases and MAPEG. Moreover, open mechanistic questions, evolutionary aspects and the physiological relevance of glutathione catalysis are discussed for each enzyme family. It is surprising how little is known about many glutathione-dependent enzymes, how often reaction geometries and acid-base catalysts are neglected, and how many mechanistic puzzles remain unsolved despite almost a century of research. On the one hand, several enzyme families with non-related protein folds recognize the glutathione moiety of their substrates. On the other hand, the thioredoxin fold is often used for glutathione catalysis. Ancient as well as recent structural changes of this fold did not only significantly alter the reaction mechanism, but also resulted in completely different protein functions. Glutathione-dependent enzymes are excellent study objects for structure-function relationships and molecular evolution. Notably, in times of systems biology, the outcome of models on glutathione metabolism and redox regulation is more than questionable as long as fundamental enzyme properties are neither studied nor understood. Furthermore, several of the presented mechanisms could have implications for drug development. This article is part of a Special Issue entitled Cellular functions of glutathione. Copyright © 2012 Elsevier B.V. All rights reserved.

The evolution of photosynthesis...again?

PubMed

Rothschild, Lynn J

2008-08-27

'Replaying the tape' is an intriguing 'would it happen again?' exercise. With respect to broad evolutionary innovations, such as photosynthesis, the answers are central to our search for life elsewhere. Photosynthesis permits a large planetary biomass on Earth. Specifically, oxygenic photosynthesis has allowed an oxygenated atmosphere and the evolution of large metabolically demanding creatures, including ourselves. There are at least six prerequisites for the evolution of biological carbon fixation: a carbon-based life form; the presence of inorganic carbon; the availability of reductants; the presence of light; a light-harvesting mechanism to convert the light energy into chemical energy; and carboxylating enzymes. All were present on the early Earth. To provide the evolutionary pressure, organic carbon must be a scarce resource in contrast to inorganic carbon. The probability of evolving a carboxylase is approached by creating an inventory of carbon-fixation enzymes and comparing them, leading to the conclusion that carbon fixation in general is basic to life and has arisen multiple times. Certainly, the evolutionary pressure to evolve new pathways for carbon fixation would have been present early in evolution. From knowledge about planetary systems and extraterrestrial chemistry, if organic carbon-based life occurs elsewhere, photosynthesis -- although perhaps not oxygenic photosynthesis -- would also have evolved.

The evolution of respiratory O2/NO reductases: an out-of-the-phylogenetic-box perspective.

PubMed

Ducluzeau, Anne-Lise; Schoepp-Cothenet, Barbara; van Lis, Robert; Baymann, Frauke; Russell, Michael J; Nitschke, Wolfgang

2014-09-06

Complex life on our planet crucially depends on strong redox disequilibria afforded by the almost ubiquitous presence of highly oxidizing molecular oxygen. However, the history of O2-levels in the atmosphere is complex and prior to the Great Oxidation Event some 2.3 billion years ago, the amount of O2 in the biosphere is considered to have been extremely low as compared with present-day values. Therefore the evolutionary histories of life and of O2-levels are likely intricately intertwined. The obvious biological proxy for inferring the impact of changing O2-levels on life is the evolutionary history of the enzyme allowing organisms to tap into the redox power of molecular oxygen, i.e. the bioenergetic O2 reductases, alias the cytochrome and quinol oxidases. Consequently, molecular phylogenies reconstructed for this enzyme superfamily have been exploited over the last two decades in attempts to elucidate the interlocking between O2 levels in the environment and the evolution of respiratory bioenergetic processes. Although based on strictly identical datasets, these phylogenetic approaches have led to diametrically opposite scenarios with respect to the history of both the enzyme superfamily and molecular oxygen on the Earth. In an effort to overcome the deadlock of molecular phylogeny, we here review presently available structural, functional, palaeogeochemical and thermodynamic information pertinent to the evolution of the superfamily (which notably also encompasses the subfamily of nitric oxide reductases). The scenario which, in our eyes, most closely fits the ensemble of these non-phylogenetic data, sees the low O2-affinity SoxM- (or A-) type enzymes as the most recent evolutionary innovation and the high-affinity O2 reductases (SoxB or B and cbb3 or C) as arising independently from NO-reducing precursor enzymes. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities.

PubMed

Rueda, Nazzoly; Dos Santos, Jose C S; Ortiz, Claudia; Torres, Rodrigo; Barbosa, Oveimar; Rodrigues, Rafael C; Berenguer-Murcia, Ángel; Fernandez-Lafuente, Roberto

2016-06-01

Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site-directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed. © 2016 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Maintaining Genetic Integrity Under Extreme Conditions: Novel DNA Damage Repair Biology in the Archaea

DTIC Science & Technology

2013-11-23

Genetic analysis of Nre DNA repair function A4 Conclusions B. Widening the net in the search for new DNA-directed enzyme activities C. New tools for H...Figure 1) were hypothesised to be novel DNA repair enzymes . The stated aims of the proposal were to use a combination of genetic, biochemical and...in E.coli Almost all proteins that interact directly with PCNA are enzymes possessing DNA-directed activities such as nucleases, glycosylases

Protein Engineering Towards Natural Product Synthesis and Diversification

PubMed Central

Zabala, Angelica O.; Cacho, Ralph A.; Tang, Yi

2014-01-01

A dazzling array of enzymes is used by nature in making structurally complex natural products. These enzymes constitute a molecular toolbox that may be used in the construction and fine-tuning of pharmaceutically active molecules. Aided by technological advancements in protein engineering, it is now possible to tailor the activities and specificities of these enzymes as biocatalysts in the production of both natural products and their unnatural derivatives. These efforts are crucial in drug discovery and development, where there is a continuous quest for more potent agents. Both rational and random evolution techniques have been utilized in engineering these enzymes. This review will highlight some examples from several large families of natural products. PMID:22006344

Artificial enzymes with protein scaffolds: structural design and modification.

PubMed

Matsuo, Takashi; Hirota, Shun

2014-10-15

Recent development in biochemical experiment techniques and bioinformatics has enabled us to create a variety of artificial biocatalysts with protein scaffolds (namely 'artificial enzymes'). The construction methods of these catalysts include genetic mutation, chemical modification using synthetic molecules and/or a combination of these methods. Designed evolution strategy based on the structural information of host proteins has become more and more popular as an effective approach to construct artificial protein-based biocatalysts with desired reactivities. From the viewpoint of application of artificial enzymes for organic synthesis, recently constructed artificial enzymes mediating oxidation, reduction and C-C bond formation/cleavage are introduced in this review article. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evolution of the Kdo2-lipid A Biosynthesis in Bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Opiyo; R Pardy; H Moriyama

BACKGROUND: Lipid A is the highly immunoreactive endotoxic center of lipopolysaccharide (LPS). It anchors the LPS into the outer membrane of most Gram-negative bacteria. Lipid A can be recognized by animal cells, triggers defense-related responses, and causes Gram-negative sepsis. The biosynthesis of Kdo2-lipid A, the LPS substructure, involves with nine enzymatic steps. RESULTS: In order to elucidate the evolutionary pathway of Kdo2-lipid A biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. We found that not all Gram-negative bacteria have all nine enzymes. Some Gram-negative bacteria have no genes encoding these enzymes and others have genesmore » only for the first four enzymes (LpxA, LpxC, LpxD, and LpxB). Among the nine enzymes, five appeared to have arisen from three independent gene duplication events. Two of such events happened within the Proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria. CONCLUSIONS: The nine-enzyme pathway, which was established based on the studies mainly in Escherichia coli K12, appears to be the most derived and optimized form. It is found only in E. coli and related Proteobacteria. Simpler and probably less efficient pathways are found in other bacterial groups, with Kdo2-lipid A variants as the likely end products. The Kdo2-lipid A biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of Proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment.« less

Hybrid schemes based on quantum mechanics/molecular mechanics simulations goals to success, problems, and perspectives.

PubMed

Ferrer, Silvia; Ruiz-Pernía, Javier; Martí, Sergio; Moliner, Vicent; Tuñón, Iñaki; Bertrán, Juan; Andrés, Juan

2011-01-01

The development of characterization techniques, advanced synthesis methods, as well as molecular modeling has transformed the study of systems in a well-established research field. The current research challenges in biocatalysis and biotransformation evolve around enzyme discovery, design, and optimization. How can we find or create enzymes that catalyze important synthetic reactions, even reactions that may not exist in nature? What is the source of enzyme catalytic power? To answer these and other related questions, the standard strategies have evolved from trial-and-error methodologies based on chemical knowledge, accumulated experience, and common sense into a clearly multidisciplinary science that allows one to reach the molecular design of tailor-made enzyme catalysts. This is even more so when one refers to enzyme catalysts, for which the detailed structure and composition are known and can be manipulated to introduce well-defined residues which can be implicated in the chemical rearrangements taking place in the active site. The methods and techniques of theoretical and computational chemistry are becoming more and more important in both understanding the fundamental biological roles of enzymes and facilitating their utilization in biotechnology. Improvement of the catalytic function of enzymes is important from scientific and industrial viewpoints, and to put this fact in the actual perspective as well as the potentialities, we recommend the very recent report of Sanderson [Sanderson, K. (2011). Chemistry: enzyme expertise. Nature 471, 397.]. Great fundamental advances have been made toward the ab initio design of enzyme catalysts based on molecular modeling. This has been based on the molecular mechanistic knowledge of the reactions to be catalyzed, together with the development of advanced synthesis and characterization techniques. The corresponding molecular mechanism can be studied by means of powerful quantum chemical calculations. The catalytic active site can be optimized to improve the transition state analogues (TSA) and to enhance the catalytic activity, even improve the active site to favor a desired direction of some promiscuous enzymes. In this chapter, we give a brief introduction, the state of the art, and future prospects and implications of enzyme design. Current computational tools to assist experimentalists for the design and engineering of proteins with desired catalytic properties are described. The interplay between enzyme design, molecular simulations, and experiments will be presented to emphasize the interdisciplinary nature of this research field. This text highlights the recent advances and examples selected from our laboratory are shown, of how the applications of these tools are a first attempt to de novo design of protein active sites. Identification of neutral/advantageous/deleterious mutation platforms can be exploited to penetrate some of Nature's closely guarded secrets of chemical reactivity. In this chapter, we give a brief introduction, the state of the art, and future prospects and implications of enzyme design. The first part describes briefly how the molecular modeling is carried out. Then, we discuss the requirements of hybrid quantum mechanical/molecular mechanics molecular dynamics (QM/MM MD) simulations, analyzing what are the basis of these theoretical methodologies, how we can use them with a view to its application in the study of enzyme catalysis, and what are the best methodologies for assessing its catalytic potential. In the second part, we focus on some selected examples, taking as a common guide the chorismate to prephenate rearrangement, studying the corresponding molecular mechanism in vacuo, in solution and in an enzyme environment. In addition, examples involving catalytic antibodies (CAs) and promiscuous enzymes will be presented. Finally, a special emphasis is made to provide some hints about the logical evolution that can be anticipated in this research field. Moreover, it helps in understanding the open directions in this area of knowledge and highlights the importance of computational approaches in discovering specific drugs and the impact on the rational design of tailor-made enzymes. Copyright © 2011 Elsevier Inc. All rights reserved.

Universal Sequence Replication, Reversible Polymerization and Early Functional Biopolymers: A Model for the Initiation of Prebiotic Sequence Evolution

PubMed Central

Walker, Sara Imari; Grover, Martha A.; Hud, Nicholas V.

2012-01-01

Many models for the origin of life have focused on understanding how evolution can drive the refinement of a preexisting enzyme, such as the evolution of efficient replicase activity. Here we present a model for what was, arguably, an even earlier stage of chemical evolution, when polymer sequence diversity was generated and sustained before, and during, the onset of functional selection. The model includes regular environmental cycles (e.g. hydration-dehydration cycles) that drive polymers between times of replication and functional activity, which coincide with times of different monomer and polymer diffusivity. Template-directed replication of informational polymers, which takes place during the dehydration stage of each cycle, is considered to be sequence-independent. New sequences are generated by spontaneous polymer formation, and all sequences compete for a finite monomer resource that is recycled via reversible polymerization. Kinetic Monte Carlo simulations demonstrate that this proposed prebiotic scenario provides a robust mechanism for the exploration of sequence space. Introduction of a polymer sequence with monomer synthetase activity illustrates that functional sequences can become established in a preexisting pool of otherwise non-functional sequences. Functional selection does not dominate system dynamics and sequence diversity remains high, permitting the emergence and spread of more than one functional sequence. It is also observed that polymers spontaneously form clusters in simulations where polymers diffuse more slowly than monomers, a feature that is reminiscent of a previous proposal that the earliest stages of life could have been defined by the collective evolution of a system-wide cooperation of polymer aggregates. Overall, the results presented demonstrate the merits of considering plausible prebiotic polymer chemistries and environments that would have allowed for the rapid turnover of monomer resources and for regularly varying monomer/polymer diffusivities. PMID:22493682

Functional Evolution of PLP-dependent Enzymes based on Active-Site Structural Similarities

PubMed Central

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-01-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5’-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the Comparison of Protein Active Site Structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. PMID:24920327

Functional evolution of PLP-dependent enzymes based on active-site structural similarities.

PubMed

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-10-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5'-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the comparison of protein active site structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional-fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. © 2014 Wiley Periodicals, Inc.

Independent Evolution of Six Families of Halogenating Enzymes.

PubMed

Xu, Gangming; Wang, Bin-Gui

2016-01-01

Halogenated natural products are widespread in the environment, and the halogen atoms are typically vital to their bioactivities. Thus far, six families of halogenating enzymes have been identified: cofactor-free haloperoxidases (HPO), vanadium-dependent haloperoxidases (V-HPO), heme iron-dependent haloperoxidases (HI-HPO), non-heme iron-dependent halogenases (NI-HG), flavin-dependent halogenases (F-HG), and S-adenosyl-L-methionine (SAM)-dependent halogenases (S-HG). However, these halogenating enzymes with similar biological functions but distinct structures might have evolved independently. Phylogenetic and structural analyses suggest that the HPO, V-HPO, HI-HPO, NI-HG, F-HG, and S-HG enzyme families may have evolutionary relationships to the α/β hydrolases, acid phosphatases, peroxidases, chemotaxis phosphatases, oxidoreductases, and SAM hydroxide adenosyltransferases, respectively. These halogenating enzymes have established sequence homology, structural conservation, and mechanistic features within each family. Understanding the distinct evolutionary history of these halogenating enzymes will provide further insights into the study of their catalytic mechanisms and halogenation specificity.

Single-molecule paleoenzymology probes the chemistry of resurrected enzymes

PubMed Central

Perez-Jimenez, Raul; Inglés-Prieto, Alvaro; Zhao, Zi-Ming; Sanchez-Romero, Inmaculada; Alegre-Cebollada, Jorge; Kosuri, Pallav; Garcia-Manyes, Sergi; Kappock, T. Joseph; Tanokura, Masaru; Holmgren, Arne; Sanchez-Ruiz, Jose M.; Gaucher, Eric A.; Fernandez, Julio M.

2011-01-01

A journey back in time is possible at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx), dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32° C more stable than modern enzymes and the oldest show significantly higher activity than extant ones at pH 5. We probed their mechanisms of reduction using single-molecule force spectroscopy. From the force-dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs utilize chemical mechanisms of reduction similar to those of modern enzymes. While Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over a 4 Gyr time span to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth. PMID:21460845

Method of generating ploynucleotides encoding enhanced folding variants

DOEpatents

Bradbury, Andrew M.; Kiss, Csaba; Waldo, Geoffrey S.

2017-05-02

The invention provides directed evolution methods for improving the folding, solubility and stability (including thermostability) characteristics of polypeptides. In one aspect, the invention provides a method for generating folding and stability-enhanced variants of proteins, including but not limited to fluorescent proteins, chromophoric proteins and enzymes. In another aspect, the invention provides methods for generating thermostable variants of a target protein or polypeptide via an internal destabilization baiting strategy. Internally destabilization a protein of interest is achieved by inserting a heterologous, folding-destabilizing sequence (folding interference domain) within DNA encoding the protein of interest, evolving the protein sequences adjacent to the heterologous insertion to overcome the destabilization (using any number of mutagenesis methods), thereby creating a library of variants. The variants in the library are expressed, and those with enhanced folding characteristics selected.

Advances in protease engineering for laundry detergents.

PubMed

Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

2015-12-25

Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. Copyright © 2015 Elsevier B.V. All rights reserved.

The TIM Barrel Architecture Facilitated the Early Evolution of Protein-Mediated Metabolism.

PubMed

Goldman, Aaron David; Beatty, Joshua T; Landweber, Laura F

2016-01-01

The triosephosphate isomerase (TIM) barrel protein fold is a structurally repetitive architecture that is present in approximately 10% of all enzymes. It is generally assumed that this ubiquity in modern proteomes reflects an essential historical role in early protein-mediated metabolism. Here, we provide quantitative and comparative analyses to support several hypotheses about the early importance of the TIM barrel architecture. An information theoretical analysis of protein structures supports the hypothesis that the TIM barrel architecture could arise more easily by duplication and recombination compared to other mixed α/β structures. We show that TIM barrel enzymes corresponding to the most taxonomically broad superfamilies also have the broadest range of functions, often aided by metal and nucleotide-derived cofactors that are thought to reflect an earlier stage of metabolic evolution. By comparison to other putatively ancient protein architectures, we find that the functional diversity of TIM barrel proteins cannot be explained simply by their antiquity. Instead, the breadth of TIM barrel functions can be explained, in part, by the incorporation of a broad range of cofactors, a trend that does not appear to be shared by proteins in general. These results support the hypothesis that the simple and functionally general TIM barrel architecture may have arisen early in the evolution of protein biosynthesis and provided an ideal scaffold to facilitate the metabolic transition from ribozymes, peptides, and geochemical catalysts to modern protein enzymes.

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

PubMed

Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

2015-07-24

The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens.

PubMed

Kaplan, J B; Merkel, W K; Nichols, B P

1985-06-05

The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathway-specific enzymes.

Targeted enzyme prodrug therapies.

PubMed

Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

2010-09-01

The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

Electrochemistry of metalloproteins: protein film electrochemistry for the study of E. coli [NiFe]-hydrogenase-1.

PubMed

Evans, Rhiannon M; Armstrong, Fraser A

2014-01-01

Protein film electrochemistry is a technique which allows the direct control of redox-active enzymes, providing particularly detailed information on their catalytic properties. The enzyme is deposited onto a working electrode tip, and through control of the applied potential the enzyme activity is monitored as electrical current, allowing for direct study of inherent activity as electrons are transferred to and from the enzyme redox center(s). No mediators are used. Because the only enzyme present in the experiment is bound at the electrode surface, gaseous and liquid phase inhibitors can be introduced and removed whilst the enzyme remains in situ. Potential control means that kinetics and thermodynamics are explored simultaneously; the kinetics of a reaction can be studied as a function of potential. Steady-state catalytic rates are observed directly as current (for a given potential) and non-steady-state rates (such as interconversions between different forms of the enzyme) are observed from the change in current with time. The more active the enzyme, the higher the current and the better the signal-to-noise. In this chapter we outline the practical aspects of PFE for studying electroactive enzymes, using the Escherichia coli [NiFe]-hydrogenase 1 (Hyd-1) as an example.

Environmental Epigenetics and a Unified Theory of the Molecular Aspects of Evolution: A Neo-Lamarckian Concept that Facilitates Neo-Darwinian Evolution.

PubMed

Skinner, Michael K

2015-04-26

Environment has a critical role in the natural selection process for Darwinian evolution. The primary molecular component currently considered for neo-Darwinian evolution involves genetic alterations and random mutations that generate the phenotypic variation required for natural selection to act. The vast majority of environmental factors cannot directly alter DNA sequence. Epigenetic mechanisms directly regulate genetic processes and can be dramatically altered by environmental factors. Therefore, environmental epigenetics provides a molecular mechanism to directly alter phenotypic variation generationally. Lamarck proposed in 1802 the concept that environment can directly alter phenotype in a heritable manner. Environmental epigenetics and epigenetic transgenerational inheritance provide molecular mechanisms for this process. Therefore, environment can on a molecular level influence the phenotypic variation directly. The ability of environmental epigenetics to alter phenotypic and genotypic variation directly can significantly impact natural selection. Neo-Lamarckian concept can facilitate neo-Darwinian evolution. A unified theory of evolution is presented to describe the integration of environmental epigenetic and genetic aspects of evolution. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Origins of Protein Functions in Cells

NASA Technical Reports Server (NTRS)

Seelig, Burchard; Pohorille, Andrzej

2011-01-01

In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis and in vitro evolution of very large libraries of random amino acid sequences. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions yet, important clues have been uncovered. In one example (Keefe and Szostak, 2001), novel ATP binding proteins were identified that appear to be unrelated in both sequence and structure to any known ATP binding proteins. One of these proteins was subsequently redesigned computationally to bind GTP through introducing several mutations that introduce targeted structural changes to the protein, improve its binding to guanine and prevent water from accessing the active center. This study facilitates further investigations of individual evolutionary steps that lead to a change of function in primordial proteins. In a second study (Seelig and Szostak, 2007), novel enzymes were generated that can join two pieces of RNA in a reaction for which no natural enzymes are known. Recently it was found that, as in the previous case, the proteins have a structure unknown among modern enzymes. In this case, in vitro evolution started from a small, non-enzymatic protein. A similar selection process initiated from a library of random polypeptides is in progress. These results not only allow for estimating the occurrence of function in random protein assemblies but also provide evidence for the possibility of alternative protein worlds. Extant proteins might simply represent a frozen accident in the world of possible proteins. Alternative collections of proteins, even with similar functions, could originate alternative evolutionary paths.

Integrating Horizontal Gene Transfer and Common Descent to Depict Evolution and Contrast It with “Common Design”1

PubMed Central

GUILLERMO PAZ-Y-MIÑO-C; ESPINOSA, AVELINA

2016-01-01

Horizontal gene transfer (HGT) and common descent interact in space and time. Because events of HGT co-occur with phylogenetic evolution, it is difficult to depict evolutionary patterns graphically. Tree-like representations of life’s diversification are useful, but they ignore the significance of HGT in evolutionary history, particularly of unicellular organisms, ancestors of multicellular life. Here we integrate the reticulated-tree model, ring of life, symbiogenesis whole-organism model, and eliminative pattern pluralism to represent evolution. Using Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2), a bifunctional enzyme in the glycolytic pathway of amoeba, we illustrate how EhADH2 could be the product of both horizontally acquired features from ancestral prokaryotes (i.e. aldehyde dehydrogenase [ALDH] and alcohol dehydrogenase [ADH]), and subsequent functional integration of these enzymes into EhADH2, which is now inherited by amoeba via common descent. Natural selection has driven the evolution of EhADH2 active sites, which require specific amino acids (cysteine 252 in the ALDH domain; histidine 754 in the ADH domain), iron- and NAD+ as cofactors, and the substrates acetyl-CoA for ALDH and acetaldehyde for ADH. Alternative views invoking “common design” (i.e. the non-naturalistic emergence of major taxa independent from ancestry) to explain the interaction between horizontal and vertical evolution are unfounded. PMID:20021546

Stereoselective aminoacylation of RNA

NASA Technical Reports Server (NTRS)

Usher, D. A.; Needels, M. C.; Brenner, T.

1986-01-01

Prebiotic chemistry is faced with a major problem: how could a controlled and selective reaction occur, when there is present in the same solution a large number of alternative possible coreactants? This problem is solved in the modern cell by the presence of enzymes, which are not only highly efficient and controllable catalysts, but which also can impose on their substrates a precise structural requirement. However, enzymes are the result of billions of years of evolution, and we cannot invoke them as prebiotic catalysts. One approach to solving this problem in the prebiotic context is to make use of template-directed reactions. These reactions increase the number of structural requirements that must be simultaneously present in a molecule for it to be able to react, and thereby increase the selectivity of the reaction. They also can give a large increase in the rate of a reaction, if the template constrains two potential coreactants to lie close together. A third benefit is that information that is present in the template molecule can be passed on to the product molecules. If the earliest organisms were based on proteins and nucleic acids, then the investigation of peptide synthesis on an oligonucleotide template is highly relevant to the study of the origin of life.

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in Escherichia coli.

PubMed

Koay, Teng Wei; Wong, Hann Ling; Lim, Boon Hoe

2016-11-26

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a rate-limiting photosynthetic enzyme that catalyzes carbon fixation in the Calvin cycle. Much interest has been devoted to engineering this ubiquitous enzyme with the goal of increasing plant growth. However, experiments that have successfully produced improved Rubisco variants, via directed evolution in Escherichia coli, are limited to bacterial Rubisco because the eukaryotic holoenzyme cannot be produced in E. coli. The present study attempts to determine the specific differences between bacterial and eukaryotic Rubisco large subunit primary structure that are responsible for preventing heterologous eukaryotic holoenzyme formation in E. coli. A series of chimeric Synechococcus Rubiscos were created in which different sections of the large subunit were swapped with those of the homologous Chlamydomonas Rubisco. Chimeric holoenzymes that can form in vivo would indicate that differences within the swapped sections do not disrupt holoenzyme formation. Large subunit residues 1-97, 198-247 and 448-472 were successfully swapped without inhibiting holoenzyme formation. In all ten chimeras, protein expression was observed for the separate subunits at a detectable level. As a first approximation, the regions that can tolerate swapping may be targets for future engineering.

Evolution of vitamin B2 biosynthesis: structural and functional similarity between pyrimidine deaminases of eubacterial and plant origin.

PubMed

Fischer, Markus; Römisch, Werner; Saller, Sabine; Illarionov, Boris; Richter, Gerald; Rohdich, Felix; Eisenreich, Wolfgang; Bacher, Adelbert

2004-08-27

The Arabidopsis thaliana open reading frame At4g20960 predicts a protein whose N-terminal part is similar to the eubacterial 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate deaminase domain. A synthetic open reading frame specifying a pseudomature form of the plant enzyme directed the synthesis of a recombinant protein which was purified to apparent homogeneity and was shown by NMR spectroscopy to convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate at a rate of 0.9 micromol mg(-1) min(-1). The substrate and product of the enzyme are both subject to spontaneous anomerization of the ribosyl side chain as shown by (13)C NMR spectroscopy. The protein contains 1 eq of Zn(2+)/subunit. The deaminase activity could be assigned to the N-terminal section of the plant protein. The deaminase domains of plants and eubacteria share a high degree of similarity, in contrast to deaminases from fungi. These data show that the riboflavin biosynthesis in plants proceeds by the same reaction steps as in eubacteria, whereas fungi use a different pathway.

Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases.

PubMed

Liu, Mira D; Warner, Elliot A; Morrissey, Charlotte E; Fick, Caitlyn W; Wu, Taia S; Ornelas, Marya Y; Ochoa, Gabriela V; Zhang, Brendan S; Rathbun, Colin M; Porterfield, William B; Prescher, Jennifer A; Leconte, Aaron M

2018-02-06

Directed evolution has proven to be an invaluable tool for protein engineering; however, there is still a need for developing new approaches to continue to improve the efficiency and efficacy of these methods. Here, we demonstrate a new method for library design that applies a previously developed bioinformatic method, Statistical Coupling Analysis (SCA). SCA uses homologous enzymes to identify amino acid positions that are mutable and functionally important and engage in synergistic interactions between amino acids. We use SCA to guide a library of the protein luciferase and demonstrate that, in a single round of selection, we can identify luciferase mutants with several valuable properties. Specifically, we identify luciferase mutants that possess both red-shifted emission spectra and improved stability relative to those of the wild-type enzyme. We also identify luciferase mutants that possess a >50-fold change in specificity for modified luciferins. To understand the mutational origin of these improved mutants, we demonstrate the role of mutations at N229, S239, and G246 in altered function. These studies show that SCA can be used to guide library design and rapidly identify synergistic amino acid mutations from a small library.

(Bio)Chemical Tailoring of Biogenic 3-D Nanopatterned Templates with Energy-Relevant Functionalities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sandhage, Kenneth H; Kroger, Nils

2014-09-08

The overall aim of this research has been to obtain fundamental understanding of (bio)chemical methodologies that will enable utilization of the unique 3-D nanopatterned architectures naturally produced by diatoms for the syntheses of advanced functional materials attractive for applications in energy harvesting/conversion and storage. This research has been conducted in three thrusts: Thrust 1 (In vivo immobilization of proteins in diatom biosilica) is directed towards elucidating the fundamental mechanism(s) underlying the cellular processes of in vivo immobilization of proteins in diatom silica. Thrust 2 (Shape-preserving reactive conversion of diatom biosilica into porous, high-surface area inorganic replicas) is aimed at understandingmore » the fundamental mechanisms of shape preservation and nanostructural evolution associated with the reactive conversion and/or coating-based conversion of diatom biosilica templates into porous inorganic replicas. Thrust 3 (Immobilization of energy-relevant enzymes in diatom biosilica and onto diatom biosilica-derived inorganic replicas) involves use of the results from both Thrust 1 and 2 to develop strategies for in vivo and in vitro immobilization of enzymes in/on diatom biosilica and diatom biosilica-derived inorganic replicas, respectively. This Final Report describes progress achieved in all 3 of these thrusts.« less

Functional Sites Induce Long-Range Evolutionary Constraints in Enzymes

PubMed Central

Jack, Benjamin R.; Meyer, Austin G.; Echave, Julian; Wilke, Claus O.

2016-01-01

Functional residues in proteins tend to be highly conserved over evolutionary time. However, to what extent functional sites impose evolutionary constraints on nearby or even more distant residues is not known. Here, we report pervasive conservation gradients toward catalytic residues in a dataset of 524 distinct enzymes: evolutionary conservation decreases approximately linearly with increasing distance to the nearest catalytic residue in the protein structure. This trend encompasses, on average, 80% of the residues in any enzyme, and it is independent of known structural constraints on protein evolution such as residue packing or solvent accessibility. Further, the trend exists in both monomeric and multimeric enzymes and irrespective of enzyme size and/or location of the active site in the enzyme structure. By contrast, sites in protein–protein interfaces, unlike catalytic residues, are only weakly conserved and induce only minor rate gradients. In aggregate, these observations show that functional sites, and in particular catalytic residues, induce long-range evolutionary constraints in enzymes. PMID:27138088

A Measure of the Broad Substrate Specificity of Enzymes Based on ‘Duplicate’ Catalytic Residues

PubMed Central

Chakraborty, Sandeep; Ásgeirsson, Bjarni; Rao, Basuthkar J.

2012-01-01

The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins. In the current work, we quantitatively characterize the active site for the ability to catalyze distinct, yet related, substrates (BRASS). A protein with known structure and active site residues provides the framework for computing ‘duplicate’ residues, each of which results in slightly modified replicas of the active site scaffold. Such spatial congruence is supplemented by Finite difference Poisson Boltzmann analysis which filters out electrostatically unfavorable configurations. The congruent configurations are used to compute an index (BrassIndex), which reflects the broad substrate profile of the active site. We identify an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of non-homologous proteins extracted from the Catalytic Site Atlas. The acetylhydrolase, a regulatory enzyme, is known to be highly specific for platelet-activating factor. In the methyltransferase (PDB: 1QAM), various combinations of glycine (Gly38/40/42), asparagine (Asn101/11) and glutamic acid (Glu59/36) residues having similar spatial and electrostatic profiles with the specified scaffold (Gly38, Asn101 and Glu59) exemplifies the broad substrate profile such an active site may provide. ‘Duplicate’ residues identified by relaxing the spatial and/or electrostatic constraints can be the target of directed evolution methodologies, like saturation mutagenesis, for modulating the substrate specificity of proteins. PMID:23166637

RubisCO selection using the vigorously aerobic and metabolically versatile bacterium Ralstonia eutropha.

PubMed

Satagopan, Sriram; Tabita, F Robert

2016-08-01

Recapturing atmospheric CO2 is key to reducing global warming and increasing biological carbon availability. Ralstonia eutropha is a biotechnologically useful aerobic bacterium that uses the Calvin-Benson-Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) for CO2 utilization, suggesting that it may be a useful host to bioselect RubisCO molecules with improved CO2 -capture capabilities. A host strain of R. eutropha was constructed for this purpose after deleting endogenous genes encoding two related RubisCOs. This strain could be complemented for CO2 -dependent growth by introducing native or heterologous RubisCO genes. Mutagenesis and suppressor selection identified amino acid substitutions in a hydrophobic region that specifically influences RubisCO's interaction with its substrates, particularly O2 , which competes with CO2 at the active site. Unlike most RubisCOs, the R. eutropha enzyme has evolved to retain optimal CO2 -fixation rates in a fast-growing host, despite the presence of high levels of competing O2 . Yet its structure-function properties resemble those of several commonly found RubisCOs, including the higher plant enzymes, allowing strategies to engineer analogous enzymes. Because R. eutropha can be cultured rapidly under harsh environmental conditions (e.g., with toxic industrial flue gas), in the presence of near saturation levels of oxygen, artificial selection and directed evolution studies in this organism could potentially impact efforts toward improving RubisCO-dependent biological CO2 utilization in aerobic environments. d-ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39; phosphoribulokinase, EC 2.7.1.19. © 2016 Federation of European Biochemical Societies.

Molecular basis of thermostability enhancement of Renilla luciferase at higher temperatures by insertion of a disulfide bridge into the structure.

PubMed

Fanaei Kahrani, Zahra; Emamzadeh, Rahman; Nazari, Mahboobeh; Rasa, Seyed Mohammad Mahdi

2017-02-01

Renilla luciferase (RLuc), also known as Renilla-luciferin 2-monooxygenase, is a light producing enzyme used in many biotechnological applications such as bioreporters. However, its kinetics stability -especially at higher temperatures- is a limiting factor for developing thermostable bioreporters. The aim of this study was to improve the stability of super Renilla luciferase 8 (SRLuc 8) which is a red-emitter variety of RLuc at higher temperatures, by introduction of a disulfide bridge into its structure. In this study, the choice of the proper disulfide bond formation was based on computational methods and enzyme functionality (active site position) which is called geometric-functional method. N45 and A71 at the N-terminal of the enzyme were selected for directed evolution. The engineered luciferase was called C-SRLuc 8 and its activity and stability were assayed. The results indicated that the kinetic stability of C-SRLuc 8 increased significantly at 60°C to 70°C as compared to SRLuc 8; the residual activity of C-SRLuc 8 was approximately 20% after incubation at 65°C for 5min. Moreover, the enzyme activity decreased compared with SRLuc 8. The molecular basis of the structural changes was considered using molecular dynamics simulations and the results indicated that the N45C/A71C crosslink was involved in a hotspot foldon which seemed to be the rate-limiting step of conformational collapse at higher temperatures. The present study may provide an opportunity for the development of the next-generation of thermostable RLuc-based biosensors. Copyright © 2016. Published by Elsevier B.V.

Insights into drug metabolism by cytochromes P450 from modelling studies of CYP2D6-drug interactions

PubMed Central

Maréchal, J-D; Kemp, C A; Roberts, G C K; Paine, M J I; Wolf, C R; Sutcliffe, M J

2008-01-01

The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity—including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O-demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes. PMID:18026129

Functional mapping of protein-protein interactions in an enzyme complex by directed evolution.

PubMed

Roderer, Kathrin; Neuenschwander, Martin; Codoni, Giosiana; Sasso, Severin; Gamper, Marianne; Kast, Peter

2014-01-01

The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84-90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84-86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.

Functional Mapping of Protein-Protein Interactions in an Enzyme Complex by Directed Evolution

PubMed Central

Roderer, Kathrin; Neuenschwander, Martin; Codoni, Giosiana; Sasso, Severin; Gamper, Marianne; Kast, Peter

2014-01-01

The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84–90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84–86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes. PMID:25551646

Evolution and function of the Mycoplasma hyopneumoniae peroxiredoxin, a 2-Cys-like enzyme with a single Cys residue.

PubMed

Gonchoroski, Taylor; Virginio, Veridiana G; Thompson, Claudia E; Paes, Jéssica A; Machado, Cláudio X; Ferreira, Henrique B

2017-04-01

The minimal genome of the mollicute Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia, encodes a limited repertoire of antioxidant enzymes that include a single and atypical peroxiredoxin (MhPrx), whose evolution and function were studied here. MhPrx has only one catalytic cysteine, in contrast with some of its possible ancestors (2-Cys peroxiredoxins), which have two. Although it is more similar to 2-Cys orthologs, MhPrx can still function with a single peroxidatic cysteine (Cys P ), using non-thiolic electron donors to reduce it. Therefore, MhPrx could be a representative of a possible group of 2-Cys peroxiredoxins, which have lost the resolving cysteine (Cys R ) residue without losing their catalytic properties. To further investigate MhPrx evolution, we performed a comprehensive phylogenetic analysis in the context of several bacterial families, including Prxs belonging to Tpx and AhpE families, shedding light on the evolutionary history of Mycoplasmataceae Prxs and giving support to the hypothesis of a relatively recent loss of the Cys R within this family. Moreover, mutational analyses provided insights into MhPrx function with one, two, or without catalytic cysteines. While removal of the MhPrx putative Cys P caused complete activity loss, confirming its catalytic role, the introduction of a second cysteine in a site correspondent to that of the Cys R of a 2-Cys orthologue, as in the MhPrx supposed ancestral form, was compatible with enzyme activity. Overall, our phylogenetic and mutational studies support that MhPrx recently diverged from a 2-Cys Prx ancestor and pave the way for future studies addressing structural, functional, and evolutive aspects of peroxiredoxin subfamilies in Mollicutes and other bacteria.

Evolution of Enzymatic Activities in the Enolase Superfamily: Stereochemically Distinct Mechanisms in Two Families of cis,cis-Muconate Lactonizing Enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakai, A.; Fedorov, A; Fedorov, E

2009-01-01

The mechanistically diverse enolase superfamily is a paradigm for elucidating Nature's strategies for divergent evolution of enzyme function. Each of the different reactions catalyzed by members of the superfamily is initiated by abstraction of the a-proton of a carboxylate substrate that is coordinated to an essential Mg2+. The muconate lactonizing enzyme (MLE) from Pseudomonas putida, a member of a family that catalyzes the syn-cycloisomerization of cis,cis-muconate to (4S)-muconolactone in the e-ketoadipate pathway, has provided critical insights into the structural bases for evolution of function within the superfamily. A second, divergent family of homologous MLEs that catalyzes anti-cycloisomerization has been identified.more » Structures of members of both families liganded with the common (4S)-muconolactone product (syn, Pseudomonas fluorescens, gi 70731221; anti, Mycobacterium smegmatis, gi 118470554) document that the conserved Lys at the end of the second e-strand in the (e/a)7e-barrel domain serves as the acid catalyst in both reactions. The different stereochemical courses (syn and anti) result from different structural strategies for determining substrate specificity: although the distal carboxylate group of the cis,cis-muconate substrate attacks the same face of the proximal double bond, opposite faces of the resulting enolate anion intermediate are presented to the conserved Lys acid catalyst. The discovery of two families of homologous, but stereochemically distinct, MLEs likely provides an example of 'pseudoconvergent' evolution of the same function from different homologous progenitors within the enolase superfamily, in which different spatial arrangements of active site functional groups and substrate specificity determinants support catalysis of the same reaction.« less

21 CFR 173.150 - Milk-clotting enzymes, microbial.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Milk-clotting enzymes, microbial. 173.150 Section... (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.150 Milk-clotting enzymes, microbial. Milk-clotting enzyme produced by pure-culture...

Nickel-centred proton reduction catalysis in a model of [NiFe] hydrogenase

NASA Astrophysics Data System (ADS)

Brazzolotto, Deborah; Gennari, Marcello; Queyriaux, Nicolas; Simmons, Trevor R.; Pécaut, Jacques; Demeshko, Serhiy; Meyer, Franc; Orio, Maylis; Artero, Vincent; Duboc, Carole

2016-11-01

Hydrogen production through water splitting is one of the most promising solutions for the storage of renewable energy. [NiFe] hydrogenases are organometallic enzymes containing nickel and iron centres that catalyse hydrogen evolution with performances that rival those of platinum. These enzymes provide inspiration for the design of new molecular catalysts that do not require precious metals. However, all heterodinuclear NiFe models reported so far do not reproduce the Ni-centred reactivity found at the active site of [NiFe] hydrogenases. Here, we report a structural and functional NiFe mimic that displays reactivity at the Ni site. This is shown by the detection of two catalytic intermediates that reproduce structural and electronic features of the Ni-L and Ni-R states of the enzyme during catalytic turnover. Under electrocatalytic conditions, this mimic displays high rates for H2 evolution (second-order rate constant of 2.5 × 104 M-1 s-1 turnover frequency of 250 s-1 at 10 mM H+ concentration) from mildly acidic solutions.

Mapping the temperature-dependent conformational landscapes of the dynamic enzymes cyclophilin A and urease

NASA Astrophysics Data System (ADS)

Thorne, Robert; Keedy, Daniel; Warkentin, Matthew; Fraser, James; Moreau, David; Atakisi, Hakan; Rau, Peter

Proteins populate complex, temperature-dependent ensembles of conformations that enable their function. Yet in X-ray crystallographic studies, roughly 98% of structures have been determined at 100 K, and most refined to only a single conformation. A combination of experimental methods enabled by studies of ice formation and computational methods for mining low-density features in electron density maps have been applied to determine the evolution of the conformational landscapes of the enzymes cyclophilin A and urease between 300 K and 100 K. Minority conformations of most side chains depopulate on cooling from 300 to ~200 K, below which subsequent conformational evolution is quenched. The characteristic temperatures for this depopulation are highly heterogeneous throughout each enzyme. The temperature-dependent ensemble of the active site flap in urease has also been mapped. These all-atom, site-resolved measurements and analyses rule out one interpretation of the protein-solvent glass transition, and give an alternative interpretation of a dynamical transition identified in site-averaged experiments. They demonstrate a powerful approach to structural characterization of the dynamic underpinnings of protein function. Supported by NSF MCB-1330685.

cis-Prenyltransferase: New Insights into Protein Glycosylation, Rubber Synthesis, and Human Diseases.

PubMed

Grabińska, Kariona A; Park, Eon Joo; Sessa, William C

2016-08-26

cis-Prenyltransferases (cis-PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. cis-PTs catalyze consecutive condensation reactions of allylic diphosphate acceptor with isopentenyl diphosphate (IPP) in the cis (Z) configuration to generate linear polyprenyl diphosphate. The chain lengths of isoprenoid carbon skeletons vary widely from neryl pyrophosphate (C10) to natural rubber (C>10,000). The homo-dimeric bacterial enzyme, undecaprenyl diphosphate synthase (UPPS), has been structurally and mechanistically characterized in great detail and serves as a model for understanding the mode of action of eukaryotic cis-PTs. However, recent experiments have revealed that mammals, fungal, and long-chain plant cis-PTs are heteromeric enzymes composed of two distantly related subunits. In this review, the classification, function, and evolution of cis-PTs will be discussed with a special emphasis on the role of the newly described NgBR/Nus1 subunit and its plants' orthologs as essential, structural components of the cis-PTs activity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Enhanced enzyme stability through site-directed covalent immobilization.

PubMed

Wu, Jeffrey Chun Yu; Hutchings, Christopher Hayden; Lindsay, Mark Jeffrey; Werner, Christopher James; Bundy, Bradley Charles

2015-01-10

Breakthroughs in enzyme immobilization have enabled increased enzyme recovery and reusability, leading to significant decreases in the cost of enzyme use and fueling biocatalysis growth. However, current enzyme immobilization techniques suffer from leaching, enzyme stability, and recoverability and reusability issues. Moreover, these techniques lack the ability to control the orientation of the immobilized enzymes. To determine the impact of orientation on covalently immobilized enzyme activity and stability, we apply our PRECISE (Protein Residue-Explicit Covalent Immobilization for Stability Enhancement) system to a model enzyme, T4 lysozyme. The PRECISE system uses non-canonical amino acid incorporation and the Huisgen 1,3-dipolar cycloaddition "click" reaction to enable directed enzyme immobilization at rationally chosen residues throughout an enzyme. Unlike previous site-specific systems, the PRECISE system is a truly covalent immobilization method. Utilizing this system, enzymes immobilized at proximate and distant locations from the active site were tested for activity and stability under denaturing conditions. Our results demonstrate that orientation control of covalently immobilized enzymes can provide activity and stability benefits exceeding that of traditional random covalent immobilization techniques. PRECISE immobilized enzymes were 50 and 73% more active than randomly immobilized enzymes after harsh freeze-thaw and chemical denaturant treatments. Copyright © 2014 Elsevier B.V. All rights reserved.

Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae.

PubMed

Zhou, Pingping; Xie, Wenping; Li, Aipeng; Wang, Fan; Yao, Zhen; Bian, Qi; Zhu, Yongqiang; Yu, Hongwei; Ye, Lidan

2017-05-01

Highly efficient biosynthesis of the commercially valuable carotenoid astaxanthin by microbial cells is an attractive alternative to chemical synthesis and microalgae extraction. With the goal of enhancing heterologous astaxanthin production in Saccharomyces cerevisiae, metabolic engineering and protein engineering were integrated to improve both the expression and activity of rate-limiting enzymes. Firstly, to increase the supply of β-carotene as a key precursor for astaxanthin, a positive mutant of GGPP synthase (CrtE03M) was overexpressed together with three other rate-limiting enzymes tHMG1, CrtI and CrtYB. Subsequently, to accelerate the conversion of β-carotene to astaxanthin, a color screening system was developed and adopted for directed evolution of β-carotene ketolase (OBKT), generating a triple mutant OBKTM (H165R/V264D/F298Y) with 2.4-fold improved activity. After adjusting copy numbers of the above-mentioned rate-limiting enzymes to further balance the metabolic flux, a diploid strain YastD-01 was generated by mating two astaxanthin-producing haploid strains carrying the same carotenogenic pathway. Finally, further overexpression of OCrtZ and OBKTM in YastD-01 resulted in accumulation of 8.10mg/g DCW (47.18mg/l) of (3S, 3'S)-astaxanthin in shake-flask cultures. This combinatorial strategy might be also applicable for alleviation of metabolic bottleneck in biosynthesis of other value-added products, especially colored metabolites. Copyright © 2017 Elsevier Inc. All rights reserved.

The Structure and Function of a Microbial Allantoin Racemase Reveal the Origin and Conservation of a Catalytic Mechanism.

PubMed

Cendron, Laura; Ramazzina, Ileana; Puggioni, Vincenzo; Maccacaro, Eleonora; Liuzzi, Anastasia; Secchi, Andrea; Zanotti, Giuseppe; Percudani, Riccardo

2016-11-22

The S enantiomer of allantoin is an intermediate of purine degradation in several organisms and the final product of uricolysis in nonhominoid mammals. Bioinformatics indicated that proteins of the Asp/Glu racemase superfamily could be responsible for the allantoin racemase (AllR) activity originally described in Pseudomonas species. In these proteins, a cysteine of the catalytic dyad is substituted with glycine, yet the recombinant enzyme displayed racemization activity with a similar efficiency (k cat /K M ≈ 5 × 10 4 M -1 s -1 ) for the R and S enantiomers of allantoin. The protein crystal structure identified a glutamate residue located three residues downstream (E78) that can functionally replace the missing cysteine; the catalytic role of E78 was confirmed by site-directed mutagenesis. Allantoin can undergo racemization through formation of a bicyclic intermediate (faster) or proton exchange at the chiral center (slower). By monitoring the two alternative mechanisms by 13 C and 1 H nuclear magnetic resonance, we found that the velocity of the faster reaction is unaffected by the enzyme, whereas the velocity of the slower reaction is increased by 7 orders of magnitude. Protein phylogenies trace the origin of the racemization mechanism in enzymes acting on glutamate, a substrate for which proton exchange is the only viable reaction mechanism. This mechanism was inherited by allantoin racemase through divergent evolution and conserved in spite of the substitution of catalytic residues.

How biochemical constraints of cellular growth shape evolutionary adaptations in metabolism.

PubMed

Berkhout, Jan; Bosdriesz, Evert; Nikerel, Emrah; Molenaar, Douwe; de Ridder, Dick; Teusink, Bas; Bruggeman, Frank J

2013-06-01

Evolutionary adaptations in metabolic networks are fundamental to evolution of microbial growth. Studies on unneeded-protein synthesis indicate reductions in fitness upon nonfunctional protein synthesis, showing that cell growth is limited by constraints acting on cellular protein content. Here, we present a theory for optimal metabolic enzyme activity when cells are selected for maximal growth rate given such growth-limiting biochemical constraints. We show how optimal enzyme levels can be understood to result from an enzyme benefit minus cost optimization. The constraints we consider originate from different biochemical aspects of microbial growth, such as competition for limiting amounts of ribosomes or RNA polymerases, or limitations in available energy. Enzyme benefit is related to its kinetics and its importance for fitness, while enzyme cost expresses to what extent resource consumption reduces fitness through constraint-induced reductions of other enzyme levels. A metabolic fitness landscape is introduced to define the fitness potential of an enzyme. This concept is related to the selection coefficient of the enzyme and can be expressed in terms of its fitness benefit and cost.

The complex allosteric and redox regulation of the fumarate hydratase and malate dehydratase reactions of Arabidopsis thaliana Fumarase 1 and 2 gives clues for understanding the massive accumulation of fumarate.

PubMed

Zubimendi, Juan P; Martinatto, Andrea; Valacco, Maria P; Moreno, Silvia; Andreo, Carlos S; Drincovich, María F; Tronconi, Marcos A

2018-06-01

Arabidopsis thaliana possesses two fumarase genes (FUM), AtFUM1 (At2g47510) encoding for the mitochondrial Krebs cycle-associated enzyme and AtFUM2 (At5g50950) for the cytosolic isoform required for fumarate massive accumulation. Here, the comprehensive biochemical studies of AtFUM1 and AtFUM2 shows that they are active enzymes with similar kinetic parameters but differential regulation. For both enzymes, fumarate hydratase (FH) activity is favored over the malate dehydratase (MD) activity; however, MD is the most regulated activity with several allosteric activators. Oxalacetate, glutamine, and/or asparagine are modulators causing the MD reaction to become preferred over the FH reaction. Activity profiles as a function of pH suggest a suboptimal FUM activity in Arabidopsis cells; moreover, the direction of the FUM reaction is sensitive to pH changes. Under mild oxidation conditions, AtFUMs form high mass molecular aggregates, which present both FUM activities decreased to a different extent. The biochemical properties of oxidized AtFUMs (oxAtFUMs) were completely reversed by NADPH-supplied Arabidopsis leaf extracts, suggesting that the AtFUMs redox regulation can be accomplished in vivo. Mass spectrometry analyses indicate the presence of an active site-associated intermolecular disulfide bridge in oxAtFUMs. Finally, a phylogenetic approach points out that other plant species may also possess cytosolic FUM2 enzymes mainly encoded by paralogous genes, indicating that the evolutionary history of this trait has been drawn through a process of parallel evolution. Overall, according to our results, a multilevel regulatory pattern of FUM activities emerges, supporting the role of this enzyme as a carbon flow monitoring point through the organic acid metabolism in plants. © 2018 Federation of European Biochemical Societies.

Development of a genetically programed vanillin-sensing bacterium for high-throughput screening of lignin-degrading enzyme libraries.

PubMed

Sana, Barindra; Chia, Kuan Hui Burton; Raghavan, Sarada S; Ramalingam, Balamurugan; Nagarajan, Niranjan; Seayad, Jayasree; Ghadessy, Farid J

2017-01-01

Lignin is a potential biorefinery feedstock for the production of value-added chemicals including vanillin. A huge amount of lignin is produced as a by-product of the paper industry, while cellulosic components of plant biomass are utilized for the production of paper pulp. In spite of vast potential, lignin remains the least exploited component of plant biomass due to its extremely complex and heterogenous structure. Several enzymes have been reported to have lignin-degrading properties and could be potentially used in lignin biorefining if their catalytic properties could be improved by enzyme engineering. The much needed improvement of lignin-degrading enzymes by high-throughput selection techniques such as directed evolution is currently limited, as robust methods for detecting the conversion of lignin to desired small molecules are not available. We identified a vanillin-inducible promoter by RNAseq analysis of Escherichia coli cells treated with a sublethal dose of vanillin and developed a genetically programmed vanillin-sensing cell by placing the 'very green fluorescent protein' gene under the control of this promoter. Fluorescence of the biosensing cell is enhanced significantly when grown in the presence of vanillin and is readily visualized by fluorescence microscopy. The use of fluorescence-activated cell sorting analysis further enhances the sensitivity, enabling dose-dependent detection of as low as 200 µM vanillin. The biosensor is highly specific to vanillin and no major response is elicited by the presence of lignin, lignin model compound, DMSO, vanillin analogues or non-specific toxic chemicals. We developed an engineered E. coli cell that can detect vanillin at a concentration as low as 200 µM. The vanillin-sensing cell did not show cross-reactivity towards lignin or major lignin degradation products including vanillin analogues. This engineered E. coli cell could potentially be used as a host cell for screening lignin-degrading enzymes that can convert lignin to vanillin.

Trends in the sand: Directional evolution in the shell shape of recessing scallops (Bivalvia: Pectinidae).

PubMed

Sherratt, Emma; Alejandrino, Alvin; Kraemer, Andrew C; Serb, Jeanne M; Adams, Dean C

2016-09-01

Directional evolution is one of the most compelling evolutionary patterns observed in macroevolution. Yet, despite its importance, detecting such trends in multivariate data remains a challenge. In this study, we evaluate multivariate evolution of shell shape in 93 bivalved scallop species, combining geometric morphometrics and phylogenetic comparative methods. Phylomorphospace visualization described the history of morphological diversification in the group; revealing that taxa with a recessing life habit were the most distinctive in shell shape, and appeared to display a directional trend. To evaluate this hypothesis empirically, we extended existing methods by characterizing the mean directional evolution in phylomorphospace for recessing scallops. We then compared this pattern to what was expected under several alternative evolutionary scenarios using phylogenetic simulations. The observed pattern did not fall within the distribution obtained under multivariate Brownian motion, enabling us to reject this evolutionary scenario. By contrast, the observed pattern was more similar to, and fell within, the distribution obtained from simulations using Brownian motion combined with a directional trend. Thus, the observed data are consistent with a pattern of directional evolution for this lineage of recessing scallops. We discuss this putative directional evolutionary trend in terms of its potential adaptive role in exploiting novel habitats. © 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.

Enhancing the biocatalytic manufacture of the key intermediate of atorvastatin by focused directed evolution of halohydrin dehalogenase.

PubMed

Luo, Yu; Chen, Yangzi; Ma, Hongmin; Tian, ZhenHua; Zhang, Yeqi; Zhang, Jian

2017-02-06

Halohydrin dehalogenases (HHDHs) are biocatalytically interesting enzymes due to their ability to form C-C, C-N, C-O, and C-S bonds. One of most important application of HHDH was the protein engineering of HheC (halohydrin dehalogenase from Agrobacterium radiobacter AD1) for the industrial manufacturing of ethyl (R)-4-cyano-3-hydroxybutanoate (HN), a key chiral synthon of a cholesterol-lowering drug of atorvastatin. During our development of an alternative, more efficient and economic route for chemo-enzymatic preparation of the intermediate of atorvastatin, we found that the HheC2360 previously reported for HN manufacture, had insufficient activity for the cyanolysis production of tert-butyl (3 R,5 S)-6-cyano-3,5-dihydroxyhexanoate (A7). Herein, we present the focused directed evolution of HheC2360 with higher activity and enhanced biocatalytic performance using active site mutagenesis. Through docking of the product, A7, into the crystal structure of HheC2360, 6 residues was selected for combined active sites testing (CASTing). After library screening, the variant V84G/W86F was identified to have a 15- fold increase in activity. Time course analysis of the cyanolysis reaction catalyzed by this variant, showed 2- fold increase in space time productivity compared with HheC2360. These results demonstrate the applicability of the variant V84G/W86F as a biocatalyst for the efficient and practical production of atorvastatin intermediate.

Effects of point mutations on the thermostability of B. subtilis lipase: investigating nonadditivity

NASA Astrophysics Data System (ADS)

Singh, Bipin; Bulusu, Gopalakrishnan; Mitra, Abhijit

2016-10-01

Molecular level understanding of mutational effects on stability and activity of enzymes is challenging particularly when several point mutations are incorporated during the directed evolution experiments. In our earlier study, we have suggested the lack of consistency in the effect of point mutations incorporated during the initial generations of directed evolution experiments, towards conformational stabilization of B. subtilis lipase mutants of later generations. Here, we report that the cumulative point mutations incorporated in mutants 2M (with two point mutations) to 6M (with six point mutations) possibly do not retain their original stabilizing nature in the most thermostable 12M mutant (with 12 point mutations). We have carried out MD simulations using structures incorporating reversal of different sets of point mutations to assess their effect on the conformational stability and activity of 12M. Our analysis has revealed that reversal of certain point mutations in 12M had little effect on its conformational stability, suggesting that these mutations were probably inconsequential towards the thermostability of the 12M mutant. Interestingly these mutations involved evolutionarily conserved residues. On the other hand, some of the other point mutations incorporated in nonconserved regions, appeared to contribute significantly towards the conformational stability and/or activity of 12M. Based on the analysis of dynamics of in silico mutants generated using the consensus sequence, we identified experimentally verifiable residue positions to further increase the conformational stability and activity of the 12M mutant.

Protein Engineering Approaches in the Post-Genomic Era.

PubMed

Singh, Raushan K; Lee, Jung-Kul; Selvaraj, Chandrabose; Singh, Ranjitha; Li, Jinglin; Kim, Sang-Yong; Kalia, Vipin C

2018-01-01

Proteins are one of the most multifaceted macromolecules in living systems. Proteins have evolved to function under physiological conditions and, therefore, are not usually tolerant of harsh experimental and environmental conditions. The growing use of proteins in industrial processes as a greener alternative to chemical catalysts often demands constant innovation to improve their performance. Protein engineering aims to design new proteins or modify the sequence of a protein to create proteins with new or desirable functions. With the emergence of structural and functional genomics, protein engineering has been invigorated in the post-genomic era. The three-dimensional structures of proteins with known functions facilitate protein engineering approaches to design variants with desired properties. There are three major approaches of protein engineering research, namely, directed evolution, rational design, and de novo design. Rational design is an effective method of protein engineering when the threedimensional structure and mechanism of the protein is well known. In contrast, directed evolution does not require extensive information and a three-dimensional structure of the protein of interest. Instead, it involves random mutagenesis and selection to screen enzymes with desired properties. De novo design uses computational protein design algorithms to tailor synthetic proteins by using the three-dimensional structures of natural proteins and their folding rules. The present review highlights and summarizes recent protein engineering approaches, and their challenges and limitations in the post-genomic era. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion.

PubMed

Zhang, N; Stewart, B G; Moore, J C; Greasham, R L; Robinson, D K; Buckland, B C; Lee, C

2000-10-01

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.

Effects of point mutations on the thermostability of B. subtilis lipase: investigating nonadditivity.

PubMed

Singh, Bipin; Bulusu, Gopalakrishnan; Mitra, Abhijit

2016-10-01

Molecular level understanding of mutational effects on stability and activity of enzymes is challenging particularly when several point mutations are incorporated during the directed evolution experiments. In our earlier study, we have suggested the lack of consistency in the effect of point mutations incorporated during the initial generations of directed evolution experiments, towards conformational stabilization of B. subtilis lipase mutants of later generations. Here, we report that the cumulative point mutations incorporated in mutants 2M (with two point mutations) to 6M (with six point mutations) possibly do not retain their original stabilizing nature in the most thermostable 12M mutant (with 12 point mutations). We have carried out MD simulations using structures incorporating reversal of different sets of point mutations to assess their effect on the conformational stability and activity of 12M. Our analysis has revealed that reversal of certain point mutations in 12M had little effect on its conformational stability, suggesting that these mutations were probably inconsequential towards the thermostability of the 12M mutant. Interestingly these mutations involved evolutionarily conserved residues. On the other hand, some of the other point mutations incorporated in nonconserved regions, appeared to contribute significantly towards the conformational stability and/or activity of 12M. Based on the analysis of dynamics of in silico mutants generated using the consensus sequence, we identified experimentally verifiable residue positions to further increase the conformational stability and activity of the 12M mutant.

A global characterization and identification of multifunctional enzymes.

PubMed

Cheng, Xian-Ying; Huang, Wei-Juan; Hu, Shi-Chang; Zhang, Hai-Lei; Wang, Hao; Zhang, Jing-Xian; Lin, Hong-Huang; Chen, Yu-Zong; Zou, Quan; Ji, Zhi-Liang

2012-01-01

Multi-functional enzymes are enzymes that perform multiple physiological functions. Characterization and identification of multi-functional enzymes are critical for communication and cooperation between different functions and pathways within a complex cellular system or between cells. In present study, we collected literature-reported 6,799 multi-functional enzymes and systematically characterized them in structural, functional, and evolutionary aspects. It was found that four physiochemical properties, that is, charge, polarizability, hydrophobicity, and solvent accessibility, are important for characterization of multi-functional enzymes. Accordingly, a combinational model of support vector machine and random forest model was constructed, based on which 6,956 potential novel multi-functional enzymes were successfully identified from the ENZYME database. Moreover, it was observed that multi-functional enzymes are non-evenly distributed in species, and that Bacteria have relatively more multi-functional enzymes than Archaebacteria and Eukaryota. Comparative analysis indicated that the multi-functional enzymes experienced a fluctuation of gene gain and loss during the evolution from S. cerevisiae to H. sapiens. Further pathway analyses indicated that a majority of multi-functional enzymes were well preserved in catalyzing several essential cellular processes, for example, metabolisms of carbohydrates, nucleotides, and amino acids. What's more, a database of known multi-functional enzymes and a server for novel multi-functional enzyme prediction were also constructed for free access at http://bioinf.xmu.edu.cn/databases/MFEs/index.htm.

Structure and function of enzymes involved in the biosynthesis of phenylpropanoids

PubMed Central

Ferrer, J.-L.; Austin, M.B.; Stewart, C.; Noel, J.P.

2010-01-01

As a major component of plant specialized metabolism, phenylpropanoid biosynthetic pathways provide anthocyanins for pigmentation, flavonoids such as flavones for protection against UV photodamage, various flavonoid and isoflavonoid inducers of Rhizobium nodulation genes, polymeric lignin for structural support and assorted antimicrobial phytoalexins. As constituents of plant-rich diets and an assortment of herbal medicinal agents, the phenylpropanoids exhibit measurable cancer chemopreventive, antimitotic, estrogenic, antimalarial, antioxidant and antiasthmatic activities. The health benefits of consuming red wine, which contains significant amounts of 3,4′,5-trihydroxystilbene (resveratrol) and other phenylpropanoids, highlight the increasing awareness in the medical community and the public at large as to the potential dietary importance of these plant derived compounds. As recently as a decade ago, little was known about the three-dimensional structure of the enzymes involved in these highly branched biosynthetic pathways. Ten years ago, we initiated X-ray crystallographic analyses of key enzymes of this pathway, complemented by biochemical and enzyme engineering studies. We first investigated chalcone synthase (CHS), the entry point of the flavonoid pathway, and its close relative stilbene synthase (STS). Work soon followed on the O-methyl transferases (OMTs) involved in modifications of chalcone, isoflavonoids and metabolic precursors of lignin. More recently, our groups and others have extended the range of phenylpropanoid pathway structural investigations to include the upstream enzymes responsible for the initial recruitment of phenylalanine and tyrosine, as well as a number of reductases, acyltransferases and ancillary tailoring enzymes of phenylpropanoid-derived metabolites. These structure–function studies collectively provide a comprehensive view of an important aspect of phenylpropanoid metabolism. More specifically, these atomic resolution insights into the architecture and mechanistic underpinnings of phenylpropanoid metabolizing enzymes contribute to our understanding of the emergence and on-going evolution of specialized phenylpropanoid products, and underscore the molecular basis of metabolic biodiversity at the chemical level. Finally, the detailed knowledge of the structure, function and evolution of these enzymes of specialized metabolism provide a set of experimental templates for the enzyme and metabolic engineering of production platforms for diverse novel compounds with desirable dietary and medicinal properties. PMID:18272377

Molecular Evolution of Phosphoprotein Phosphatases in Drosophila

PubMed Central

Miskei, Márton; Ádám, Csaba; Kovács, László; Karányi, Zsolt; Dombrádi, Viktor

2011-01-01

Phosphoprotein phosphatases (PPP), these ancient and important regulatory enzymes are present in all eukaryotic organisms. Based on the genome sequences of 12 Drosophila species we traced the evolution of the PPP catalytic subunits and noted a substantial expansion of the gene family. We concluded that the 18–22 PPP genes of Drosophilidae were generated from a core set of 8 indispensable phosphatases that are present in most of the insects. Retropositons followed by tandem gene duplications extended the phosphatase repertoire, and sporadic gene losses contributed to the species specific variations in the PPP complement. During the course of these studies we identified 5, up till now uncharacterized phosphatase retrogenes: PpY+, PpD5+, PpD6+, Pp4+, and Pp6+ which are found only in some ancient Drosophila. We demonstrated that all of these new PPP genes exhibit a distinct male specific expression. In addition to the changes in gene numbers, the intron-exon structure and the chromosomal localization of several PPP genes was also altered during evolution. The G−C content of the coding regions decreased when a gene moved into the heterochromatic region of chromosome Y. Thus the PPP enzymes exemplify the various types of dynamic rearrangements that accompany the molecular evolution of a gene family in Drosophilidae. PMID:21789237

Evolution in vitro of an RNA enzyme with altered metal dependence

NASA Technical Reports Server (NTRS)

Lehman, N.; Joyce, G. F.

1993-01-01

The Tetrahymena group I ribozyme catalyses a sequence-specific phosphodiester cleavage reaction on an external RNA oligonucleotide substrate in the presence of a divalent metal cation cofactor. This reaction proceeds readily with either Mg2+ or Mn2+, but no detectable reaction has been reported when other divalent cations are used as the sole cofactor. Cations such as Ca2+, Sr2+ and Ba2+ can stabilize the correct folded conformation of the ribozyme, thereby partially alleviating the Mg2+ or Mn2+ requirement. But catalysis by the ribozyme involves coordination of either Mg2+ or Mn2+ at the active site, resulting in an overall requirement for one of these two cations. Here we use an in vitro evolution process to obtain variants of the Tetrahymena ribozyme that are capable of cleaving an RNA substrate in reaction mixtures containing Ca2+ as the divalent cation. These findings extend the range of different chemical environments available to RNA enzymes and illustrate the power of in vitro evolution in generating macromolecular catalysts with desired properties.

Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schenck, Craig A.; Holland, Cynthia K.; Schneider, Matthew R.

L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrAp) or arogenate dehydrogenase (ADH, also known as TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that definesmore » TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.« less

Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function

PubMed Central

Lisi, George P.; Loria, J. Patrick

2015-01-01

Conformational motions play an essential role in enzyme function, often facilitating the formation of enzyme-substrate complexes and/or product release. Although considerable debate remains regarding the role of molecular motions in the conversion of enzymatic substrates to products, numerous examples have found motions to be crucial for optimization of enzyme scaffolds, effective substrate binding, and product dissociation. Conformational fluctuations are often rate-limiting to enzyme catalysis, primarily through product release, with the chemical reaction occurring much more quickly. As a result, the direct involvement of motions at various stages along the enzyme reaction coordinate remains largely unknown and untested. In the following review, we describe the use of solution NMR techniques designed to probe various timescales of molecular motions and detail examples in which motions play a role in propagating catalytic effects from the active site and directly participate in essential aspects of enzyme function. PMID:26952190

SpyRings Declassified: A Blueprint for Using Isopeptide-Mediated Cyclization to Enhance Enzyme Thermal Resilience.

PubMed

Schoene, C; Bennett, S P; Howarth, M

2016-01-01

Enzymes often have marginal stability, with unfolding typically leading to irreversible denaturation. This sensitivity is a major barrier, both for de novo enzyme development and for expanding enzyme impact beyond the laboratory. Seeking an approach to enhance resilience to denaturation that could be applied to a range of different enzymes, we developed SpyRing cyclization. SpyRings contain genetically encoded SpyTag (13 amino acids) on the N-terminus and SpyCatcher (12kDa) on the C-terminus of the enzyme, so that the Spy partners spontaneously react together through an irreversible isopeptide bond. SpyRing cyclization gave major increases in thermal resilience, including on a model for enzyme evolution, β-lactamase, and an industrially important enzyme in agriculture and nutrition, phytase. We outline the SpyRing rationale, including comparison of SpyRing cyclization to other cyclization strategies. The cloning strategy is presented for the simple insertion of enzyme genes for recombinant expression. We discuss structure-based approaches to select suitable enzyme cyclization targets. Approaches to evaluate the cyclization reaction and its effect on enzyme resilience are described. We also highlight the use of differential scanning calorimetry to understand how SpyRing cyclization promotes enzyme refolding. Efficiently searching sequence space will continue to be important for enzyme improvement, but the SpyRing platform may be a valuable rational adjunct for conferring resilience. © 2016 Elsevier Inc. All rights reserved.

[NiFeSe]-hydrogenase chemistry.

PubMed

Wombwell, Claire; Caputo, Christine A; Reisner, Erwin

2015-11-17

The development of technology for the inexpensive generation of the renewable energy vector H2 through water splitting is of immediate economic, ecological, and humanitarian interest. Recent interest in hydrogenases has been fueled by their exceptionally high catalytic rates for H2 production at a marginal overpotential, which is presently only matched by the nonscalable noble metal platinum. The mechanistic understanding of hydrogenase function guides the design of synthetic catalysts, and selection of a suitable hydrogenase enables direct applications in electro- and photocatalysis. [FeFe]-hydrogenases display excellent H2 evolution activity, but they are irreversibly damaged upon exposure to O2, which currently prevents their use in full water splitting systems. O2-tolerant [NiFe]-hydrogenases are known, but they are typically strongly biased toward H2 oxidation, while H2 production by [NiFe]-hydrogenases is often product (H2) inhibited. [NiFeSe]-hydrogenases are a subclass of [NiFe]-hydrogenases with a selenocysteine residue coordinated to the active site nickel center in place of a cysteine. They exhibit a combination of unique properties that are highly advantageous for applications in water splitting compared with other hydrogenases. They display a high H2 evolution rate with marginal inhibition by H2 and tolerance to O2. [NiFeSe]-hydrogenases are therefore one of the most active molecular H2 evolution catalysts applicable in water splitting. Herein, we summarize our recent progress in exploring the unique chemistry of [NiFeSe]-hydrogenases through biomimetic model chemistry and the chemistry with [NiFeSe]-hydrogenases in semiartificial photosynthetic systems. We gain perspective from the structural, spectroscopic, and electrochemical properties of the [NiFeSe]-hydrogenases and compare them with the chemistry of synthetic models of this hydrogenase active site. Our synthetic models give insight into the effects on the electronic properties and reactivity of the active site upon the introduction of selenium. We have utilized the exceptional properties of the [NiFeSe]-hydrogenase from Desulfomicrobium baculatum in a number of photocatalytic H2 production schemes, which are benchmark systems in terms of single site activity, tolerance toward O2, and in vitro water splitting with biological molecules. Each system comprises a light-harvesting component, which allows for light-driven electron transfer to the hydrogenase in order for it to catalyze H2 production. A system with [NiFeSe]-hydrogenase on a dye-sensitized TiO2 nanoparticle gives an enzyme-semiconductor hybrid for visible light-driven generation of H2 with an enzyme-based turnover frequency of 50 s(-1). A stable and inexpensive polymeric carbon nitride as a photosensitizer in combination with the [NiFeSe]-hydrogenase shows good activity for more than 2 days. Light-driven H2 evolution with the enzyme and an organic dye under high O2 levels demonstrates the excellent robustness and feasibility of water splitting with a hydrogenase-based scheme. This has led, most recently, to the development of a light-driven full water splitting system with a [NiFeSe]-hydrogenase wired to the water oxidation enzyme photosystem II in a photoelectrochemical cell. In contrast to the other systems, this photoelectrochemical system does not rely on a sacrificial electron donor and allowed us to establish the long sought after light-driven water splitting with an isolated hydrogenase.

Molecular relationships between closely related strains and species of nematodes

NASA Technical Reports Server (NTRS)

Butler, M. H.; Wall, S. M.; Luehrsen, K. R.; Fox, G. E.; Hecht, R. M.

1981-01-01

Electrophoretic comparisons have been made for 24 enzymes in the Bergerac and Bristol strains of Caenorhabditis elegans and the related species, Caenorhabditis briggsae. No variation was detected between the two strains of C. elegans. In contrast, the two species, C. elegans and C. briggsae exhibited electrophoretic differences in 22 of 24 enzymes. A consensus 5S rRNA sequence was determined for C. elegans and found to be identical to that from C. briggsae. By analogy with other species with relatively well established fossil records it can be inferred that the time of divergence between the two nematode species is probably in the tens of millions of years. The limited anatomical evolution during a time period in which proteins undergo extensive changes supports the hypothesis that anatomical evolution is not dependent on overall protein changes.

Rapid Evolution of Sex Pheromone-Producing Enzyme Expression in Drosophila

PubMed Central

Williams, Thomas M.; Carroll, Sean B.

2009-01-01

A wide range of organisms use sex pheromones to communicate with each other and to identify appropriate mating partners. While the evolution of chemical communication has been suggested to cause sexual isolation and speciation, the mechanisms that govern evolutionary transitions in sex pheromone production are poorly understood. Here, we decipher the molecular mechanisms underlying the rapid evolution in the expression of a gene involved in sex pheromone production in Drosophilid flies. Long-chain cuticular hydrocarbons (e.g., dienes) are produced female-specifically, notably via the activity of the desaturase DESAT-F, and are potent pheromones for male courtship behavior in Drosophila melanogaster. We show that across the genus Drosophila, the expression of this enzyme is correlated with long-chain diene production and has undergone an extraordinary number of evolutionary transitions, including six independent gene inactivations, three losses of expression without gene loss, and two transitions in sex-specificity. Furthermore, we show that evolutionary transitions from monomorphism to dimorphism (and its reversion) in desatF expression involved the gain (and the inactivation) of a binding-site for the sex-determination transcription factor, DOUBLESEX. In addition, we documented a surprising example of the gain of particular cis-regulatory motifs of the desatF locus via a set of small deletions. Together, our results suggest that frequent changes in the expression of pheromone-producing enzymes underlie evolutionary transitions in chemical communication, and reflect changing regimes of sexual selection, which may have contributed to speciation among Drosophila. PMID:19652700

21 CFR 173.110 - Amyloglucosidase derived from Rhizopus niveus.

Code of Federal Regulations, 2014 CFR

2014-04-01

... SERVICES (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme... enzyme product, consisting of enzyme derived from Rhizopus niveus, and diatomaceous silica as a carrier... enzyme is produced by a process which completely removes the organism Rhizopus niveus from the...

Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

PubMed

Altman, Amnon; Kong, Kok-Fai

2016-05-20

The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

A DNA enzyme with N-glycosylase activity

NASA Technical Reports Server (NTRS)

Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F.

2000-01-01

In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

Evolution of TEM-type enzymes: biochemical and genetic characterization of two new complex mutant TEM enzymes, TEM-151 and TEM-152, from a single patient.

PubMed

Robin, Frédéric; Delmas, Julien; Schweitzer, Cédric; Tournilhac, Olivier; Lesens, Olivier; Chanal, Catherine; Bonnet, Richard

2007-04-01

Two clinical isolates of Escherichia coli, CF1179 and CF1295, were isolated from a patient hospitalized in the hematology unit of the University Hospital of Clermont-Ferrand, Clermont-Ferrand, France. They were resistant to penicillin-clavulanate combinations and to ceftazidime. The double-disk synergy test was positive only for isolate CF1179. Molecular comparison of the isolates showed that they were clonally related. E. coli recombinant strains exhibiting the resistance phenotype of the clinical strains were obtained by cloning. The clones corresponding to strains CF1179 and CF1295 produced TEM-type beta-lactamases with pI values of 5.7 and 5.3, respectively. Sequencing analysis revealed two novel blaTEM genes encoding closely related complex mutant TEM enzymes, designated TEM-151 (pI 5.3) and TEM-152 (pI 5.7). These two genes also harbored a new promoter region which presented a 9-bp deletion. The two novel beta-lactamases differed from the parental enzyme, TEM-1, by the substitution Arg164His, previously observed in extended-spectrum beta-lactamases (ESBLs), and by the substitutions Met69Val and Asn276Asp, previously observed in the inhibitor-resistant penicillinase TEM-36/IRT-7. They differed by two amino acid substitutions: TEM-152 harbored a Glu240Lys ESBL-type substitution and TEM-151 had an Ala284Gly substitution. Functional analysis of TEM-151 and TEM-152 showed that both enzymes had hydrolytic activity against ceftazidime (kcat, 5 and 16 s-1, respectively). TEM-152 was more resistant than TEM-151 to the inhibitor clavulanic acid (50% inhibitory concentrations, 1 versus 0.17 microM). These results confirm the evolution of TEM-type enzymes toward complex enzymes harboring the two kinds of substitutions which confer an extended spectrum of action against beta-lactam antibiotics and resistance to inhibitors.

Dynamic Reorganization of Metabolic Enzymes into Intracellular Bodies

PubMed Central

O’Connell, Jeremy D.; Zhao, Alice; Ellington, Andrew D.; Marcotte, Edward M.

2013-01-01

Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci—such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis—to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality—and dysfunctionality—of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable. PMID:23057741

Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

NASA Technical Reports Server (NTRS)

Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

2016-01-01

Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

Independent Evolution of Six Families of Halogenating Enzymes

PubMed Central

Xu, Gangming; Wang, Bin-Gui

2016-01-01

Halogenated natural products are widespread in the environment, and the halogen atoms are typically vital to their bioactivities. Thus far, six families of halogenating enzymes have been identified: cofactor-free haloperoxidases (HPO), vanadium-dependent haloperoxidases (V-HPO), heme iron-dependent haloperoxidases (HI-HPO), non-heme iron-dependent halogenases (NI-HG), flavin-dependent halogenases (F-HG), and S-adenosyl-L-methionine (SAM)-dependent halogenases (S-HG). However, these halogenating enzymes with similar biological functions but distinct structures might have evolved independently. Phylogenetic and structural analyses suggest that the HPO, V-HPO, HI-HPO, NI-HG, F-HG, and S-HG enzyme families may have evolutionary relationships to the α/β hydrolases, acid phosphatases, peroxidases, chemotaxis phosphatases, oxidoreductases, and SAM hydroxide adenosyltransferases, respectively. These halogenating enzymes have established sequence homology, structural conservation, and mechanistic features within each family. Understanding the distinct evolutionary history of these halogenating enzymes will provide further insights into the study of their catalytic mechanisms and halogenation specificity. PMID:27153321

[Cutaneo-viscero-hemolytic loxoscelism with acute renal failure].

PubMed

Alfaro, Flavia V; Dotto, Beatriz; Sesin, Ana M; Prettini, Viviana; Sesin, Jorge; Aliciardi, Enrique; Vergottini, Juan C; Gonzalez, Mauricio

2008-01-01

The Loxoscelism is caused by the bite of spider Loxosceles laeta gender, of worldwide distribution. The poisoning can cause lesions dermonecrotic and less frequently a systemic illness that can be fatal. The mechanism of venom action is multifactorial. The characteristic dermonecrotic lesion results from the direct effects of the venom on the celular and basal membrane components, as well as the extracelular matrix. The initial interaction between the poison and tissues, causes complement activation, migration of polymorphic neutrophils, liberation of proteolytic enzymes, cytoquines, aggregation platelet, and blood flow alterations that result in edema and ischemia, with development of necrosis. There is no a definitive treatment for loxoscelism. However, the value of specific antivenom, to decrease lesion size and limit systemic illness even when such administration is delayed. We present a case of cutaneous-visceral loxoscelismo with unfavorable evolution.

Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases.

PubMed

Meziane-Cherif, Djalal; Stogios, Peter J; Evdokimova, Elena; Egorova, Olga; Savchenko, Alexei; Courvalin, Patrice

2015-08-11

Vancomycin resistance in Gram-positive bacteria results from the replacement of the D-alanyl-D-alanine target of peptidoglycan precursors with D-alanyl-D-lactate or D-alanyl-D-serine (D-Ala-D-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of D-Ala-D-Ser-terminating precursors by converting L-Ser to D-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in L-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5'-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the L-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against L-Ser versus L-Ala implied that this enzyme relies on its membrane-bound domain for L-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria. Vancomycin is one of the drugs of last resort against Gram-positive antibiotic-resistant pathogens. However, bacteria have evolved a sophisticated mechanism which remodels the drug target, the D-alanine ending precursors in cell wall synthesis, into precursors terminating with D-lactate or D-serine, to which vancomycin has less affinity. D-Ser is synthesized by VanT serine racemase, which has two unusual characteristics: (i) it is one of the few serine racemases identified in bacteria and (ii) it contains a membrane-bound domain involved in L-Ser uptake. The structure of the catalytic domain of VanTG showed high similarity to alanine racemases, and we identified three specific active site substitutions responsible for L-Ser specificity. The data provide the molecular basis for VanT evolution to a bifunctional enzyme coordinating both transport and racemization. Our findings also illustrate the evolution of the essential alanine racemase into a vancomycin resistance enzyme in response to antibiotic pressure. Copyright © 2015 Meziane-Cherif et al.

Enzymatic aspects in ENT cancer-Matrix metalloproteinases

PubMed Central

Zamfir Chiru, AA; Popescu, CR; Gheorghe, DC

2014-01-01

Abstract The study of ENT cancer allows the implementation of molecular biology methods in diagnosis, predicting the evolution of the disease and suggesting a certain treatment. MMPs are proteolytic enzymes, zinc dependent endopeptidases, secreted by tissues and proinflammatory cells that play a role in the clearance of cell surface receptors. They are expressed as zymogens (inactive forms). Proteolytic enzymes cleave zymogens generating active forms. They are involved in cell proliferation, adhesion, differentiation, migration, angiogenesis, apoptosis and host defense. PMID:25408759

Restoration of areas degraded by alluvial sand mining: use of soil microbiological activity and plant biomass growth to assess evolution of restored riparian vegetation.

PubMed

Venson, Graziela R; Marenzi, Rosemeri C; Almeida, Tito César M; Deschamps-Schmidt, Alexandre; Testolin, Renan C; Rörig, Leonardo R; Radetski, Claudemir M

2017-03-01

River or alluvial sand mining is causing a variety of environmental problems in the Itajaí-açú river basin in Santa Catarina State (south of Brazil). When this type of commercial activity degrades areas around rivers, environmental restoration programs need to be executed. In this context, the aim of this study was to assess the evolution of a restored riparian forest based on data on the soil microbial activity and plant biomass growth. A reference site and three sites with soil degradation were studied over a 3-year period. Five campaigns were performed to determine the hydrolysis of the soil enzyme fluorescein diacetate (FDA), and the biomass productivity was determined at the end of the studied period. The variation in the enzyme activity for the different campaigns at each site was low, but this parameter did differ significantly according to the site. Well-managed sites showed the highest biomass productivity, and this, in turn, showed a strong positive correlation with soil enzyme activity. In conclusion, soil enzyme activity could form the basis for monitoring and the early prediction of the success of vegetal restoration programs, since responses at the higher level of biological organization take longer, inhibiting the assessment of the project within an acceptable time frame.

FunTree: advances in a resource for exploring and contextualising protein function evolution.

PubMed

Sillitoe, Ian; Furnham, Nicholas

2016-01-04

FunTree is a resource that brings together protein sequence, structure and functional information, including overall chemical reaction and mechanistic data, for structurally defined domain superfamilies. Developed in tandem with the CATH database, the original FunTree contained just 276 superfamilies focused on enzymes. Here, we present an update of FunTree that has expanded to include 2340 superfamilies including both enzymes and proteins with non-enzymatic functions annotated by Gene Ontology (GO) terms. This allows the investigation of how novel functions have evolved within a structurally defined superfamily and provides a means to analyse trends across many superfamilies. This is done not only within the context of a protein's sequence and structure but also the relationships of their functions. New measures of functional similarity have been integrated, including for enzymes comparisons of overall reactions based on overall bond changes, reaction centres (the local environment atoms involved in the reaction) and the sub-structure similarities of the metabolites involved in the reaction and for non-enzymes semantic similarities based on the GO. To identify and highlight changes in function through evolution, ancestral character estimations are made and presented. All this is accessible through a new re-designed web interface that can be found at http://www.funtree.info. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Digestive peptidase evolution in holometabolous insects led to a divergent group of enzymes in Lepidoptera.

PubMed

Dias, Renata O; Via, Allegra; Brandão, Marcelo M; Tramontano, Anna; Silva-Filho, Marcio C

2015-03-01

Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic L-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera. Copyright © 2015 Elsevier Ltd. All rights reserved.

Catalysis by a de novo zinc-mediated protein interface: implications for natural enzyme evolution and rational enzyme engineering.

PubMed

Der, Bryan S; Edwards, David R; Kuhlman, Brian

2012-05-08

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

Biochemical characterization in Norway spruce (Picea abies) of SABATH methyltransferases that methylate phytohormones.

PubMed

Chaiprasongsuk, Minta; Zhang, Chi; Qian, Ping; Chen, Xinlu; Li, Guanglin; Trigiano, Robert N; Guo, Hong; Chen, Feng

2018-05-01

Indole-3-acetic acid (IAA), gibberellins (GAs), salicylic acid (SA) and jasmonic acid (JA) exist in methyl ester forms in plants in addition to their free acid forms. The enzymes that catalyze methylation of these carboxylic acid phytohormones belong to a same protein family, the SABATH methyltransferases. While the genes encoding these enzymes have been isolated from a small number of flowering plants, little is known about their occurrence and evolution in non-flowering plants. Here, we report the systematic characterization of the SABATH family from Norway spruce (Picea abies), a gymnosperm. The Norway spruce genome contains ten SABATH genes (PaSABATH1-10). Full-length cDNA for each of the ten PaSABATH genes was cloned and expressed in Escherichia coli. Recombinant PaSABATHs were tested for activity with IAA, GA, SA, and JA. Among the ten PaSABATHs, five had activity with one or more of the four substrates. PaSABATH1 and PaSABATH2 had the highest activities with IAA and SA, respectively. PaSABATH4, PaSABATH5 and PaSABATH10 all had JA as a preferred substrate but with notable differences in biochemical properties. The structural basis of PaSABATHs in discriminating various phytohormone substrates was inferred based on structural models of the enzyme-substrate complexes. The phylogeny of PaSABATHs with selected SABATHs from other plants implies that the enzymes methylating IAA are conserved in seed plants whereas the enzymes methylating JA and SA have independent evolution in gymnosperms and angiosperms. Copyright © 2018 Elsevier Ltd. All rights reserved.

An evolutionary insight into the hatching strategies of pipefish and seahorse embryos.

PubMed

Kawaguchi, Mari; Nakano, Yuko; Kawahara-Miki, Ryouka; Inokuchi, Mayu; Yorifuji, Makiko; Okubo, Ryohei; Nagasawa, Tatsuki; Hiroi, Junya; Kono, Tomohiro; Kaneko, Toyoji

2016-03-01

Syngnathiform fishes carry their eggs in a brood structure found in males. The brood structure differs from species to species: seahorses carry eggs within enclosed brood pouch, messmate pipefish carry eggs in the semi-brood pouch, and alligator pipefish carry eggs in the egg compartment on abdomen. These egg protection strategies were established during syngnathiform evolution. In the present study, we compared the hatching mode of protected embryos of three species. Electron microscopic observations revealed that alligator pipefish and messmate pipefish egg envelopes were thicker than those of seahorses, suggesting that the seahorse produces a weaker envelope. Furthermore, molecular genetic analysis revealed that these two pipefishes possessed the egg envelope-digesting enzymes, high choriolytic enzyme (HCE), and low choriolytic enzyme (LCE), as do many euteleosts. In seahorses, however, only HCE gene expression was detected. When searching the entire seahorse genome by high-throughput DNA sequencing, we did not find a functional LCE gene and only a trace of the LCE gene exon was found, confirming that the seahorse LCE gene was pseudogenized during evolution. Finally, we estimated the size and number of hatching gland cells expressing hatching enzyme genes by whole-mount in situ hybridization. The seahorse cells were the smallest of the three species, while they had the greatest number. These results suggest that the isolation of eggs from the external environment by paternal bearing might bring the egg envelope thin, and then, the hatching enzyme genes became pseudogenized. J. Exp. Zool. (Mol. Dev. Evol.) 9999B:XX-XX, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Search for the Evolution of Steroid Biosynthesis in the Geological Record

NASA Astrophysics Data System (ADS)

Brocks, J. J.

2004-12-01

To study the evolution of the structure of organisms we can directly examine fossilized shells, skeletons and petrified cells. In contrast, for the tentative reconstruction of the phylogeny of biosynthetic pathways, such as steroid anabolism, we rely entirely on the comparative molecular biology of living organisms. Thus, without fossil evidence, the times in geological history when successive steps of a metabolic pathway evolved remain particularly elusive. Molecular clocks of genes coding for the enzymes involved in a biosynthetic pathway might provide a rough guess when a natural product first appeared in geological time, but they are intrinsically unreliable without calibration points in the distant past. However, it might be possible to trace the evolutionary history of some biosynthetic pathways directly in the geological record by searching for hydrocarbon biomarkers of anabolic intermediates. Biomarkers are molecular fossils of natural products. They often retain the diagnostic carbon skeleton of their biological precursor and remain stable over hundreds of millions of years enclosed in organic-rich sedimentary rocks. Sterane hydrocarbons are particularly abundant biomarkers and potentially suitable for the search of biosynthetic intermediates. Steranes are the fossil equivalents of functionalized steroids found in eukaryotes and certain bacteria. The biosynthesis of typical eukaryotic steroids such as cholesterol (C27), ergosterol (C28) and sitosterol (C29) from the acyclic precursor squalene (C30) involves more than 20 enzymatic steps. The most crucial steps include modification of the carbon skeleton by removal of several methyl groups from the ring system and addition of alkyl groups to the steroid side chain. The evolution of this complex pathway must have occurred over geologically significant periods of time and likely involved several preadaptive intermediates that represented structurally less derived but fully functional lipids. Thus, if a molecular corollary of `ontogeny recapitulates phylogeny' applies, it might be possible to detect a sequence of increasingly modified fossil steroids in the geological record and to create a time frame for the evolution of this fundamental biosynthetic pathway. Here we present first results of an extensive search for the fossil remains of evolutionary intermediate steroids in sedimentary successions of Precambrian age.

When does female multiple mating evolve to adjust inbreeding? Effects of inbreeding depression, direct costs, mating constraints, and polyandry as a threshold trait.

PubMed

Duthie, A Bradley; Bocedi, Greta; Reid, Jane M

2016-09-01

Polyandry is often hypothesized to evolve to allow females to adjust the degree to which they inbreed. Multiple factors might affect such evolution, including inbreeding depression, direct costs, constraints on male availability, and the nature of polyandry as a threshold trait. Complex models are required to evaluate when evolution of polyandry to adjust inbreeding is predicted to arise. We used a genetically explicit individual-based model to track the joint evolution of inbreeding strategy and polyandry defined as a polygenic threshold trait. Evolution of polyandry to avoid inbreeding only occurred given strong inbreeding depression, low direct costs, and severe restrictions on initial versus additional male availability. Evolution of polyandry to prefer inbreeding only occurred given zero inbreeding depression and direct costs, and given similarly severe restrictions on male availability. However, due to its threshold nature, phenotypic polyandry was frequently expressed even when strongly selected against and hence maladaptive. Further, the degree to which females adjusted inbreeding through polyandry was typically very small, and often reflected constraints on male availability rather than adaptive reproductive strategy. Evolution of polyandry solely to adjust inbreeding might consequently be highly restricted in nature, and such evolution cannot necessarily be directly inferred from observed magnitudes of inbreeding adjustment. © 2016 The Author(s). Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.

EVOLUTION OF ENVIRONMENTAL IMMUNOCHEMISTRY

EPA Science Inventory

Enzyme-linked immunosorbent assays (ELISAs), initially developed for clinical applications, have made a tremendous impact as clinical diagnostic indicators. Pesticide chemists became attracted to the potential of these sensitive and selective methods in the 1970s. Thus, beg...

Science Notes.

ERIC Educational Resources Information Center

School Science Review, 1990

1990-01-01

Presented are 25 science activities on colorations of prey, evolution, blood, physiology, nutrition, enzyme kinetics, leaf pigments, analytical chemistry, milk, proteins, fermentation, surface effects of liquids, magnetism, drug synthesis, solvents, wintergreen synthesis, chemical reactions, multicore cables, diffraction, air resistance,…

The Causality of Evolution on Different Fitness Landscapes

NASA Astrophysics Data System (ADS)

Vyawahare, Saurabh; Austin, Robert; Zhang, Qiucen; Kim, Hyunsung; Bestoso, John

2013-03-01

Evolution of antibiotic resistance is a growing problem. One major reason why most antibiotics fail is because of mutations on drug targets (e.g. essential enzymes). Sequencing of clinically resistant isolates have shown that multiple mutational-hotspots exist in coding regions, which could potentially prohibit the binding of drugs. However, it is not clear whether the appearance of each mutation is random or influenced by other factors. In this paper, we compare evolution of resistance to ciprofloxacin from two distinct but well characterized genetic backgrounds. By combining our recently developed evolution reactor and deep whole-genome sequencing, we show different alleles of σs factor lead to fixation of different mutations in gyrA gene that confer ciprofloxacin resistance to bacteria Escherichia coli. Such causality of evolution in different genes provides an opportunity to control the evolution of antibiotic resistance. Sponsored by the NCI/NIH Physical Sciences Oncology Centers

Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production

PubMed Central

Trollope, K. M.; Görgens, J. F.

2015-01-01

The Aspergillus japonicus β-fructofuranosidase catalyzes the industrially important biotransformation of sucrose to fructooligosaccharides. Operating at high substrate loading and temperatures between 50 and 60°C, the enzyme activity is negatively influenced by glucose product inhibition and thermal instability. To address these limitations, the solvent-exposed loop regions of the β-fructofuranosidase were engineered using a combined crystal structure- and evolutionary-guided approach. This semirational approach yielded a functionally enriched first-round library of 36 single-amino-acid-substitution variants with 58% retaining activity, and of these, 71% displayed improved activities compared to the parent. The substitutions yielding the five most improved variants subsequently were exhaustively combined and evaluated. A four-substitution combination variant was identified as the most improved and reduced the time to completion of an efficient industrial-like reaction by 22%. Characterization of the top five combination variants by isothermal denaturation assays indicated that these variants displayed improved thermostability, with the most thermostable variant displaying a 5.7°C increased melting temperature. The variants displayed uniquely altered, concentration-dependent substrate and product binding as determined by differential scanning fluorimetry. The altered catalytic activity was evidenced by increased specific activities of all five variants, with the most improved variant doubling that of the parent. Variant homology modeling and computational analyses were used to rationalize the effects of amino acid changes lacking direct interaction with substrates. Data indicated that targeting substitutions to loop regions resulted in improved enzyme thermostability, specific activity, and relief from product inhibition. PMID:26253664

Engineered Proteins: Redox Properties and Their Applications

PubMed Central

Prabhulkar, Shradha; Tian, Hui; Wang, Xiaotang; Zhu, Jun-Jie

2012-01-01

Abstract Oxidoreductases and metalloproteins, representing more than one third of all known proteins, serve as significant catalysts for numerous biological processes that involve electron transfers such as photosynthesis, respiration, metabolism, and molecular signaling. The functional properties of the oxidoreductases/metalloproteins are determined by the nature of their redox centers. Protein engineering is a powerful approach that is used to incorporate biological and abiological redox cofactors as well as novel enzymes and redox proteins with predictable structures and desirable functions for important biological and chemical applications. The methods of protein engineering, mainly rational design, directed evolution, protein surface modifications, and domain shuffling, have allowed the creation and study of a number of redox proteins. This review presents a selection of engineered redox proteins achieved through these methods, resulting in a manipulation in redox potentials, an increase in electron-transfer efficiency, and an expansion of native proteins by de novo design. Such engineered/modified redox proteins with desired properties have led to a broad spectrum of practical applications, ranging from biosensors, biofuel cells, to pharmaceuticals and hybrid catalysis. Glucose biosensors are one of the most successful products in enzyme electrochemistry, with reconstituted glucose oxidase achieving effective electrical communication with the sensor electrode; direct electron-transfer-type biofuel cells are developed to avoid thermodynamic loss and mediator leakage; and fusion proteins of P450s and redox partners make the biocatalytic generation of drug metabolites possible. In summary, this review includes the properties and applications of the engineered redox proteins as well as their significance and great potential in the exploration of bioelectrochemical sensing devices. Antioxid. Redox Signal. 17, 1796–1822. PMID:22435347

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Code of Federal Regulations, 2014 CFR

2014-04-01

... (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme... animals. (c) The enzyme is produced by a process which completely removes the organism Mucor miehei var...

Transition-State Ensembles Navigate the Pathways of Enzyme Catalysis.

PubMed

Mickert, Matthias J; Gorris, Hans H

2018-06-07

Transition-state theory (TST) provides an important framework for analyzing and explaining the reaction rates of enzymes. TST, however, needs to account for protein dynamic effects and heterogeneities in enzyme catalysis. We have analyzed the reaction rates of β-galactosidase and β-glucuronidase at the single molecule level by using large arrays of femtoliter-sized chambers. Heterogeneities in individual reaction rates yield information on the intrinsic distribution of the free energy of activation (Δ G ‡ ) in an enzyme ensemble. The broader distribution of Δ G ‡ in β-galactosidase compared to β-glucuronidase is attributed to β-galactosidase's multiple catalytic functions as a hydrolase and a transglycosylase. Based on the catalytic mechanism of β-galactosidase, we show that transition-state ensembles do not only contribute to enzyme catalysis but can also channel the catalytic pathway to the formation of different products. We conclude that β-galactosidase is an example of natural evolution, where a new catalytic pathway branches off from an established enzyme function. The functional division of work between enzymatic substates explains why the conformational space represented by the enzyme ensemble is larger than the conformational space that can be sampled by any given enzyme molecule during catalysis.

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

PubMed

Beneyton, Thomas; Thomas, Stéphane; Griffiths, Andrew D; Nicaud, Jean-Marc; Drevelle, Antoine; Rossignol, Tristan

2017-01-31

Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement). Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (10 5 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.

Lignin degradation: microorganisms, enzymes involved, genomes analysis and evolution.

PubMed

Janusz, Grzegorz; Pawlik, Anna; Sulej, Justyna; Swiderska-Burek, Urszula; Jarosz-Wilkolazka, Anna; Paszczynski, Andrzej

2017-11-01

Extensive research efforts have been dedicated to describing degradation of wood, which is a complex process; hence, microorganisms have evolved different enzymatic and non-enzymatic strategies to utilize this plentiful plant material. This review describes a number of fungal and bacterial organisms which have developed both competitive and mutualistic strategies for the decomposition of wood and to thrive in different ecological niches. Through the analysis of the enzymatic machinery engaged in wood degradation, it was possible to elucidate different strategies of wood decomposition which often depend on ecological niches inhabited by given organism. Moreover, a detailed description of low molecular weight compounds is presented, which gives these organisms not only an advantage in wood degradation processes, but seems rather to be a new evolutionatory alternative to enzymatic combustion. Through analysis of genomics and secretomic data, it was possible to underline the probable importance of certain wood-degrading enzymes produced by different fungal organisms, potentially giving them advantage in their ecological niches. The paper highlights different fungal strategies of wood degradation, which possibly correlates to the number of genes coding for secretory enzymes. Furthermore, investigation of the evolution of wood-degrading organisms has been described. © FEMS 2017.

Lignin degradation: microorganisms, enzymes involved, genomes analysis and evolution

PubMed Central

Pawlik, Anna; Sulej, Justyna; Świderska-Burek, Urszula; Jarosz-Wilkołazka, Anna; Paszczyński, Andrzej

2017-01-01

Abstract Extensive research efforts have been dedicated to describing degradation of wood, which is a complex process; hence, microorganisms have evolved different enzymatic and non-enzymatic strategies to utilize this plentiful plant material. This review describes a number of fungal and bacterial organisms which have developed both competitive and mutualistic strategies for the decomposition of wood and to thrive in different ecological niches. Through the analysis of the enzymatic machinery engaged in wood degradation, it was possible to elucidate different strategies of wood decomposition which often depend on ecological niches inhabited by given organism. Moreover, a detailed description of low molecular weight compounds is presented, which gives these organisms not only an advantage in wood degradation processes, but seems rather to be a new evolutionatory alternative to enzymatic combustion. Through analysis of genomics and secretomic data, it was possible to underline the probable importance of certain wood-degrading enzymes produced by different fungal organisms, potentially giving them advantage in their ecological niches. The paper highlights different fungal strategies of wood degradation, which possibly correlates to the number of genes coding for secretory enzymes. Furthermore, investigation of the evolution of wood-degrading organisms has been described. PMID:29088355

The modules of trans-acyltransferase assembly lines redefined with a central acyl carrier protein.

PubMed

Vander Wood, Drew A; Keatinge-Clay, Adrian T

2018-06-01

Here, the term "module" is redefined for trans-acyltransferase (trans-AT) assembly lines to agree with how its domains cooperate and evolutionarily co-migrate. The key domain in both the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) modules of assembly lines is the acyl carrier protein (ACP). ACPs not only relay growing acyl chains through the assembly line but also collaborate with enzymes in modules, both in cis and in trans, to add a specific chemical moiety. A ketosynthase (KS) downstream of ACP often plays the role of gatekeeper, ensuring that only a single intermediate generated by the enzymes of a module is passed downstream. Bioinformatic analysis of 526 ACPs from 33 characterized trans-AT assembly lines reveals ACPs from the same module type generally clade together, reflective of the co-evolution of these domains with their cognate enzymes. While KSs downstream of ACPs from the same module type generally also clade together, KSs upstream of ACPs do not-in disagreement with the traditional definition of a module. Beyond nomenclature, the presented analysis impacts our understanding of module function, the evolution of assembly lines, pathway prediction, and assembly line engineering. © 2018 Wiley Periodicals, Inc.

Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803.

PubMed

Liang, Feiyan; Lindblad, Peter

2016-11-01

Synechocystis PCC 6803 is a model unicellular cyanobacterium used in e.g. photosynthesis and CO 2 assimilation research. In the present study we examined the effects of overexpressing Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7-biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the Calvin-Bassham-Benson (CBB) cycle in higher plants, in Synechocystis PCC 6803. Overexpressing RuBisCO, SBPase and FBA resulted in increased in vivo oxygen evolution (maximal 115%), growth rate and biomass accumulation (maximal 52%) under 100μmolphotonsm -2 s -1 light condition. Cells overexpressing TK showed a chlorotic phenotype but increased biomass by approximately 42% under 100μmolphotonsm -2 s -1 light condition. Under 15μmolphotonsm -2 s -1 light condition, cells overexpressing TK showed enhanced in vivo oxygen evolution. This study demonstrates increased growth and biomass accumulation when overexpressing selected enzymes of the CBB cycle. RuBisCO, SBPase, FBA and TK are identified as four potential targets to improve growth and subsequently also yield of valuable products from Synechocystis PCC 6803. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function.

PubMed

Lisi, George P; Loria, J Patrick

2016-02-01

Conformational motions play an essential role in enzyme function, often facilitating the formation of enzyme-substrate complexes and/or product release. Although considerable debate remains regarding the role of molecular motions in the conversion of enzymatic substrates to products, numerous examples have found motions to be crucial for optimization of enzyme scaffolds, effective substrate binding, and product dissociation. Conformational fluctuations are often rate-limiting to enzyme catalysis, primarily through product release, with the chemical reaction occurring much more quickly. As a result, the direct involvement of motions at various stages along the enzyme reaction coordinate remains largely unknown and untested. In the following review, we describe the use of solution NMR techniques designed to probe various timescales of molecular motions and detail examples in which motions play a role in propagating catalytic effects from the active site and directly participate in essential aspects of enzyme function. Copyright © 2015 Elsevier B.V. All rights reserved.

ENZYME-CATALYZED MUTATION IN BREAST CANCER

DTIC Science & Technology

2015-10-01

ATCC)  and  hTERT-­‐HMEC  (a  gift   from  the  lab  of  Dr.   Vitaly  Polunovsky)  to  over-­‐express  A3B   by...PMC3978439 Swanton, C., N . McGranahan, G.J. Starrett & R.S. Harris (2015) APOBEC enzymes: mutagenic fuel for cancer evolution and heterogeneity. Cancer... N , Brady JJ, Damian M, Sacco A, Corbel SY, Blau HM. Reprogramming towards pluripotency requires AID-dependent DNA demethylation. Nature. 2010;463

Insights into an evolutionary strategy leading to antibiotic resistance

NASA Astrophysics Data System (ADS)

Hou, Chun-Feng D.; Liu, Jian-Wei; Collyer, Charles; Mitić, Nataša; Pedroso, Marcelo Monteiro; Schenk, Gerhard; Ollis, David L.

2017-01-01

Metallo-β-lactamases (MBLs) with activity towards a broad-spectrum of β-lactam antibiotics have become a major threat to public health, not least due to their ability to rapidly adapt their substrate preference. In this study, the capability of the MBL AIM-1 to evade antibiotic pressure by introducing specific mutations was probed by two alternative methods, i.e. site-saturation mutagenesis (SSM) of active site residues and in vitro evolution. Both approaches demonstrated that a single mutation in AIM-1 can greatly enhance a pathogen’s resistance towards broad spectrum antibiotics without significantly compromising the catalytic efficiency of the enzyme. Importantly, the evolution experiments demonstrated that relevant amino acids are not necessarily in close proximity to the catalytic centre of the enzyme. This observation is a powerful demonstration that MBLs have a diverse array of possibilities to adapt to new selection pressures, avenues that cannot easily be predicted from a crystal structure alone.

Repeated Evolution of the Pyrrolizidine Alkaloid–Mediated Defense System in Separate Angiosperm LineagesW⃞

PubMed Central

Reimann, Andreas; Nurhayati, Niknik; Backenköhler, Anita; Ober, Dietrich

2004-01-01

Species of several unrelated families within the angiosperms are able to constitutively produce pyrrolizidine alkaloids as a defense against herbivores. In pyrrolizidine alkaloid (PA) biosynthesis, homospermidine synthase (HSS) catalyzes the first specific step. HSS was recruited during angiosperm evolution from deoxyhypusine synthase (DHS), an enzyme involved in the posttranslational activation of eukaryotic initiation factor 5A. Phylogenetic analysis of 23 cDNA sequences coding for HSS and DHS of various angiosperm species revealed at least four independent recruitments of HSS from DHS: one within the Boraginaceae, one within the monocots, and two within the Asteraceae family. Furthermore, sequence analyses indicated elevated substitution rates within HSS-coding sequences after each gene duplication, with an increased level of nonsynonymous mutations. However, the contradiction between the polyphyletic origin of the first enzyme in PA biosynthesis and the structural identity of the final biosynthetic PA products needs clarification. PMID:15466410

Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity.

PubMed

Fan, Pengxiang; Miller, Abigail M; Liu, Xiaoxiao; Jones, A Daniel; Last, Robert L

2017-12-12

Plants produce hundreds of thousands of structurally diverse specialized metabolites via multistep biosynthetic networks, including compounds of ecological and therapeutic importance. These pathways are restricted to specific plant groups, and are excellent systems for understanding metabolic evolution. Tomato and other plants in the nightshade family synthesize protective acylated sugars in the tip cells of glandular trichomes on stems and leaves. We describe a metabolic innovation in wild tomato species that contributes to acylsucrose structural diversity. A small number of amino acid changes in two acylsucrose acyltransferases alter their acyl acceptor preferences, resulting in reversal of their order of reaction and increased product diversity. This study demonstrates how small numbers of amino acid changes in multiple pathway enzymes can lead to diversification of specialized metabolites in plants. It also highlights the power of a combined genetic, genomic and in vitro biochemical approach to identify the evolutionary mechanisms leading to metabolic novelty.

21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant...

21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant...

21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant...

21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme...) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant...

Redesigning the specificity of protein-DNA interactions with Rosetta.

PubMed

Thyme, Summer; Baker, David

2014-01-01

Building protein tools that can selectively bind or cleave specific DNA sequences requires efficient technologies for modifying protein-DNA interactions. Computational design is one method for accomplishing this goal. In this chapter, we present the current state of protein-DNA interface design with the Rosetta macromolecular modeling program. The LAGLIDADG endonuclease family of DNA-cleaving enzymes, under study as potential gene therapy reagents, has been the main testing ground for these in silico protocols. At this time, the computational methods are most useful for designing endonuclease variants that can accommodate small numbers of target site substitutions. Attempts to engineer for more extensive interface changes will likely benefit from an approach that uses the computational design results in conjunction with a high-throughput directed evolution or screening procedure. The family of enzymes presents an engineering challenge because their interfaces are highly integrated and there is significant coordination between the binding and catalysis events. Future developments in the computational algorithms depend on experimental feedback to improve understanding and modeling of these complex enzymatic features. This chapter presents both the basic method of design that has been successfully used to modulate specificity and more advanced procedures that incorporate DNA flexibility and other properties that are likely necessary for reliable modeling of more extensive target site changes.

Cloning, expression and decoding of the cold adaptation of a new widely represented thermolabile subtilisin-like protease.

PubMed

Acevedo, J P; Rodriguez, V; Saavedra, M; Muñoz, M; Salazar, O; Asenjo, J A; Andrews, B A

2013-02-01

Cloning, expression and characterization of a new cold-adapted protease with potential biotechnological application, isolated from Antarctic bacteria. A subtilisin-like gene was isolated from several Antarctic bacterial genus using CODPEHOP-designed primers and a genome walking method. This gene encodes a precursor protein, which undergoes an autocatalytic cleavage resulting in a 34.6 kDa active cold-adapted protease with a maximum activity at 25-35°C and optimum pH of 8.0-9.0. It showed a higher catalytic efficiency at lower temperatures compared to its mesophilic counterpart. Heat-induced inactivation resulted in a very low melting point. Local packing analysis using the homology model indicated Ala284 as an important cold-adaptation determinant, which was corroborated by the site-directed mutagenesis. A new thermolabile subtilisin-like protease has been successfully cloned and analysed, and an important hot spot in the evolution of the cold adaptation and substrate specificity of this enzyme was identified and tested. This work reports a new cold-adapted protease with a vast representation amongst Antarctic genus, suggesting therefore its evolutionary success in this cold environment. Likewise, important sites for genetic potentiation have been identified, which are extrapolated to other enzymes of the same kind. © 2012 The Society for Applied Microbiology.

Joint scaling laws in functional and evolutionary categories in prokaryotic genomes

PubMed Central

Grilli, J.; Bassetti, B.; Maslov, S.; Cosentino Lagomarsino, M.

2012-01-01

We propose and study a class-expansion/innovation/loss model of genome evolution taking into account biological roles of genes and their constituent domains. In our model, numbers of genes in different functional categories are coupled to each other. For example, an increase in the number of metabolic enzymes in a genome is usually accompanied by addition of new transcription factors regulating these enzymes. Such coupling can be thought of as a proportional ‘recipe’ for genome composition of the type ‘a spoonful of sugar for each egg yolk’. The model jointly reproduces two known empirical laws: the distribution of family sizes and the non-linear scaling of the number of genes in certain functional categories (e.g. transcription factors) with genome size. In addition, it allows us to derive a novel relation between the exponents characterizing these two scaling laws, establishing a direct quantitative connection between evolutionary and functional categories. It predicts that functional categories that grow faster-than-linearly with genome size to be characterized by flatter-than-average family size distributions. This relation is confirmed by our bioinformatics analysis of prokaryotic genomes. This proves that the joint quantitative trends of functional and evolutionary classes can be understood in terms of evolutionary growth with proportional recipes. PMID:21937509

Re-engineering Cytochrome P450 2B11dH for Enhanced Metabolism of Several Substrates Including the Anti-cancer Prodrugs Cyclophosphamide and Ifosfamide

PubMed Central

Sun, Ling; Chen, Chong S.; Waxman, David J.; Liu, Hong; Halpert, James R.; Kumar, Santosh

2007-01-01

Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced kcat/Km for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in Km (0.72 vs. 18 μM). I209A also demonstrated enhanced selectivity for testosterone 16β-hydroxylation over 16α-hydroxylation. In contrast, V183L showed a 4-fold increased kcat for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased kcat/Km for testosterone 16α-hydroxylation. V183L also displayed a 1.7-fold higher kcat/Km than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a ~4-fold decrease in Km. Introduction of the V183L mutation into full-length P450 2B11 did not enhance the kcat/Km. Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs. PMID:17254539

New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria.

PubMed

Selmer, Thorsten; Pierik, Antonio J; Heider, Johann

2005-10-01

During the last decade, an increasing number of new enzymes containing glycyl radicals in their active sites have been identified and biochemically characterised. These include benzylsuccinate synthase (Bss), 4-hydroxyphenylacetate decarboxylase (Hpd) and the coenzyme B12-independent glycerol dehydratase (Gdh). These are involved in metabolic pathways as different as anaerobic toluene metabolism, fermentative production of p-cresol and glycerol fermentation. Some features of these newly discovered enzymes are described and compared with those of the previously known glycyl radical enzymes pyruvate formate-lyase (Pfl) and anaerobic ribonucleotide reductase (Nrd). Among the new enzymes, Bss and Hpd share the presence of small subunits, the function of which in the catalytic mechanisms is still enigmatic, and both enzymes contain metal centres in addition to the glycyl radical prosthetic group. The activating enzymes of the novel systems also deviate from the standard type, containing at least one additional Fe-S cluster. Finally, the available whole-genome sequences of an increasing number of strictly or facultative anaerobic bacteria revealed the presence of many more hitherto unknown glycyl radical enzyme (GRE) systems. Recent studies suggest that the particular types of these enzymes represent the ends of different evolutionary lines, which emerged early in evolution and diversified to yield remarkably versatile biocatalysts for chemical reactions that are otherwise difficult to perform in anoxic environments.

Recruitment of pre-existing networks during the evolution of C4 photosynthesis.

PubMed

Reyna-Llorens, Ivan; Hibberd, Julian M

2017-09-26

During C 4 photosynthesis, CO 2 is concentrated around the enzyme RuBisCO. The net effect is to reduce photorespiration while increasing water and nitrogen use efficiencies. Species that use C 4 photosynthesis have evolved independently from their C 3 ancestors on more than 60 occasions. Along with mimicry and the camera-like eye, the C 4 pathway therefore represents a remarkable example of the repeated evolution of a highly complex trait. In this review, we provide evidence that the polyphyletic evolution of C 4 photosynthesis is built upon pre-existing metabolic and genetic networks. For example, cells around veins of C 3 species show similarities to those of the C 4 bundle sheath in terms of C 4 acid decarboxylase activity and also the photosynthetic electron transport chain. Enzymes of C 4 photosynthesis function together in gluconeogenesis during early seedling growth of C 3 Arabidopsis thaliana Furthermore, multiple C 4 genes appear to be under control of both light and chloroplast signals in the ancestral C 3 state. We, therefore, hypothesize that relatively minor rewiring of pre-existing genetic and metabolic networks has facilitated the recurrent evolution of this trait. Understanding how these changes are likely to have occurred could inform attempts to install C 4 traits into C 3 crops.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'. © 2017 The Author(s).

The Vanadium Iodoperoxidase from the Marine Flavobacteriaceae Species Zobellia galactanivorans Reveals Novel Molecular and Evolutionary Features of Halide Specificity in the Vanadium Haloperoxidase Enzyme Family

PubMed Central

Fournier, Jean-Baptiste; Rebuffet, Etienne; Delage, Ludovic; Grijol, Romain; Meslet-Cladière, Laurence; Rzonca, Justyna; Potin, Philippe; Michel, Gurvan; Czjzek, Mirjam

2014-01-01

Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family. PMID:25261522

Molecular system identification for enzyme directed evolution and design

NASA Astrophysics Data System (ADS)

Guan, Xiangying; Chakrabarti, Raj

2017-09-01

The rational design of chemical catalysts requires methods for the measurement of free energy differences in the catalytic mechanism for any given catalyst Hamiltonian. The scope of experimental learning algorithms that can be applied to catalyst design would also be expanded by the availability of such methods. Methods for catalyst characterization typically either estimate apparent kinetic parameters that do not necessarily correspond to free energy differences in the catalytic mechanism or measure individual free energy differences that are not sufficient for establishing the relationship between the potential energy surface and catalytic activity. Moreover, in order to enhance the duty cycle of catalyst design, statistically efficient methods for the estimation of the complete set of free energy differences relevant to the catalytic activity based on high-throughput measurements are preferred. In this paper, we present a theoretical and algorithmic system identification framework for the optimal estimation of free energy differences in solution phase catalysts, with a focus on one- and two-substrate enzymes. This framework, which can be automated using programmable logic, prescribes a choice of feasible experimental measurements and manipulated input variables that identify the complete set of free energy differences relevant to the catalytic activity and minimize the uncertainty in these free energy estimates for each successive Hamiltonian design. The framework also employs decision-theoretic logic to determine when model reduction can be applied to improve the duty cycle of high-throughput catalyst design. Automation of the algorithm using fluidic control systems is proposed, and applications of the framework to the problem of enzyme design are discussed.

Beam width evolution of astigmatic hollow Gaussian beams in highly nonlocal nonlinear media

NASA Astrophysics Data System (ADS)

Yang, Zhen-Feng; Jiang, Xue-Song; Yang, Zhen-Jun; Li, Jian-Xing; Zhang, Shu-Min

We investigate the beam width evolution of astigmatic hollow Gaussian beams propagating in highly nonlocal nonlinear media. The input-power-induced different evolutions of the beam width are illustrated: (i) the beam widths in two transverse directions are compressed or broadened at the same time; (ii) the beam width in one transverse direction keeps invariant, and the other is compressed or broadened; (iii) furthermore, the beam width in one transverse direction is compressed, whereas it in the other transverse direction is broadened.

Comparative Genomics of Early-Diverging Mushroom-Forming Fungi Provides Insights into the Origins of Lignocellulose Decay Capabilities.

PubMed

Nagy, László G; Riley, Robert; Tritt, Andrew; Adam, Catherine; Daum, Chris; Floudas, Dimitrios; Sun, Hui; Yadav, Jagjit S; Pangilinan, Jasmyn; Larsson, Karl-Henrik; Matsuura, Kenji; Barry, Kerrie; Labutti, Kurt; Kuo, Rita; Ohm, Robin A; Bhattacharya, Sukanta S; Shirouzu, Takashi; Yoshinaga, Yuko; Martin, Francis M; Grigoriev, Igor V; Hibbett, David S

2016-04-01

Evolution of lignocellulose decomposition was one of the most ecologically important innovations in fungi. White-rot fungi in the Agaricomycetes (mushrooms and relatives) are the most effective microorganisms in degrading both cellulose and lignin components of woody plant cell walls (PCW). However, the precise evolutionary origins of lignocellulose decomposition are poorly understood, largely because certain early-diverging clades of Agaricomycetes and its sister group, the Dacrymycetes, have yet to be sampled, or have been undersampled, in comparative genomic studies. Here, we present new genome sequences of ten saprotrophic fungi, including members of the Dacrymycetes and early-diverging clades of Agaricomycetes (Cantharellales, Sebacinales, Auriculariales, and Trechisporales), which we use to refine the origins and evolutionary history of the enzymatic toolkit of lignocellulose decomposition. We reconstructed the origin of ligninolytic enzymes, focusing on class II peroxidases (AA2), as well as enzymes that attack crystalline cellulose. Despite previous reports of white rot appearing as early as the Dacrymycetes, our results suggest that white-rot fungi evolved later in the Agaricomycetes, with the first class II peroxidases reconstructed in the ancestor of the Auriculariales and residual Agaricomycetes. The exemplars of the most ancient clades of Agaricomycetes that we sampled all lack class II peroxidases, and are thus concluded to use a combination of plesiomorphic and derived PCW degrading enzymes that predate the evolution of white rot. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Higher-level classification of the Archaea: evolution of methanogenesis and methanogens

PubMed Central

Bapteste, Éric; Brochier, Céline; Boucher, Yan

2005-01-01

We used a phylogenetic approach to analyze the evolution of methanogenesis and methanogens. We show that 23 vertically transmitted ribosomal proteins do not support the monophyly of methanogens, and propose instead that there are two distantly related groups of extant archaea that produce methane, which we have named Class I and Class II. Based on this finding, we subsequently investigated the uniqueness of the origin of methanogenesis by studying both the enzymes of methanogenesis and the proteins that synthesize its specific coenzymes. We conclude that hydrogenotrophic methanogenesis appeared only once during evolution. Genes involved in the seven central steps of the methanogenic reduction of carbon dioxide (CO2) are ubiquitous in methanogens and share a common history. This suggests that, although extant methanogens produce methane from various substrates (CO2, formate, acetate, methylated C-1 compounds), these archaea have a core of conserved enzymes that have undergone little evolutionary change. Furthermore, this core of methanogenesis enzymes seems to originate (as a whole) from the last ancestor of all methanogens and does not appear to have been horizontally transmitted to other organisms or between members of Class I and Class II. The observation of a unique and ancestral form of methanogenesis suggests that it was preserved in two independent lineages, with some instances of specialization or added metabolic flexibility. It was likely lost in the Halobacteriales, Thermoplasmatales and Archaeoglobales. Given that fossil evidence for methanogenesis dates back 2.8 billion years, a unique origin of this process makes the methanogenic archaea a very ancient taxon. PMID:15876569

Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

PubMed Central

Li, Min; Zhang, Zhi-Jun; Kong, Xu-Dong; Yu, Hui-Lei

2017-01-01

ABSTRACT Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/I158V/P168S, whose thermostability (T5015, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C5015, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter−1 day−1. These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor. IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis. PMID:28389544

Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

PubMed

Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

2015-12-18

A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique and adenylating enzymes together using a combination of active site-directed probes for the A domains in NRPSs should accelerate both the functional characterization and manipulation of the A domains in NRPSs.

DC-Analyzer-facilitated combinatorial strategy for rapid directed evolution of functional enzymes with multiple mutagenesis sites.

PubMed

Wang, Xiong; Zheng, Kai; Zheng, Huayu; Nie, Hongli; Yang, Zujun; Tang, Lixia

2014-12-20

Iterative saturation mutagenesis (ISM) has been shown to be a powerful method for directed evolution. In this study, the approach was modified (termed M-ISM) by combining the single-site saturation mutagenesis method with a DC-Analyzer-facilitated combinatorial strategy, aiming to evolve novel biocatalysts efficiently in the case where multiple sites are targeted simultaneously. Initially, all target sites were explored individually by constructing single-site saturation mutagenesis libraries. Next, the top two to four variants in each library were selected and combined using the DC-Analyzer-facilitated combinatorial strategy. In addition to site-saturation mutagenesis, iterative saturation mutagenesis also needed to be performed. The advantages of M-ISM over ISM were that the screening effort is greatly reduced, and the entire M-ISM procedure was less time-consuming. The M-ISM strategy was successfully applied to the randomization of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) when five interesting sites were targeted simultaneously. After screening 900 clones in total, six positive mutants were obtained. These mutants exhibited 4.0- to 9.3-fold higher k(cat) values than did the wild-type HheC toward 1,3-dichloro-2-propanol. However, with the ISM strategy, the best hit showed a 5.9-fold higher k(cat) value toward 1,3-DCP than the wild-type HheC, which was obtained after screening 4000 clones from four rounds of mutagenesis. Therefore, M-ISM could serve as a simple and efficient version of ISM for the randomization of target genes with multiple positions of interest.

When does female multiple mating evolve to adjust inbreeding? Effects of inbreeding depression, direct costs, mating constraints, and polyandry as a threshold trait

PubMed Central

Duthie, A. Bradley; Bocedi, Greta; Reid, Jane M.

2016-01-01

Polyandry is often hypothesized to evolve to allow females to adjust the degree to which they inbreed. Multiple factors might affect such evolution, including inbreeding depression, direct costs, constraints on male availability, and the nature of polyandry as a threshold trait. Complex models are required to evaluate when evolution of polyandry to adjust inbreeding is predicted to arise. We used a genetically explicit individual‐based model to track the joint evolution of inbreeding strategy and polyandry defined as a polygenic threshold trait. Evolution of polyandry to avoid inbreeding only occurred given strong inbreeding depression, low direct costs, and severe restrictions on initial versus additional male availability. Evolution of polyandry to prefer inbreeding only occurred given zero inbreeding depression and direct costs, and given similarly severe restrictions on male availability. However, due to its threshold nature, phenotypic polyandry was frequently expressed even when strongly selected against and hence maladaptive. Further, the degree to which females adjusted inbreeding through polyandry was typically very small, and often reflected constraints on male availability rather than adaptive reproductive strategy. Evolution of polyandry solely to adjust inbreeding might consequently be highly restricted in nature, and such evolution cannot necessarily be directly inferred from observed magnitudes of inbreeding adjustment. PMID:27464756

Partitioning the Fitness Components of RNA Populations Evolving In Vitro

PubMed Central

Díaz Arenas, Carolina; Lehman, Niles

2013-01-01

All individuals in an evolving population compete for resources, and their performance is measured by a fitness metric. The performance of the individuals is relative to their abilities and to the biotic surroundings – the conditions under which they are competing – and involves many components. Molecules evolving in a test tube can also face complex environments and dynamics, and their fitness measurements should reflect the complexity of various contributing factors as well. Here, the fitnesses of a set of ligase ribozymes evolved by the continuous in vitro evolution system were measured. During these evolution cycles there are three different catalytic steps, ligation, reverse transcription, and forward transcription, each with a potential differential influence on the total fitness of each ligase. For six distinct ligase ribozyme genotypes that resulted from continuous evolution experiments, the rates of reaction were measured for each catalytic step by tracking the kinetics of enzymes reacting with their substrates. The reaction products were analyzed for the amount of product formed per time. Each catalytic step of the evolution cycle was found to have a differential incidence in the total fitness of the ligases, and therefore the total fitness of any ligase cannot be inferred from only one catalytic step of the evolution cycle. Generally, the ribozyme-directed ligation step tends to impart the largest effect on overall fitness. Yet it was found that the ligase genotypes have different absolute fitness values, and that they exploit different stages of the overall cycle to gain a net advantage. This is a new example of molecular niche partitioning that may allow for coexistence of more than one species in a population. The dissection of molecular events into multiple components of fitness provides new insights into molecular evolutionary studies in the laboratory, and has the potential to explain heretofore counterintuitive findings. PMID:24391957

Metabolically Coupled Replicator Systems: Overview of an RNA-world model concept of prebiotic evolution on mineral surfaces.

PubMed

Czárán, Tamás; Könnyű, Balázs; Szathmáry, Eörs

2015-09-21

Metabolically Coupled Replicator Systems (MCRS) are a family of models implementing a simple, physico-chemically and ecologically feasible scenario for the first steps of chemical evolution towards life. Evolution in an abiotically produced RNA-population sets in as soon as any one of the RNA molecules become autocatalytic by engaging in template directed self-replication from activated monomers, and starts increasing exponentially. Competition for the finite external supply of monomers ignites selection favouring RNA molecules with catalytic activity helping self-replication by any possible means. One way of providing such autocatalytic help is to become a replicase ribozyme. An additional way is through increasing monomer supply by contributing to monomer synthesis from external resources, i.e., by evolving metabolic enzyme activity. Retroevolution may build up an increasingly autotrophic, cooperating community of metabolic ribozymes running an increasingly complicated and ever more efficient metabolism. Maintaining such a cooperating community of metabolic replicators raises two serious ecological problems: one is keeping the system coexistent in spite of the different replicabilities of the cooperating replicators; the other is constraining parasitism, i.e., keeping "cheaters" in check. Surface-bound MCRS provide an automatic solution to both problems: coexistence and parasite resistance are the consequences of assuming the local nature of metabolic interactions. In this review we present an overview of results published in previous articles, showing that these effects are, indeed, robust in different MCRS implementations, by considering different environmental setups and realistic chemical details in a few different models. We argue that the MCRS model framework naturally offers a suitable starting point for the future modelling of membrane evolution and extending the theory to cover the emergence of the first protocell in a self-consistent manner. The coevolution of metabolic, genetic and membrane functions is hypothesized to follow the progressive sequestration scenario, the conceptual blueprint for the earliest steps of protocell evolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

Probing the mutational interplay between primary and promiscuous protein functions: a computational-experimental approach.

PubMed

Garcia-Seisdedos, Hector; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

2012-01-01

Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes.

Probing the Mutational Interplay between Primary and Promiscuous Protein Functions: A Computational-Experimental Approach

PubMed Central

Garcia-Seisdedos, Hector; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

2012-01-01

Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes. PMID:22719242

Molecular phylogeny and intricate evolutionary history of the three isofunctional enzymes involved in the oxidation of protoporphyrinogen IX.

PubMed

Kobayashi, Koichi; Masuda, Tatsuru; Tajima, Naoyuki; Wada, Hajime; Sato, Naoki

2014-08-01

Tetrapyrroles such as heme and chlorophyll are essential for biological processes, including oxygenation, respiration, and photosynthesis. In the tetrapyrrole biosynthesis pathway, protoporphyrinogen IX oxidase (Protox) catalyzes the formation of protoporphyrin IX, the last common intermediate for the biosynthesis of heme and chlorophyll. Three nonhomologous isofunctional enzymes, HemG, HemJ, and HemY, for Protox have been identified. To reveal the distribution and evolution of the three Protox enzymes, we identified homologs of each along with other heme biosynthetic enzymes by whole-genome clustering across three domains of life. Most organisms possess only one of the three Protox types, with some exceptions. Detailed phylogenetic analysis revealed that HemG is mostly limited to γ-Proteobacteria whereas HemJ may have originated within α-Proteobacteria and transferred to other Proteobacteria and Cyanobacteria. In contrast, HemY is ubiquitous in prokaryotes and is the only Protox in eukaryotes, so this type may be the ancestral Protox. Land plants have a unique HemY homolog that is also shared by Chloroflexus species, in addition to the main HemY homolog originating from Cyanobacteria. Meanwhile, organisms missing any Protox can be classified into two groups; those lacking most heme synthetic genes, which necessarily depend on external heme supply, and those lacking only genes involved in the conversion of uroporphyrinogen III into heme, which would use a precorrin2-dependent alternative pathway. However, hemN encoding coproporphyrinogen IX oxidase was frequently found in organisms lacking Protox enzyme, which suggests a unique role of this gene other than in heme biosynthesis. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Enzyme Efficiency but Not Thermostability Drives Cefotaxime Resistance Evolution in TEM-1 β-Lactamase.

PubMed

Knies, Jennifer L; Cai, Fei; Weinreich, Daniel M

2017-05-01

A leading intellectual challenge in evolutionary genetics is to identify the specific phenotypes that drive adaptation. Enzymes offer a particularly promising opportunity to pursue this question, because many enzymes' contributions to organismal fitness depend on a comparatively small number of experimentally accessible properties. Moreover, on first principles the demands of enzyme thermostability stand in opposition to the demands of catalytic activity. This observation, coupled with the fact that enzymes are only marginally thermostable, motivates the widely held hypothesis that mutations conferring functional improvement require compensatory mutations to restore thermostability. Here, we explicitly test this hypothesis for the first time, using four missense mutations in TEM-1 β-lactamase that jointly increase cefotaxime Minimum Inhibitory Concentration (MIC) ∼1500-fold. First, we report enzymatic efficiency (kcat/KM) and thermostability (Tm, and thence ΔG of folding) for all combinations of these mutations. Next, we fit a quantitative model that predicts MIC as a function of kcat/KM and ΔG. While kcat/KM explains ∼54% of the variance in cefotaxime MIC (∼92% after log transformation), ΔG does not improve explanatory power of the model. We also find that cefotaxime MIC rises more slowly in kcat/KM than predicted. Several explanations for these discrepancies are suggested. Finally, we demonstrate substantial sign epistasis in MIC and kcat/KM, and antagonistic pleiotropy between phenotypes, in spite of near numerical additivity in the system. Thus constraints on selectively accessible trajectories, as well as limitations in our ability to explain such constraints in terms of underlying mechanisms are observed in a comparatively "well-behaved" system. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Simultaneous measurement of two enzyme activities using infrared spectroscopy: A comparative evaluation of PARAFAC, TUCKER and N-PLS modeling.

PubMed

Baum, Andreas; Hansen, Per Waaben; Meyer, Anne S; Mikkelsen, Jørn Dalgaard

2013-08-06

Enzymes are used in many processes to release fermentable sugars for green production of biofuel, or the refinery of biomass for extraction of functional food ingredients such as pectin or prebiotic oligosaccharides. The complex biomasses may, however, require a multitude of specific enzymes which are active on specific substrates generating a multitude of products. In this paper we use the plant polymer, pectin, to present a method to quantify enzyme activity of two pectolytic enzymes by monitoring their superimposed spectral evolutions simultaneously. The data is analyzed by three chemometric multiway methods, namely PARAFAC, TUCKER3 and N-PLS, to establish simultaneous enzyme activity assays for pectin lyase and pectin methyl esterase. Correlation coefficients Rpred(2) for prediction test sets are 0.48, 0.96 and 0.96 for pectin lyase and 0.70, 0.89 and 0.89 for pectin methyl esterase, respectively. The retrieved models are compared and prediction test sets show that especially TUCKER3 performs well, even in comparison to the supervised regression method N-PLS. Copyright © 2013 Elsevier B.V. All rights reserved.

Enzyme Active Site Interactions by Raman/FTIR, NMR, and Ab Initio Calculations

PubMed Central

Deng, Hua

2017-01-01

Characterization of enzyme active site structure and interactions at high resolution is important for the understanding of the enzyme catalysis. Vibrational frequency and NMR chemical shift measurements of enzyme-bound ligands are often used for such purpose when X-ray structures are not available or when higher resolution active site structures are desired. This review is focused on how ab initio calculations may be integrated with vibrational and NMR chemical shift measurements to quantitatively determine high-resolution ligand structures (up to 0.001 Å for bond length and 0.01 Å for hydrogen bonding distance) and how interaction energies between bound ligand and its surroundings at the active site may be determined. Quantitative characterization of substrate ionic states, bond polarizations, tautomeric forms, conformational changes and its interactions with surroundings in enzyme complexes that mimic ground state or transition state can provide snapshots for visualizing the substrate structural evolution along enzyme-catalyzed reaction pathway. Our results have shown that the integration of spectroscopic studies with theoretical computation greatly enhances our ability to interpret experimental data and significantly increases the reliability of the theoretical analysis. PMID:24018325

Design and application of a lactulose biosensor.

PubMed

Wu, Jieyuan; Jiang, Peixia; Chen, Wei; Xiong, Dandan; Huang, Linglan; Jia, Junying; Chen, Yuanyuan; Jin, Jian-Ming; Tang, Shuang-Yan

2017-04-07

In this study the repressor of Escherichia coli lac operon, LacI, has been engineered for altered effector specificity. A LacI saturation mutagenesis library was subjected to Fluorescence Activated Cell Sorting (FACS) dual screening. Mutant LacI-L5 was selected and it is specifically induced by lactulose but not by other disaccharides tested (lactose, epilactose, maltose, sucrose, cellobiose and melibiose). LacI-L5 has been successfully used to construct a whole-cell lactulose biosensor which was then applied in directed evolution of cellobiose 2-epimerase (C2E) for elevated lactulose production. The mutant C2E enzyme with ~32-fold enhanced expression level was selected, demonstrating the high efficiency of the lactulose biosensor. LacI-L5 can also be used as a novel regulatory tool. This work explores the potential of engineering LacI for customized molecular biosensors which can be applied in practice.

Bringing inorganic chemistry to life with inspiration from R. J. P. Williams.

PubMed

Hill, H Allen O; Sadler, Peter J

2016-03-01

Our appreciation of the scholarly ideas and thinking of Bob Williams is illustrated here by a few of the areas in which he inspired us. His journey to bring inorganic chemistry to life began with an early interest in analytical chemistry, rationalising the relative stabilities of metal coordination complexes (The Irving-Williams Series), and elucidating the organometallic redox chemistry of vitamin B12. He (and Vallee) recognised that metal ions are in energised (entatic) states in proteins and enzymes, which themselves are dynamic structures of rods and springs. He played a key role in helping Rosenberg to pave the road toward the clinic for the anticancer drug cisplatin. He believed that evolution is not just dependent on DNA, but also on the metallome. Organisms and the environment are one system: does DNA code directly for all the essential elements of life?

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

PubMed Central

Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

2015-01-01

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution. PMID:26673816

Metallothionein Gene Duplications and Metal Tolerance in Natural Populations of Drosophila melanogaster

PubMed Central

Maroni, G.; Wise, J.; Young, J. E.; Otto, E.

1987-01-01

A search for duplications of the Drosophila melanogaster metallothionein gene (Mtn) yielded numerous examples of this type of chromosomal rearrangement. These duplications are distributed widely—we found them in samples from four continents, and they are functional—larvae carrying Mtn duplications produce more Mtn RNA and tolerate increased cadmium and copper concentrations. Six different duplication types were characterized by restriction-enzyme analyses using probes from the Mtn region. The restriction maps show that in four cases the sequences, ranging in size between 2.2 and 6.0 kb, are arranged as direct, tandem repeats; in two other cases, this basic pattern is modified by the insertion of a putative transposable element into one of the repeated units. Duplications of the D. melanogaster metallothionein gene such as those that we found in natural populations may represent early stages in the evolution of a gene family. PMID:2828157

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

NASA Astrophysics Data System (ADS)

Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

2015-12-01

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.

Fundamental studies of glucose oxidase deposition on a Pt electrode.

PubMed

Matsumoto, Norio; Chen, Xiaohong; Wilson, George S

2002-01-15

The direct electrodeposition of glucose oxidase (EC 1.1.3.4) on a platinum electrode was investigated as a means for controlled immobilization. The presence of a nonionic detergent, Triton X-100, was found essential to produce a multilayered deposit. Moreover, to work properly, the detergent must be present above its critical micelle concentration. Under these conditions, a deposit of approximately 50 enzyme layers (480 nm), with surface uniformity of +/-20 nm, was verified using an electrochemical quartz crystal microbalance and by atomic force microscopy. In the absence of detergent, a layer of 25 nm is formed. Contrary to most previous claims, the deposition, which is potential dependent but optimal at 1.3 V versus AgCl/Ag electrode, is not electrophoretically driven, but is instead controlled by a lowering of the pH at the electrode surface due to concomitant oxygen evolution.

Bioactive compounds synthesized by non-ribosomal peptide synthetases and type-I polyketide synthases discovered through genome-mining and metagenomics.

PubMed

Nikolouli, Katerina; Mossialos, Dimitris

2012-08-01

Non-ribosomal peptide synthetases (NRPS) and type-I polyketide synthases (PKS-I) are multimodular enzymes involved in biosynthesis of oligopeptide and polyketide secondary metabolites produced by microorganisms such as bacteria and fungi. New findings regarding the mechanisms underlying NRPS and PKS-I evolution illustrate how microorganisms expand their metabolic potential. During the last decade rapid development of bioinformatics tools as well as improved sequencing and annotation of microbial genomes led to discovery of novel bioactive compounds synthesized by NRPS and PKS-I through genome-mining. Taking advantage of these technological developments metagenomics is a fast growing research field which directly studies microbial genomes or specific gene groups and their products. Discovery of novel bioactive compounds synthesized by NRPS and PKS-I will certainly be accelerated through metagenomics, allowing the exploitation of so far untapped microbial resources in biotechnology and medicine.

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

DOE PAGES

Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; ...

2013-06-17

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymesmore » having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.« less

Graphene oxide-assisted non-immobilized SELEX of okdaic acid aptamer and the analytical application of aptasensor

NASA Astrophysics Data System (ADS)

Gu, Huajie; Duan, Nuo; Wu, Shijia; Hao, Liling; Xia, Yu; Ma, Xiaoyuan; Wang, Zhouping

2016-02-01

Okadaic acid (OA) is a low-molecular-weight marine toxin from shellfish that causes abdominal pain, vomiting and diarrhea, i.e., diarrheic shellfish poisoning. In this study, a ssDNA aptamer that specifically binds to OA with high affinity was obtained via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assisted by graphene oxide (GO). This aptamer was then applied to fabricate a novel direct competitive enzyme-linked aptamer assay (ELAA). At the optimized conditions, this ELAA method showed a low detection limit (LOD of 0.01 ng/mL), wide linear range (from 0.025 to 10 ng/mL), good recovery rate (92.86-103.34% in OA-spiked clam samples) and repeatability (RSD of 2.28-4.53%). The proposed method can be used to detect OA in seafood products with high sensitivity and can potentially be adapted for the determination of other small molecular analytes.

Micromotors Powered by Enzyme Catalysis.

PubMed

Dey, Krishna K; Zhao, Xi; Tansi, Benjamin M; Méndez-Ortiz, Wilfredo J; Córdova-Figueroa, Ubaldo M; Golestanian, Ramin; Sen, Ayusman

2015-12-09

Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymatic reactions and their directed movement in substrate concentration gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the respective substrates, using optical microscopy and dynamic light scattering analysis. The diffusion of the particles was found to increase in a substrate concentration dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture.

Novel Architectures for Achieving Direct Electron Transfer in Enzymatic Biofuel Cells

NASA Astrophysics Data System (ADS)

Blaik, Rita A.

Enzymatic biofuel cells are a promising source of alternative energy for small device applications, but still face the challenge of achieving direct electron transfer with high enzyme concentrations in a simple system. In this dissertation, methods of constructing electrodes consisting of enzymes attached to nanoparticle-enhanced substrates that serve as high surface area templates are evaluated. In the first method described, glucose oxidase is covalently attached to gold nanoparticles that are assembled onto genetically engineered M13 bacteriophage. The resulting anodes achieve a high peak current per area and a significant improvement in enzyme surface coverage. In the second system, fructose dehydrogenase, a membrane-bound enzyme that has the natural ability to achieve direct electron transfer, is immobilized into a matrix consisting of binders and carbon nanotubes to extend the lifetime of the anode. For the cathode, bilirubin oxidase is immobilized in a carbon nanotube and sol-gel matrix to achieve direct electron transfer. Finally, a full fuel cell consisting of both an anode and cathode is constructed and evaluated with each system described.

Relief of autoinhibition by conformational switch explains enzyme activation by a catalytically dead paralog

DOE Office of Scientific and Technical Information (OSTI.GOV)

Volkov, Oleg A.; Kinch, Lisa; Ariagno, Carson

Catalytically inactive enzyme paralogs occur in many genomes. Some regulate their active counterparts but the structural principles of this regulation remain largely unknown. We report X-ray structures ofTrypanosoma brucei S-adenosylmethionine decarboxylase alone and in functional complex with its catalytically dead paralogous partner, prozyme. We show monomericTbAdoMetDC is inactive because of autoinhibition by its N-terminal sequence. Heterodimerization with prozyme displaces this sequence from the active site through a complex mechanism involving acis-to-transproline isomerization, reorganization of a β-sheet, and insertion of the N-terminal α-helix into the heterodimer interface, leading to enzyme activation. We propose that the evolution of this intricate regulatory mechanismmore » was facilitated by the acquisition of the dimerization domain, a single step that can in principle account for the divergence of regulatory schemes in the AdoMetDC enzyme family. These studies elucidate an allosteric mechanism in an enzyme and a plausible scheme by which such complex cooperativity evolved.« less

Chromogenic detection of aminoglycoside phosphotransferases.

PubMed

Amoroso, A M; Gutkind, G O

2001-01-01

Acquired resistance to aminoglycosides is most frequently due to the presence of the so-called aminoglycoside modifying enzymes (AGME) (1) able to catalyze one or more of three general reactions: N-acetylation, O-nucleotidylation and O-phosphorylation (2). Although resistance phenotype (to different (substrate or not for enzymatic modification) may serve as an approach for identifying actual enzymes present in a given isolate (3), results can be obscured or confusing, particularly when several different enzymes (4) (even, isoenzymes with different affinities) are superimposing their action in a single microorganism with potential "permeability" or target alterations. Thus, identification of the AGME content of a given strain also requires screening at the DNA level using probes specific to all the known AGME (5). However, the complete set of probes is available only to a few laboratories around the world, making surveillance for the appearance of novel enzymes, or the unlikely evolution of those already known, a relatively nonfeasible goal, as search for new enzymes may begin only after failing to hybridize to all known probes.

To what extent do structural changes in catalytic metal sites affect enzyme function?

PubMed

Valasatava, Yana; Rosato, Antonio; Furnham, Nicholas; Thornton, Janet M; Andreini, Claudia

2018-02-01

About half of known enzymatic reactions involve metals. Enzymes belonging to the same superfamily often evolve to catalyze different reactions on the same structural scaffold. The work presented here investigates how functional differentiation, within superfamilies that contain metalloenzymes, relates to structural changes at the catalytic metal site. In general, when the catalytic metal site is unchanged across the enzymes of a superfamily, the functional differentiation within the superfamily tends to be low and the mechanism conserved. Conversely, all types of structural changes in the metal binding site are observed for superfamilies with high functional differentiation. Overall, the catalytic role of the metal ions appears to be one of the most conserved features of the enzyme mechanism within metalloenzyme superfamilies. In particular, when the catalytic role of the metal ion does not involve a redox reaction (i.e. there is no exchange of electrons with the substrate), this role is almost always maintained even when the site undergoes significant structural changes. In these enzymes, functional diversification is most often associated with modifications in the surrounding protein matrix, which has changed so much that the enzyme chemistry is significantly altered. On the other hand, in more than 50% of the examples where the metal has a redox role in catalysis, changes at the metal site modify its catalytic role. Further, we find that there are no examples in our dataset where metal sites with a redox role are lost during evolution. In this paper we investigate how functional diversity within superfamilies of metalloenzymes relates to structural changes at the catalytic metal site. Evolution tends to strictly conserve the metal site. When changes occur, they do not modify the catalytic role of non-redox metals whereas they affect the role of redox-active metals. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Direct measurement of catalase activity in living cells and tissue biopsies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scaglione, Christine N.; Xu, Qijin; Ramanujan, V. Krishnan, E-mail: Ramanujanv@csmc.edu

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Usingmore » catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear demonstration of the applicability in cancer cells and aging animal tissues.« less

Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer

PubMed Central

2016-01-01

The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer. PMID:27187273

Enzyme clustering accelerates processing of intermediates through metabolic channeling

PubMed Central

Castellana, Michele; Wilson, Maxwell Z.; Xu, Yifan; Joshi, Preeti; Cristea, Ileana M.; Rabinowitz, Joshua D.; Gitai, Zemer; Wingreen, Ned S.

2015-01-01

We present a quantitative model to demonstrate that coclustering multiple enzymes into compact agglomerates accelerates the processing of intermediates, yielding the same efficiency benefits as direct channeling, a well-known mechanism in which enzymes are funneled between enzyme active sites through a physical tunnel. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with previously reported spacings between coclusters in mammalian cells. For direct validation, we study a metabolic branch point in Escherichia coli and experimentally confirm the model prediction that enzyme agglomerates can accelerate the processing of a shared intermediate by one branch, and thus regulate steady-state flux division. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation. PMID:25262299

Crosstalk between poly(ADP-ribose) polymerase and sirtuin enzymes

PubMed Central

Cantó, Carles; Sauve, Anthony A.; Bai, Peter

2013-01-01

Poly(ADP-ribose) polymerases (PARPs) are NAD+ dependent enzymes that were identified as DNA repair proteins, however, today it seems clear that PARPs are responsible for a plethora of biological functions. Sirtuins (SIRTs) are NAD+-dependent deacetylase enzymes involved in the same biological processes as PARPs raising the question whether PARP and SIRT enzymes may interact with each other in physiological and pathophysiological conditions. Hereby we review the current understanding of the SIRT-PARP interplay in regard to the biochemical nature of the interaction (competition for the common NAD+ substrate, mutual posttranslational modifications and direct transcriptional effects) and the physiological, or pathophysiological consequences of the interactions (metabolic events, oxidative stress response, genomic stability and ageing). Finally, we give an overview of the possibilities of pharmacological intervention to modulate PARP and SIRT enzymes either directly, or through modulating NAD+ homeostasis. PMID:23357756

Zinc irreversibly damages major enzymes of energy production and antioxidant defense prior to mitochondrial permeability transition.

PubMed

Gazaryan, Irina G; Krasinskaya, Inna P; Kristal, Bruce S; Brown, Abraham M

2007-08-17

Recent observations point to the role played by Zn2+ as an inducer of neuronal death. Two Zn2+ targets have been identified that result in inhibition of mitochondrial respiration: the bc1 center and, more recently, alpha-ketoglutarate dehydrogenase. Zn2+ is also a mediator of oxidative stress, leading to mitochondrial failure, release of apoptotic peptides, and neuronal death. We now present evidence, by means of direct biochemical assays, that Zn2+ is imported through the Ca2+ uniporter and directly targets major enzymes of energy production (lipoamide dehydrogenase) and antioxidant defense (thioredoxin reductase and glutathione reductase). We demonstrate the following. (a) These matrix enzymes are rapidly inhibited by application of Zn2+ to intact mitochondria. (b) Delayed treatment with membrane-impermeable chelators has no effect, indicating rapid transport of biologically relevant quantities of Zn2+ into the matrix. (c) Membrane-permeable chelators stop but do not reverse enzyme inactivation. (d) Enzyme inhibition is rapid and irreversible and precedes the major changes associated with the mitochondrial permeability transition (MPT). (e) The extent and rate of enzyme inactivation linearly correlates with the MPT onset and propagation. (f) The Ca2+ uniporter blocker, Ruthenium Red, protects enzyme activities and delays pore opening up to 2 microm Zn2+. An additional, unidentified import route functions at higher Zn2+ concentrations. (g) No enzyme inactivation is observed for Ca2+-induced MPT. These observations strongly suggest that, unlike Ca2+, exogenous Zn2+ interferes with mitochondrial NADH production and directly alters redox protection in the matrix, contributing to mitochondrial dysfunction. Inactivation of these enzymes by Zn2+ is irreversible, and thus only their de novo synthesis can restore function, which may underlie persistent loss of oxidative carbohydrate metabolism following transient ischemia.

Wine Flavonoids in Health and Disease Prevention.

PubMed

Fernandes, Iva; Pérez-Gregorio, Rosa; Soares, Susana; Mateus, Nuno; de Freitas, Victor

2017-02-14

Wine, and particularly red wine, is a beverage with a great chemical complexity that is in continuous evolution. Chemically, wine is a hydroalcoholic solution (~78% water) that comprises a wide variety of chemical components, including aldehydes, esters, ketones, lipids, minerals, organic acids, phenolics, soluble proteins, sugars and vitamins. Flavonoids constitute a major group of polyphenolic compounds which are directly associated with the organoleptic and health-promoting properties of red wine. However, due to the insufficient epidemiological and in vivo evidences on this subject, the presence of a high number of variables such as human age, metabolism, the presence of alcohol, the complex wine chemistry, and the wide array of in vivo biological effects of these compounds suggest that only cautious conclusions may be drawn from studies focusing on the direct effect of wine and any specific health issue. Nevertheless, there are several reports on the health protective properties of wine phenolics for several diseases such as cardiovascular diseases, some cancers, obesity, neurodegenerative diseases, diabetes, allergies and osteoporosis. The different interactions that wine flavonoids may have with key biological targets are crucial for some of these health-promoting effects. The interaction between some wine flavonoids and some specific enzymes are one example. The way wine flavonoids may be absorbed and metabolized could interfere with their bioavailability and therefore in their health-promoting effect. Hence, some reports have focused on flavonoids absorption, metabolism, microbiota effect and overall on flavonoids bioavailability. This review summarizes some of these major issues which are directly related to the potential health-promoting effects of wine flavonoids. Reports related to flavonoids and health highlight some relevant scientific information. However, there is still a gap between the knowledge of wine flavonoids bioavailability and their health-promoting effects. More in vivo results as well as studies focused on flavonoid metabolites are still required. Moreover, it is also necessary to better understand how biological interactions (with microbiota and cells, enzymes or general biological systems) could interfere with flavonoid bioavailability.

Phylogenomic Relationships between Amylolytic Enzymes from 85 Strains of Fungi

PubMed Central

Chen, Wanping; Xie, Ting; Shao, Yanchun; Chen, Fusheng

2012-01-01

Fungal amylolytic enzymes, including α-amylase, gluocoamylase and α-glucosidase, have been extensively exploited in diverse industrial applications such as high fructose syrup production, paper making, food processing and ethanol production. In this paper, amylolytic genes of 85 strains of fungi from the phyla Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota were annotated on the genomic scale according to the classification of glycoside hydrolase (GH) from the Carbohydrate-Active enZymes (CAZy) Database. Comparisons of gene abundance in the fungi suggested that the repertoire of amylolytic genes adapted to their respective lifestyles. Amylolytic enzymes in family GH13 were divided into four distinct clades identified as heterologous α- amylases, eukaryotic α-amylases, bacterial and fungal α-amylases and GH13 α-glucosidases. Family GH15 had two branches, one for gluocoamylases, and the other with currently unknown function. GH31 α-glucosidases showed diverse branches consisting of neutral α-glucosidases, lysosomal acid α-glucosidases and a new clade phylogenetically related to the bacterial counterparts. Distribution of starch-binding domains in above fungal amylolytic enzymes was related to the enzyme source and phylogeny. Finally, likely scenarios for the evolution of amylolytic enzymes in fungi based on phylogenetic analyses were proposed. Our results provide new insights into evolutionary relationships among subgroups of fungal amylolytic enzymes and fungal evolutionary adaptation to ecological conditions. PMID:23166747

Nature's inordinate fondness for metabolic enzymes: why metabolic enzyme loci are so frequently targets of selection.

PubMed

Marden, James H

2013-12-01

Metabolic enzyme loci were some of the first genes accessible for molecular evolution and ecology research. New technologies now make the whole genome, transcriptome or proteome readily accessible, allowing unbiased scans for loci exhibiting significant differences in allele frequency or expression level and associated with phenotypes and/or responses to natural selection. With surprising frequency and in many cases in proportions greater than chance relative to other genes, glycolysis and TCA cycle enzyme loci appear among the genes with significant associations in these studies. Hence, there is an ongoing need to understand the basis for fitness effects of metabolic enzyme polymorphisms. Allele-specific effects on the binding affinity and catalytic rate of individual enzymes are well known, but often of uncertain significance because metabolic control theory and in vivo studies indicate that many individual metabolic enzymes do not affect pathway flux rate. I review research, so far little used in evolutionary biology, showing that metabolic enzyme substrates affect signalling pathways that regulate cell and organismal biology, and that these enzymes have moonlighting functions. To date there is little knowledge of how alleles in natural populations affect these phenotypes. I discuss an example in which alleles of a TCA enzyme locus associate with differences in a signalling pathway and development, organismal performance, and ecological dynamics. Ultimately, understanding how metabolic enzyme polymorphisms map to phenotypes and fitness remains a compelling and ongoing need for gaining robust knowledge of ecological and evolutionary processes. © 2013 John Wiley & Sons Ltd.

Direct detection of male quality can facilitate the evolution of female choosiness and indicators of good genes: Evolution across a continuum of indicator mechanisms.

PubMed

Dhole, Sumit; Stern, Caitlin A; Servedio, Maria R

2018-04-01

The evolution of mating displays as indicators of male quality has been the subject of extensive theoretical and empirical research for over four decades. Research has also addressed the evolution of female mate choice favoring such indicators. Yet, much debate still exists about whether displays can evolve through the indirect benefits of female mate choice. Here, we use a population genetic model to investigate how the extent to which females can directly detect male quality influences the evolution of female choosiness and male displays. We use a continuum framework that incorporates indicator mechanisms that are traditionally modeled separately. Counter to intuition, we find that intermediate levels of direct detection of male quality can facilitate, rather than impede, the evolution of female choosiness and male displays in broad regions of this continuum. We examine how this evolution is driven by selective forces on genetic quality and on the display, and find that direct detection of male quality results in stronger indirect selection favoring female choosiness. Our results imply that displays maybe more likely to evolve when female choosiness has already evolved to discriminate perceptible forms of male quality. They also highlight the importance of considering general female choosiness, as well as preference, in studies of "good genes." © 2018 The Author(s). Evolution © 2018 The Society for the Study of Evolution.

Sex in a test tube: testing the benefits of in vitro recombination.

PubMed

Pesce, Diego; Lehman, Niles; de Visser, J Arjan G M

2016-10-19

The origin and evolution of sex, and the associated role of recombination, present a major problem in biology. Sex typically involves recombination of closely related DNA or RNA sequences, which is fundamentally a random process that creates but also breaks up beneficial allele combinations. Directed evolution experiments, which combine in vitro mutation and recombination protocols with in vitro or in vivo selection, have proved to be an effective approach for improving functionality of nucleic acids and enzymes. As this approach allows extreme control over evolutionary conditions and parameters, it also facilitates the detection of small or position-specific recombination benefits and benefits associated with recombination between highly divergent genotypes. Yet, in vitro approaches have been largely exploratory and motivated by obtaining improved end products rather than testing hypotheses of recombination benefits. Here, we review the various experimental systems and approaches used by in vitro studies of recombination, discuss what they say about the evolutionary role of recombination, and sketch their potential for addressing extant questions about the evolutionary role of sex and recombination, in particular on complex fitness landscapes. We also review recent insights into the role of 'extracellular recombination' during the origin of life.This article is part of the themed issue 'Weird sex: the underappreciated diversity of sexual reproduction'. © 2016 The Author(s).

Downregulation of the evolutionary capacitor Hsp90 is mediated by social cues

PubMed Central

Eggert, Hendrik

2015-01-01

The relationship between robustness and evolvability is a long-standing question in evolution. Heat shock protein 90 (HSP90), a molecular chaperone, has been identified as a potential capacitor for evolution, since it allows for the accumulation and release of cryptic genetic variation, and also for the regulation of novel genetic variation through transposon activity. However, to date, it is unknown whether Hsp90 expression is regulated upon demand (i.e. when the release of cryptic genetic variation is most needed). Here, we show that Hsp90 has reduced transcription under conditions where the mobilization of genetic variation could be advantageous. We designed a situation that indicates a stressful environment but avoids the direct effects of stress, by placing untreated (focal) red flour beetles, Tribolium castaneum, into groups together with wounded conspecifics, and found a consistent reduction in expression of two Hsp90 genes (Hsp83 and Hsp90) in focal beetles. We moreover observed a social transfer of immunity in this non-eusocial insect: there was increased activity of the phenoloxidase enzyme and downregulation of the immune regulator, imd. Our study poses the exciting question of whether evolvability might be regulated through the use of information derived from the social environment. PMID:26582024

Copy number polymorphism of the salivary amylase gene: implications in human nutrition research.

PubMed

Santos, J L; Saus, E; Smalley, S V; Cataldo, L R; Alberti, G; Parada, J; Gratacòs, M; Estivill, X

2012-01-01

The salivary α-amylase is a calcium-binding enzyme that initiates starch digestion in the oral cavity. The α-amylase genes are located in a cluster on the chromosome that includes salivary amylase genes (AMY1), two pancreatic α-amylase genes (AMY2A and AMY2B) and a related pseudogene. The AMY1 genes show extensive copy number variation which is directly proportional to the salivary α-amylase content in saliva. The α-amylase amount in saliva is also influenced by other factors, such as hydration status, psychosocial stress level, and short-term dietary habits. It has been shown that the average copy number of AMY1 gene is higher in populations that evolved under high-starch diets versus low-starch diets, reflecting an intense positive selection imposed by diet on amylase copy number during evolution. In this context, a number of different aspects can be considered in evaluating the possible impact of copy number variation of the AMY1 gene on nutrition research, such as issues related to human diet gene evolution, action on starch digestion, effect on glycemic response after starch consumption, modulation of the action of α-amylases inhibitors, effect on taste perception and satiety, influence on psychosocial stress and relation to oral health. Copyright © 2012 S. Karger AG, Basel.

De novo active sites for resurrected Precambrian enzymes

NASA Astrophysics Data System (ADS)

Risso, Valeria A.; Martinez-Rodriguez, Sergio; Candel, Adela M.; Krüger, Dennis M.; Pantoja-Uceda, David; Ortega-Muñoz, Mariano; Santoyo-Gonzalez, Francisco; Gaucher, Eric A.; Kamerlin, Shina C. L.; Bruix, Marta; Gavira, Jose A.; Sanchez-Ruiz, Jose M.

2017-07-01

Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

Directional Communication in Evolved Multiagent Teams

DTIC Science & Technology

2013-06-10

decentralized localization proposed by Franchi et al. [9]. Overall, the significant advantage of directional communication over non- directional...reception benefits the evolution of communicating autonomous agents because it simplifies the language required to express positional information, which...systems. This paper hypothesizes that such directional reception benefits the evolution of communicating autonomous agents because it simplifies the

Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16.

PubMed

Lu, Jingnan; Brigham, Christopher J; Plassmeier, Jens K; Sinskey, Anthony J

2015-01-01

2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by L-valine (IC50=1.2 mM), L-isoleucine (IC50=2.3 mM), and L-leucine (IC50=5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (KM=10.5 μM) and is highly selective towards 2-ketobutyrate (R=140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for value-added materials.

Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, JN; Brigham, CJ; Plassmeier, JK

2014-08-01

2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the threemore » R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by l-valine (IC50 = 1.2 mM), l-isoleucine (IC50 = 2.3 mM), and l-leucine (IC50 = 5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (K-M = 10.5 mu M) and is highly selective towards 2-ketobutyrate (R = 140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for value-added materials.« less