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Sample records for l-sorbose dehydrogenase accelerative

  1. Crystal structure of L-sorbose dehydrogenase, a pyrroloquinoline quinone-dependent enzyme with homodimeric assembly, from Ketogulonicigenium vulgare.

    PubMed

    Han, Xiaodong; Xiong, Xianghua; Jiang, Dunquan; Chen, Sihan; Huang, Enyu; Zhang, Weicai; Liu, Xinqi

    2014-05-01

    The crystal structure of the L-sorbose dehydrogenase (SDH) from Ketogulonicigenium vulgare Y25 has been determined at 2.7 Å resolution using the molecular replacement method. The overall structure of SDH is similar to that of other quinoprotein dehydrogenases; consisting of an eight bladed β-propeller PQQ domain and protrusion loops. We identified a stable homodimer in crystal and demonstrated its existence in solution by sedimentation velocity measurement. By biochemical characterization of the SDH in vitro, using L-sorbose as substrate and cytochrome c551 as electron acceptor, we revealed cytochrome c551 acting as physiological primary electron acceptor for SDH. PMID:24557074

  2. Characterization of a group of pyrroloquinoline quinone-dependent dehydrogenases that are involved in the conversion of L-sorbose to 2-Keto-L-gulonic acid in Ketogulonicigenium vulgare WSH-001.

    PubMed

    Gao, Lili; Du, Guocheng; Zhou, Jingwen; Chen, Jian; Liu, Jie

    2013-01-01

    Ketogulonicigenium vulgare WSH-001 is an industrial strain used for vitamin C production. Based on genome sequencing and pathway analysis of the bacterium, some of its potential pyrroloquinoline quinone (PQQ)-dependent dehydrogenases were predicted, including KVU_pmdA_0245, KVU_2142, KVU_2159, KVU_1366, KVU_0203, KVU_0095, and KVU_pmdB_0115. BLAST and function domain searches showed that enzymes encoded by these genes may act as putative PQQ-dependent L-sorbose dehydrogenases (SDH) or L-sorbosone dehydrogenases (SNDH). To validate whether these dehydrogenases are PQQ-dependent or not, these seven putative dehyrogenases were overexpressed in Escherichia coli BL21 (DE3) and purified for characterization. Biochemical and kinetic characterization of the purified proteins have led to the identification of seven enzymes that possess the ability to oxidize L-sorbose or L-sorbosone to varying degrees. In addition, the dehydrogenation of sorbose in K. vulgare is validated to be PQQ dependent, identification of these PQQ-dependent dehydrogenases expanded the PQQ-dependent dehydrogenase family. Besides, the optimal combination of enzymes that could more efficiently catalyze the conversion of sorbose to gulonic acid was proposed. These are important in supporting the development of metabolic engineering strategies and engineering of efficient strains for one-step production of vitamin C in the future. PMID:23970495

  3. Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain.

    PubMed Central

    Saito, Y; Ishii, Y; Hayashi, H; Imao, Y; Akashi, T; Yoshikawa, K; Noguchi, Y; Soeda, S; Yoshida, M; Niwa, M; Hosoda, J; Shimomura, K

    1997-01-01

    We have purified L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) from Gluconobacter oxydans T-100 that showed an ability to convert D-sorbitol to 2-keto-L-gulonate (2-KLGA). A genomic library of Gluconobacter oxydans T-100 was screened with a probe, a 180-bp PCR product which was obtained from degenerate oligodeoxyribonucleotides based on the elucidated sequence of the purified SDH (used as primers) and the genomic DNA of G. oxydans T-100 (used as a template). From sequencing of the DNA from a clone positive to the probe, the SNDH and the SDH were estimated to be coded in sequential open reading frames with 1,497 and 1,599 nucleotides, respectively, which was confirmed by expression of the DNA in Escherichia coli that showed both enzymatic activities. The DNA was introduced to a shuttle vector which was prepared from a plasmid of G. oxydans T-100 and pHSG298 to obtain an expression vector designated pSDH155. The production of 2-KLGA by pSDH155 in G. oxydans G624, an L-sorbose-accumulating strain, was improved to 230% compared to that of G. oxydans T-100. Chemical mutation of the host strain to suppress the L-idonate pathway and replacement of the original promoter with that of E. coli tufB resulted in improving the production of 2-KLGA. Consequently, high-level production from D-sorbitol to 2-KLGA (130 mg/ml) was achieved by simple fermentation of the recombinant Gluconobacter. PMID:9023923

  4. A two-step strategy for the preparation of 6-deoxy-l-sorbose.

    PubMed

    Wen, Liuqing; Huang, Kenneth; Zheng, Yuan; Liu, Yunpeng; Zhu, He; Wang, Peng George

    2016-09-01

    A two-step enzymatic strategy for the efficient and convenient synthesis of 6-deoxy-l-sorbose was reported herein. In the first reaction step, the isomerization of l-fucose (6-deoxy-l-galactose) to l-fuculose (6-deoxy-l-tagatose) catalyzed by l-fucose isomerase (FucI), and the epimerization of l-fuculose to 6-deoxy-l-sorbose catalyzed by d-tagatose 3-epimerase (DTE) were coupled with the targeted phosphorylation of 6-deoxy-l-sorbose by fructose kinase from human (HK) in a one-pot reaction. The resultant 6-deoxy-l-sorbose 1-phosphate was purified by silver nitrate precipitation method. In the second reaction step, the phosphate group of the 6-deoxy-l-sorbose 1-phosphate was hydrolyzed with acid phosphatase (AphA) to produce 6-deoxy-l-sorbose in 81% yield with regard to l-fucose. PMID:27485385

  5. Biosynthesis of l-Sorbose and l-Psicose Based on C—C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain

    PubMed Central

    Yang, Jiangang; Li, Jitao; Men, Yan; Zhu, Yueming; Zhang, Ying; Ma, Yanhe

    2015-01-01

    The property of loose stereochemical control at aldol products from aldolases helped to synthesize multiple polyhydroxylated compounds with nonnatural stereoconfiguration. In this study, we discovered for the first time that some fructose 1,6-diphosphate aldolases (FruA) and tagatose 1,6-diphosphate (TagA) aldolases lost their strict stereoselectivity when using l-glyceraldehyde and synthesized not only l-sorbose but also a high proportion of l-psicose. Among the aldolases tested, TagA from Bacillus licheniformis (BGatY) showed the highest enzyme activity with l-glyceraldehyde. Subsequently, a “one-pot” reaction based on BGatY and fructose-1-phosphatase (YqaB) generated 378 mg/liter l-psicose and 199 mg/liter l-sorbose from dihydroxyacetone-phosphate (DHAP) and l-glyceraldehyde. Because of the high cost and instability of DHAP, a microbial fermentation strategy was used further to produce l-sorbose/l-psicose from glucose and l-glyceraldehyde, in which DHAP was obtained from glucose through the glycolytic pathway, and some recombination pathways based on FruA or TagA and YqaB were constructed in Escherichia coli and Corynebacterium glutamicum strains. After evaluation of different host cells and combinations of FruA or TagA with YqaB and optimization of gene expression, recombinant C. glutamicum strain WT(pXFTY) was selected and produced 2.53 g/liter total ketoses, with a yield of 0.50 g/g l-glyceraldehyde. Moreover, deletion of gene cgl0331, encoding the Zn-dependent alcohol dehydrogenase in C. glutamicum, was confirmed for the first time to significantly decrease conversion of l-glyceraldehyde to glycerol and to increase yield of target products. Finally, fed-batch culture of strain SY14(pXFTY) produced 3.5 g/liter l-sorbose and 2.3 g/liter l-psicose, with a yield of 0.61 g/g l-glyceraldehyde. This microbial fermentation strategy also could be applied to efficiently synthesize other l-sugars. PMID:25888171

  6. Titanium-Beta Zeolites Catalyze the Stereospecific Isomerization of D-Glucose to L-Sorbose via Intramolecular C5-C1 Hydride Shift

    SciTech Connect

    Gounder, Rajamani; Davis, Mark E.

    2013-06-03

    Pure-silica zeolite beta containing Lewis acidic framework Ti4+ centers (Ti-Beta) is shown to catalyze the isomerization of D-glucose to L-sorbose via an intramolecular C5–C1 hydride shift. Glucose–sorbose isomerization occurs in parallel to glucose–fructose isomerization on Ti-Beta in both water and methanol solvents, with fructose formed as the predominant product in water and sorbose as the predominant product in methanol (at 373 K) at initial times and over the course of >10 turnovers. Isotopic tracer studies demonstrate that 13C and D labels placed respectively at the C1 and C2 positions of glucose are retained respectively at the C6 and C5 positions of sorbose, consistent with its formation via an intramolecular C5–C1 hydride shift isomerization mechanism. This direct Lewis acid-mediated pathway for glucose–sorbose isomerization appears to be unprecedented among heterogeneous or biological catalysts and sharply contrasts indirect base-mediated glucose–sorbose isomerization via 3,4-enediol intermediates or via retro-aldol fragmentation and recombination of sugar fragments. Measured first-order glucose–sorbose isomerization rate constants (per total Ti; 373 K) for Ti-Beta in methanol are similar for glucose and glucose deuterated at the C2 position (within a factor of ~1.1), but are a factor of ~2.3 lower for glucose deuterated at each carbon position, leading to H/D kinetic isotope effects expected for kinetically relevant intramolecular C5–C1 hydride shift steps. Optical rotation measurements show that isomerization of D-(+)-glucose (92% enantiomeric purity) with Ti-Beta in water (373 K) led to the formation of L-(-)-sorbose (73% enantiomeric purity) and D-(-)-fructose (87% enantiomeric purity) as the predominant stereoisomers, indicating that stereochemistry is preserved at carbon centers not directly involved in intramolecular C5–C1 or C2–C1 hydride shift steps, respectively. This new Lewis acid

  7. Crystal structure of Pseudomonas fluorescens mannitol 2-dehydrogenase: evidence for a very divergent long-chain dehydrogenase family.

    PubMed

    Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

    2003-02-01

    Mannitol 2-dehydrogenase from Pseudomonas fluorescens (pfMDH) is a secondary alcohol dehydrogenase that catalyzes the reversible NAD(P)-dependent oxidation of D-mannitol to D-fructose, D-arabinitol to D-xylulose, and D-sorbitol to L-sorbose. It is a member of the mostly prokaryotic family of long-chain mannitol dehydrogenases that so far includes 66 members. Unlike other alcohol and polyol dehydrogenases that utilize metal cofactors or a conserved active-site tyrosine for catalysis, an invariant lysine is the general base. The crystal structure of pfMDH in a binary complex with NAD(H) and a ternary complex with NAD(H) and D-mannitol have been determined to 1.7 and 1.8 A resolution respectively. Comparison of secondary structure assignment to sequence alignments suggest the shortest members of this family, mannitol-1-phosphate 5-dehydrogenases, retain core elements but lack secondary structural components found on the surface of pfMDH. The elements predicted to be absent are distributed throughout the primary sequence, implying that a simple truncation or fusion did not occur. The closest structural neighbors are 6-phosphogluconate dehydrogenase, UDP-glucose dehydrogenase, N-(1-D-carboxyethyl)-L-norvaline dehydrogenase, and glycerol-3-phosphate dehydrogenase. Although sequence identity is only a barely recognizable 7-10%, conservation of secondary structural elements as well as homologous residues that are contributed to the active site indicates they may be related by divergent evolution. PMID:12604241

  8. The first step in polyethylene glycol degradation by sphingomonads proceeds via a flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide.

    PubMed

    Sugimoto, M; Tanabe, M; Hataya, M; Enokibara, S; Duine, J A; Kawai, F

    2001-11-01

    Several Sphingomonas spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound. The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E. coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%). PMID:11673442

  9. Suggestive-Accelerative Learning and Teaching in Foreign Languages.

    ERIC Educational Resources Information Center

    Herr, Kay U.

    The suggestive-accelerative approach to foreign language instruction is described. This method, first used in Bulgaria by Georgi Lozanov, emphasizes bringing the imagination to bear on the learning task, in a relaxed classroom environment. After establishing a calm atmosphere through direct and indirect suggestion, the teacher proceeds to…

  10. Percutaneous Coronary Intervention Enhances Accelerative Wave Intensity in Coronary Arteries

    PubMed Central

    Narayan, Om; Leung, Michael C. H.; Wong, Dennis T. L.; Meredith, Ian T.; Cameron, James D.

    2015-01-01

    Background The systolic forward travelling compression wave (sFCW) and diastolic backward travelling decompression waves (dBEW) predominantly accelerate coronary blood flow. The effect of a coronary stenosis on the intensity of these waves in the distal vessel is unknown. We investigated the relationship between established physiological indices of hyperemic coronary flow and the intensity of the two major accelerative coronary waves identified by Coronary Wave Intensity analysis (CWIA). Methodology / Principal Findings Simultaneous intracoronary pressure and velocity measurement was performed during adenosine induced hyperemia in 17 patients with pressure / Doppler flow wires positioned distal to the target lesion. CWI profiles were generated from this data. Fractional Flow Reserve (FFR) and Coronary Flow Velocity Reserve (CFVR) were calculated concurrently. The intensity of the dBEW was significantly correlated with FFR (R = -0.70, P = 0.003) and CFVR (R = -0.73, P = 0.001). The intensity of the sFCW was also significantly correlated with baseline FFR (R = 0.71, p = 0.002) and CFVR (R = 0.59, P = 0.01). Stenting of the target lesion resulted in a median 178% (interquartile range 55–280%) (P<0.0001) increase in sFCW intensity and a median 117% (interquartile range 27–509%) (P = 0.001) increase in dBEW intensity. The increase in accelerative wave intensity following PCI was proportionate to the baseline FFR and CFVR, such that stenting of lesions associated with the greatest flow limitation (lowest FFR and CFVR) resulted in the largest increases in wave intensity. Conclusions Increasing ischemia severity is associated with proportionate reductions in cumulative intensity of both major accelerative coronary waves. Impaired diastolic microvascular decompression may represent a novel, important pathophysiologic mechanism driving the reduction in coronary blood flow in the setting of an epicardial stenosis. PMID:26658896

  11. The Journal of Suggestive-Accelerative Learning and Teaching, Volume 3, Number 1, Spring 1978.

    ERIC Educational Resources Information Center

    Schuster, Donald H., Ed.

    1978-01-01

    Contents of this issue are as follows: "Applied Suggestology in the United States: Suggestive-Accelerative Learning and Teaching" by Dean F. Held; "A Study of Suggestopedia Features that Can Be Omitted Once Students Learn How to Learn" by D.H. Schuster and P.J. Wardell; "Evaluation of the Suggestive-Accelerative Lozanov Teaching (SALT) Method in…

  12. The Journal of the Society for Accelerative Learning and Teaching, Volume 7.

    ERIC Educational Resources Information Center

    Journal of the Society for Accelerative Learning and Teaching, 1982

    1982-01-01

    The four 1982 numbers of the Journal of the Society for Accelerative Learning and Teaching (SALT) include articles on: a comparison of the Tomatis Method and Suggestopedia; the CLC system of accelerated learning; Suggestopedia in the English-as-a-second-language classroom; experiments with SALT techniques; accelerative learning techniques for…

  13. Accelerative forces associated with routine inhouse transportation of rodent cages.

    PubMed

    Hurst, Keriann; Litwak, Kenneth N

    2012-01-01

    Transportation of rodents has repeatedly been demonstrated to potentially affect research outcomes. In addition, rapid acceleration and deceleration have marked physiologic effects. The current study determined the accelerative forces associated with common types of animal transportation within the institution and means of reducing these effects. A rodent-sized (24 g) accelerometer was placed in a standard polycarbonate mouse cage, which then was hand-carried or loaded onto a plastic, small metal, or large metal cart. The cage then moved along a set path that included several flooring types and obstacles. Accelerative forces within the mouse cage varied by as much as 35 m/s(2) in as little as 1 s, primarily along the vertical axis (Z-axis). Measured acceleration was greatest with the plastic cart and lowest during hand-carrying. The placement of a towel under the cage dampened in-cage acceleration due to cart use by more than 50%, whereas a similarly located underpad had no significant effect. These data document that small rodents typically are exposed to considerable motion during transportation. The resulting physical and physiologic effects could affect study outcomes. PMID:23312081

  14. High charge state, ion-atom collision experiments using accel-decel

    SciTech Connect

    Bernstein, E.M.; Clark, M.W.; Tanis, J.A.; Graham, W.G.

    1987-01-01

    Recent studies of /sub 16/S/sup 13 +/ + He collisions between 2.5 and 200 MeV, which were made using the accel-decel technique with the Brookhaven National Laboratory coupled MP tandem Van de Graaff accelerators, are discussed. Cross sections were measured for single electron-capture and -loss as well as K x rays correlated to electron-capture. Other planned ion-atom collision experiments requiring accel-decel are also presented. 18 refs., 3 figs.

  15. Increased energy harvesting and reduced accelerative load for backpacks via frequency tuning

    NASA Astrophysics Data System (ADS)

    Xie, Longhan; Cai, Mingjing

    2015-06-01

    In this research, a backpack-based frequency-tuneable harvesting device was developed to harvest part of the human kinetic energy during walking and to relieve part of the accelerative load of the backpack from the bearer. The harvester employed a tuning mechanism to adjust the stretch ratio of the springs to adjust the system's stiffness so that the harvesting device can work in an appropriate status to generate more power and relieve a greater load from the bearer. The analysis indicates that adjusting the stiffness harvesting system to fit well with various external excitation conditions, can not only achieve more power output but also relieve part of the accelerative load from the bearer; and the experimental results agreed with the simulation. Compared with previous work, the harvester in this work had a higher efficiency in energy harvesting and could relieve an increased accelerative load from the bearer.

  16. A Meta-Analysis of Suggestopedia, Suggestology, Suggestive-accelerative Learning and Teaching (SALT), and Super-learning.

    ERIC Educational Resources Information Center

    Moon, Charles E.; And Others

    Forty studies using one or more components of Lozanov's method of suggestive-accelerative learning and teaching were identified from a search of all issues of the "Journal of Suggestive-Accelerative Learning and Teaching." Fourteen studies contained sufficient statistics to compute effect sizes. The studies were coded according to substantive and…

  17. Suggestive, Accelerative Learning and Teaching: A Manual of Classroom Procedures Based on the Lozanov Method.

    ERIC Educational Resources Information Center

    Schuster, Donald H.; And Others

    The Suggestive Accelerative Learning and Teaching Method uses aspects of suggestion and unusual styles of presenting material to accelerate classroom learning. The essence of this technique is the use of a combination of physical relaxation exercises, mental concentration and suggestive principles to strengthen a person's ego and expand his memory…

  18. The Journal of the Society for Accelerative Learning and Teaching. Volume 15, 1990.

    ERIC Educational Resources Information Center

    Journal of the Society for Accelerative Learning and Teaching, 1990

    1990-01-01

    Articles in this volume of the Journal of the Society for Accelerative Learning and Teaching (SALT) include the following: "Accelerated Learning Components in Elementary Classrooms"; "Ball-Stick Bird: Teaching with the Story Engram"; "A SALT Pilot Study in College Developmental Mathematics"; "Black Education in South Africa: Issues, Problems and…

  19. The Journal of the Society for Accelerative Learning and Teaching, Volume 10, 1985.

    ERIC Educational Resources Information Center

    Schuster, Don H., Ed.

    1985-01-01

    Four numbers of the journal contain a variety of articles on methods and programs of accelerative learning and teaching, including: "Music Therapy and Education"; "The Effects of Background Music on Vocabulary Learning"; "Terminating the Tyranny of Time from 21st Century Education"; "An Example of Limbic Learning"; "Orchestration of Internal…

  20. The Journal of the Society for Accelerative Learning and Teaching, Volume 9, 1984.

    ERIC Educational Resources Information Center

    Journal of the Society for Accelerative Learning and Teaching, 1984

    1984-01-01

    The four 1984 numbers of the journal contain articles on the following topics: reading and visual imagery; script preparation for accelerative learning and teaching; guided relaxation and baroque music's effects on student test performance; imagery in the creative process; suggestopedia in English as a second language (ESL) instruction;…

  1. The Journal of Suggestive-Accelerative Learning and Teaching, Volume 3, Number 2, Summer 1978.

    ERIC Educational Resources Information Center

    Schuster, Donald H., Ed.

    1978-01-01

    Contents of this issue are as follows: "Suggestopedia: The Use of Music and Suggestion in Learning and Hypermnesia:" by Elizabeth Risova Philipov; "A Two Year Evaluation of the Suggestive-Accelerative Learning and Teaching (SALT) Method in Central Iowa Public Schools" by D.H. Schuster and R.A. Prichard; "Improving Lecturer Performance Through…

  2. Brookhaven four-stage accel-decel production of low-energy highly stripped heavy ions

    SciTech Connect

    Barrette, J.; Thieberger, P.

    1981-01-01

    The dual MP tandem facility at Brookhaven has been used in a four-stage accel-decel mode to produce highly stripped S ion beams (Q = 10-16/sup +/). Fully stripped S ions were obtained at energies down to 8 MeV. The low energy limit is presently due to the inclined field configuration of the last acceleration tube.

  3. Journal of the Society for Accelerative Learning and Teaching; Volume 12, Number 1-4.

    ERIC Educational Resources Information Center

    Schuster, Don, Ed.

    1987-01-01

    The basic focus on this journal, which publishes a wide variety of articles on suggestive and accelerative learning, is Suggestopedia theory, research, and application. Articles in the volume presented here include: "The Effects of Superlearning on Retention/Hypermnesia of Rare English Words in College Students" (Lynn D. Anderson); "SALT in…

  4. The Journal of the Society for Accelerative Learning and Teaching. Volume 13, 1988.

    ERIC Educational Resources Information Center

    Journal of the Society for Accelerative Learning and Teaching, 1988

    1988-01-01

    These four 1988 issues of the journal of the Society for Accelerated Learning and Teaching (SALT) include the following articles: "Educating the Children of Changing Cultures"; "All Stars to Center Stage: Accelerative Learning in the School of Business"; "SALT in the First Grade Classroom"; "Unlearning Technologies: Coping with Anti-Suggestive…

  5. The Journal of Suggestive-Accelerative Learning and Teaching, Volume 5, Number 2.

    ERIC Educational Resources Information Center

    Journal of Suggestive-Accelerative Learning and Teaching, 1980

    1980-01-01

    A collection of articles concerning suggestive-accelerative learning and teaching (SALT) methods includes: "Suggestive Teaching Methods in the Soviet Union" (Eva Szalontai); "SALT Applied to Remedial Reading: A Critical Review" (Allyn Prichard and Jean Taylor); "The Waldorf Schools: An Artistic Approach to Education" (Earl J. Ogletree); "Effects…

  6. Detonative propagation and accelerative expansion of the Crab Nebula shock front.

    PubMed

    Gao, Yang; Law, Chung K

    2011-10-21

    The accelerative expansion of the Crab Nebula's outer envelope is a mystery in dynamics, as a conventional expanding blast wave decelerates when bumping into the surrounding interstellar medium. Here we show that the strong relativistic pulsar wind bumping into its surrounding nebula induces energy-generating processes and initiates a detonation wave that propagates outward to form the current outer edge, namely, the shock front, of the nebula. The resulting detonation wave, with a reactive downstream, then provides the needed power to maintain propagation of the shock front. Furthermore, relaxation of the curvature-induced reduction of the propagation velocity from the initial state of formation to the asymptotic, planar state of Chapman-Jouguet propagation explains the observed accelerative expansion. Potential richness in incorporating reactive fronts in the description of various astronomical phenomena is expected. PMID:22107499

  7. Experiments with highly ionized low-energy tandem accel-decel beams

    SciTech Connect

    Jones, K.W.; Johnson, B.M.; Meron, M.; Da-Hai, W.; Thieberger, P.; Barrette, J.; Schuch, R.; Schmidt-Boecking, H.; Tserruya, I.; Kruse, T.H.

    1982-01-01

    The present status of the production of low-energy highly ionized beams by the 4-stage tandem accel-decel method is surveyed, and their use in three atomic physics experiments is summarized. The experiments are: the measurement of the charge state dependence of K-x-ray production in S/sup +q/-Ar collisions at 10 MeV, the impact-parameter dependence of quasimolecular x rays in S/sup +15/-Ar collisions again at 10 MeV, and the determination of charge transfer cross sections in S/sup +q/ + Ar, He collisions. The results of the three experiments give evidence that the 4-stage accel-decel ion production technique is extremely powerful and useful.

  8. The Journal for the Society of Accelerative Learning and Teaching, Volume 11, Numbers 1-4, 1986.

    ERIC Educational Resources Information Center

    The Journal for the Society of Accelerative Learning and Teaching, 1986

    1986-01-01

    The four 1986 issues of the journal on suggestive and accelerative learning and teaching (SALT) include articles on these topics: SALT components in English composition instruction; music therapy for moderately retarded students; the brain and accelerated learning; accelerated learning and self-concept; remedial reading; versions of the concert…

  9. The Journal of Suggestive-Accelerative Learning and Teaching, Volume 5, Number 1. Proceedings of the SALT Conference (1980).

    ERIC Educational Resources Information Center

    Schuster, Donald, Ed.

    1980-01-01

    The proceedings of the 1980 Suggestive-Accelerative Learning and Teaching (SALT) conference include: "Creative Power in Action: Discovering Your 'Right' Mind" (Carole Austen and Hilary Johnson); "The Co-Creative Teacher: A Key Dimension in Accelerated Learning in the Classroom" (Susan M. Campbell); "The Application of Psychosynthesis to…

  10. The Journal of the Society for Accelerative Learning and Teaching. (Volume 5, Numbers 3 through Volume 6, Number 4).

    ERIC Educational Resources Information Center

    Journal of the Society for Accelerative Learning and Teaching, 1981

    1981-01-01

    Numbers 3 and 4 of volume 5 and numbers 1 through 4 of volume 6 of the journal, spanning fall 1980 through winter 1981, include articles concerning the individualized study center; consciousness, psychology, and education; suggestive-accelerative learning and suggestopedia; creativity; brain lateralization; the Lozanov method; biofeedback and…

  11. Cloning, Expression, and Characterization of a Thermophilic Endoglucanase, AcCel12B from Acidothermus cellulolyticus 11B

    PubMed Central

    Wang, Junling; Gao, Gui; Li, Yuwei; Yang, Liangzhen; Liang, Yanli; Jin, Hanyong; Han, Weiwei; Feng, Yan; Zhang, Zuoming

    2015-01-01

    The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U·mg−1, respectively. The Km and Vmax of AcCel12B for CMC were 25.47 mg·mL−1 and 131.75 U·mg−1, respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future. PMID:26506341

  12. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a type of ...

  13. Desirable limits of accelerative forces in a space-based materials processing facility

    NASA Technical Reports Server (NTRS)

    Naumann, Robert J.

    1990-01-01

    There are three categories of accelerations to be encountered on orbiting spacecraft: (1) quasi-steady accelerations, caused by atmospheric drag or by gravity gradients, 10(exp -6) to 10(exp -7) g sub o; (2) transient accelerations, caused by movements of the astronauts, mass translocations, landing and departure of other spacecraft, etc.; and (3) oscillary accelerations, caused by running machinery (fans, pumps, generators). Steady accelerations cause continuing displacements; transients cause time-limited displacements. The important aspect is the area under the acceleration curve, measured over a certain time interval. Note that this quantity is not equivalent to a velocity because of friction effects. Transient motions are probably less important than steady accelerations because they only produce constant displacements. If the accelerative forces were not equal and opposite, the displacement would increase with time. A steady acceleration will produce an increasing velocity of a particle, but eventually an equilibrium value will be reached where drag and acceleration forces are equal. From then on, the velocity will remain constant, and the displacement will increase linearly with time.

  14. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  15. Separation of dehydrogenases on polyaminomethylstyrene.

    PubMed

    Schöpp, W; Meinert, S; Thyfronitou, J; Aurich, H

    1975-01-29

    The binding of dehydrogenases, especially alcohol dehydrogenase, and other proteins to several ion exchangers and hydrophobic polymers was investigated. Quantitative parameters for the stability of the polymer-protein complexes (obtained form double reciprocal plots) indicate a high but different affinity of many proteins for polyaminomethylstyrene. The chromatography of a mixture of five dehydrogenases and human serum albumin on polyaminomethylstyrene is described. PMID:237012

  16. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  17. The Microbial Conversion of D-Sorbitol to L-Sorbose: An Interdisciplinary Experiment Illustrating an Industrial Process.

    ERIC Educational Resources Information Center

    Volker, Eugene J.; Schultz, Clyde

    1978-01-01

    Describes an experiment that has been introduced in the organic chemistry laboratory program at Shepherd College, Shepherdstown, West Virginia. It illustrates microbial reaction and its role in the synthesis of vitamin C. (HM)

  18. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... control the activity of the complex: pyruvate dehydrogenase phosphatase turns on (activates) the complex, while pyruvate dehydrogenase ... binding protein (the PDHX gene), and pyruvate dehydrogenase phosphatase (the PDP1 gene) have been identified in people ...

  19. 77 FR 53913 - Accellent

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-04

    ... Management Group (CMG), and Marathon Staffing. The Department's notice of determination was published in the Federal Register on July 10, 2012 (77 FR 40641). At the request of a company official, the Department... Management Group (CMG), Marathon Staffing, And Excel Personnel, Inc., Englewood, Colorado;...

  20. Lactate dehydrogenase-elevating virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This book chapter describes the taxonomic classification of Lactate dehydrogenase-elevating virus (LDV). Included are: host, genome, classification, morphology, physicochemical and physical properties, nucleic acid, proteins, lipids, carbohydrates, geographic range, phylogenetic properties, biologic...

  1. Alcohol Dehydrogenase from Methylobacterium organophilum

    PubMed Central

    Wolf, H. J.; Hanson, R. S.

    1978-01-01

    The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10−5 M for methanol and 8.2 × 10−5 M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum. Images PMID:80974

  2. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  3. Genetics Home Reference: lactate dehydrogenase deficiency

    MedlinePlus

    ... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

  4. Formaldehyde dehydrogenase preparations from Methylococcus capsulatus (Bath) comprise methanol dehydrogenase and methylene tetrahydromethanopterin dehydrogenase.

    PubMed

    Adeosun, Ekundayo K; Smith, Thomas J; Hoberg, Anne-Mette; Velarde, Giles; Ford, Robert; Dalton, Howard

    2004-03-01

    In methylotrophic bacteria, formaldehyde is an important but potentially toxic metabolic intermediate that can be assimilated into biomass or oxidized to yield energy. Previously reported was the purification of an NAD(P)(+)-dependent formaldehyde dehydrogenase (FDH) from the obligate methane-oxidizing methylotroph Methylococcus capsulatus (Bath), presumably important in formaldehyde oxidation, which required a heat-stable factor (known as the modifin) for FDH activity. Here, the major protein component of this FDH preparation was shown by biophysical techniques to comprise subunits of 64 and 8 kDa in an alpha(2)beta(2) arrangement. N-terminal sequencing of the subunits of FDH, together with enzymological characterization, showed that the alpha(2)beta(2) tetramer was a quinoprotein methanol dehydrogenase of the type found in other methylotrophs. The FDH preparations were shown to contain a highly active NAD(P)(+)-dependent methylene tetrahydromethanopterin dehydrogenase that was the probable source of the NAD(P)(+)-dependent formaldehyde oxidation activity. These results support previous findings that methylotrophs possess multiple pathways for formaldehyde dissimilation. PMID:14993320

  5. Cellobiose dehydrogenase in cellulose degradation

    SciTech Connect

    Eriksson, L.; Igarashi, Kiyohiko; Samejima, Masahiro

    1996-10-01

    Cellobiose dehydrogenase is produced by a variety of fungi. Although it was already discovered during the 70`s, it`s role in cellulose and lignin degradation is yet ambiguous. The enzyme contains both heme and FAD as prosthetic groups, and seems to have a domain specifically designed to bind the enzyme to cellulose. It`s affinity to amorphous cellulose is higher than to crystalline cellulose. We will report on the binding behavior of the enzyme, its usefulness in elucidation of cellulose structures and also, possibilities for applications such as its use in measuring individual and synergistic mechanisms for cellulose degradation by endo- and exo-glucanases.

  6. Betaine aldehyde dehydrogenase in sorghum.

    PubMed Central

    Wood, A J; Saneoka, H; Rhodes, D; Joly, R J; Goldsbrough, P B

    1996-01-01

    The ability to synthesize and accumulate glycine betaine is wide-spread among angiosperms and is thought to contribute to salt and drought tolerance. In plants glycine betaine is synthesized by the two-step oxidation of choline via the intermediate betaine aldehyde, catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). Two sorghum (Sorghum bicolor) cDNA clones, BADH1 and BADH15, putatively encoding betaine aldehyde dehydrogenase were isolated and characterized. BADH1 is a truncated cDNA of 1391 bp. BADH15 is a full-length cDNA clone, 1812 bp in length, predicted to encode a protein of 53.6 kD. The predicted amino acid sequences of BADH1 and BADH15 share significant homology with other plant BADHs. The effects of water deficit on BADH mRNA expression, leaf water relations, and glycine betaine accumulation were investigated in leaves of preflowering sorghum plants. BADH1 and BADH15 mRNA were both induced by water deficit and their expression coincided with the observed glycine betaine accumulation. During the course of 17 d, the leaf water potential in stressed sorghum plants reached -2.3 MPa. In response to water deficit, glycine betaine levels increased 26-fold and proline levels increased 108-fold. In severely stressed plants, proline accounted for > 60% of the total free amino acid pool. Accumulation of these compatible solutes significantly contributed to osmotic potential and allowed a maximal osmotic adjustment of 0.405 MPa. PMID:8934627

  7. Single motoneuron succinate dehydrogenase activity.

    PubMed

    Chalmers, G R; Edgerton, V R

    1989-07-01

    We have developed a quantitative histochemical assay for measurement of succinate dehydrogenase (SDH) activity in single motoneurons. A computer image processing system was used to quantify the histochemical enzyme reaction product and to follow the time course of the reaction. The optimal concentration for each of the ingredients of the incubation medium for the SDH reaction was determined and the importance of using histochemical "blanks" in the determination of enzymatic activity was demonstrated. The enzymatic activity was linear with respect to reaction time and tissue thickness. The procedure described meets the criteria generally considered essential for establishment of a quantitative histochemical assay. The assay was then used to examine the SDH activity of cat and rat motoneurons. It was found that motoneurons with a small soma size had a wide range of SDH activity, whereas those with a large soma size were restricted to low SDH activity. PMID:2732457

  8. Glucose-6-Phosphate Dehydrogenase Deficiency.

    PubMed

    Luzzatto, Lucio; Nannelli, Caterina; Notaro, Rosario

    2016-04-01

    G6PD is a housekeeping gene expressed in all cells. Glucose-6-phosphate dehydrogenase (G6PD) is part of the pentose phosphate pathway, and its main physiologic role is to provide NADPH. G6PD deficiency, one of the commonest inherited enzyme abnormalities in humans, arises through one of many possible mutations, most of which reduce the stability of the enzyme and its level as red cells age. G6PD-deficient persons are mostly asymptomatic, but they can develop severe jaundice during the neonatal period and acute hemolytic anemia when they ingest fava beans or when they are exposed to certain infections or drugs. G6PD deficiency is a global health issue. PMID:27040960

  9. AuPt Alloy on TiO2: A Selective and Durable Catalyst for L-Sorbose Oxidation to 2-Keto-Gulonic Acid.

    PubMed

    Chan-Thaw, Carine E; Chinchilla, Lidia E; Campisi, Sebastian; Botton, Gianluigi A; Prati, Laura; Dimitratos, Nikolaos; Villa, Alberto

    2015-12-21

    Pt nanoparticles were prepared by a sol immobilization route, deposited on supports with different acid/base properties (MgO, activated carbon, TiO2 , Al2O3, H-Mordenite), and tested in the selective oxidation of sorbose to 2-keto-gulonic acid (2-KGUA), an important precursor for vitamin C. In general, as the basicity of the support increased, a higher catalytic activity occurred. However, in most cases, a strong deactivation was observed. The best selectivity to 2-KGUA was observed with acidic supports (TiO2 and H-Mordenite) that were able to minimize the formation of C1/C2 products. We also demonstrated that, by alloying Pt to Au, it is possible to enhance significantly the selectivity of Pt-based catalysts. Moreover, the AuPt catalyst, unlike monometallic Pt, showed good stability in recycling because of the prevention of metal leaching during the reaction. PMID:26611807

  10. Genetics Home Reference: succinic semialdehyde dehydrogenase deficiency

    MedlinePlus

    ... a chemical that transmits signals in the brain (neurotransmitter) called gamma-amino butyric acid (GABA). The primary ... Diseases National Organization for Rare Disorders (NORD) Pediatric Neurotransmitter Disease Association GeneReviews (1 link) Succinic Semialdehyde Dehydrogenase ...

  11. Phosphorylation site on yeast pyruvate dehydrogenase complex

    SciTech Connect

    Uhlinger, D.J.

    1986-01-01

    The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the /sup 32/P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation.

  12. Proline dehydrogenase (oxidase) in cancer.

    PubMed

    Liu, Wei; Phang, James M

    2012-01-01

    Proline dehydrogenase (oxidase, PRODH/POX), the first enzyme in the proline degradative pathway, plays a special role in tumorigenesis and tumor development. Proline metabolism catalyzed by PRODH/POX is closely linked with the tricarboxylic acid (TCA) cycle and urea cycle. The proline cycle formed by the interconversion of proline and Δ(1) -pyrroline-5-carboxylate (P5C) between mitochondria and cytosol interlocks with pentose phosphate pathway. Importantly, by catalyzing proline to P5C, PRODH/POX donates electrons into the electron transport chain to generate ROS or ATP. In earlier studies, we found that PRODH/POX functions as a tumor suppressor to initiate apoptosis, inhibit tumor growth, and block the cell cycle, all by ROS signaling. It also suppresses hypoxia inducible factor signaling by increasing α-ketoglutarate. During tumor progression, PRODH/POX is under the control of various tumor-associated factors, such as tumor suppressor p53, inflammatory factor peroxisome proliferator-activated receptor gamma (PPARγ), onco-miRNA miR-23b*, and oncogenic transcription factor c-MYC. Recent studies revealed the two-sided features of PRODH/POX-mediated regulation. Under metabolic stress such as oxygen and glucose deprivation, PRODH/POX can be induced to serve as a tumor survival factor through ATP production or ROS-induced autophagy. The paradoxical roles of PRODH/POX can be understood considering the temporal and spatial context of the tumor. Further studies will provide additional insights into this protein and on its metabolic effects in tumors, which may lead to new therapeutic strategies. PMID:22886911

  13. An autosomal glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) polymorphism in human saliva.

    PubMed

    Tan, S G; Ashton, G C

    1976-01-01

    Glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) from human saliva has been demonstrated by the zymogram technique. Three phenotypes were found. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles Sgd-1 and Sgd-2. PMID:950237

  14. Benzene toxicity: emphasis on cytosolic dihydrodiol dehydrogenases

    SciTech Connect

    Bolcsak, L.E.

    1982-01-01

    Blood dyscrasias such as leukopenia and anemia have been clearly identified as consequences of chronic benzene exposure. The metabolites, phenol, catechol, and hydroquinone produced inhibition of /sup 59/Fe uptake in mice which followed the same time course as that produced by benzene. The inhibitor of benzene oxidation, 3-amino-1,2,4-triazole, mitigated the inhibitory effects of benzene and phenol only. These data support the contention that benzene toxicity is mediated by a metabolite and suggest that the toxicity of phenol is a consequence of its metabolism to hydroquinone and that the route of metabolism to catechol may also contribute to the production of toxic metabolite(s). The properties of mouse liver cytosolic dihydrodiol dehydrogenases were examined. These enzymes catalyze the NADP/sup +/-dependent oxidation of trans-1,2-dihydro-1,2-dihydroxybenzene (BDD) to catechol, a possible toxic metabolite of benzene produced via this metabolic route. Four distinct dihydrodiol dehydrogenases (DD1, DD2, DD3, and DD4) were purified to apparent homogeneity as judged by SDS polyacrylamide gel electrophoresis and isoelectric focusing. DD1 appeared to be identical to the major ketone reductase and 17..beta..-hydroxysteroid dehydrogenase activity in the liver. DD2 exhibited aldehyde reductase activity. DD3 and DD4 oxidized 17..beta..-hydroxysteroids, but no carbonyl reductase activity was detected. These relationships between BDD dehydrogenases and carbonyl reductase and/or 17..beta..-hydroxysteroid dehydrogenase activities were supported by several lines of evidence.

  15. Alteration of substrate specificity of alanine dehydrogenase

    PubMed Central

    Fernandes, Puja; Aldeborgh, Hannah; Carlucci, Lauren; Walsh, Lauren; Wasserman, Jordan; Zhou, Edward; Lefurgy, Scott T.; Mundorff, Emily C.

    2015-01-01

    The l-alanine dehydrogenase (AlaDH) has a natural history that suggests it would not be a promising candidate for expansion of substrate specificity by protein engineering: it is the only amino acid dehydrogenase in its fold family, it has no sequence or structural similarity to any known amino acid dehydrogenase, and it has a strong preference for l-alanine over all other substrates. By contrast, engineering of the amino acid dehydrogenase superfamily members has produced catalysts with expanded substrate specificity; yet, this enzyme family already contains members that accept a broad range of substrates. To test whether the natural history of an enzyme is a predictor of its innate evolvability, directed evolution was carried out on AlaDH. A single mutation identified through molecular modeling, F94S, introduced into the AlaDH from Mycobacterium tuberculosis (MtAlaDH) completely alters its substrate specificity pattern, enabling activity toward a range of larger amino acids. Saturation mutagenesis libraries in this mutant background additionally identified a double mutant (F94S/Y117L) showing improved activity toward hydrophobic amino acids. The catalytic efficiencies achieved in AlaDH are comparable with those that resulted from similar efforts in the amino acid dehydrogenase superfamily and demonstrate the evolvability of MtAlaDH specificity toward other amino acid substrates. PMID:25538307

  16. NAD + -dependent Formate Dehydrogenase from Plants

    PubMed Central

    Alekseeva, A.A.; Savin, S.S.; Tishkov, V.I.

    2011-01-01

    NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) widely occurs in nature. FDH consists of two identical subunits and contains neither prosthetic groups nor metal ions. This type of FDH was found in different microorganisms (including pathogenic ones), such as bacteria, yeasts, fungi, and plants. As opposed to microbiological FDHs functioning in cytoplasm, plant FDHs localize in mitochondria. Formate dehydrogenase activity was first discovered as early as in 1921 in plant; however, until the past decade FDHs from plants had been considerably less studied than the enzymes from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehydrogenases. PMID:22649703

  17. Two different dihydroorotate dehydrogenases in Lactococcus lactis.

    PubMed Central

    Andersen, P S; Jansen, P J; Hammer, K

    1994-01-01

    The pyrimidine de novo biosynthesis pathway has been characterized for a number of organisms. The general pathway consists of six enzymatic steps. In the characterization of the pyrimidine pathway of Lactococcus lactis, two different pyrD genes encoding dihydroorotate dehydrogenase were isolated. The nucleotide sequences of the two genes, pyrDa and pyrDb, have been determined. One of the deduced amino acid sequences has a high degree of homology to the Saccharomyces cerevisiae dihydroorotate dehydrogenase, and the other resembles the dihydroorotate dehydrogenase from Bacillus subtilis. It is possible to distinguish between the two enzymes in crude extracts by using different electron acceptors. We constructed mutants containing a mutated form of either one or the other or both of the pyrD genes. Only the double mutant is pyrimidine auxotrophic. Images PMID:8021180

  18. Fundamental molecular differences between alcohol dehydrogenase classes.

    PubMed Central

    Danielsson, O; Atrian, S; Luque, T; Hjelmqvist, L; Gonzàlez-Duarte, R; Jörnvall, H

    1994-01-01

    Two types of alcohol dehydrogenase in separate protein families are the "medium-chain" zinc enzymes (including the classical liver and yeast forms) and the "short-chain" enzymes (including the insect form). Although the medium-chain family has been characterized in prokaryotes and many eukaryotes (fungi, plants, cephalopods, and vertebrates), insects have seemed to possess only the short-chain enzyme. We have now also characterized a medium-chain alcohol dehydrogenase in Drosophila. The enzyme is identical to insect octanol dehydrogenase. It is a typical class III alcohol dehydrogenase, similar to the corresponding human form (70% residue identity), with mostly the same residues involved in substrate and coenzyme interactions. Changes that do occur are conservative, but Phe-51 is of functional interest in relation to decreased coenzyme binding and increased overall activity. Extra residues versus the human enzyme near position 250 affect the coenzyme-binding domain. Enzymatic properties are similar--i.e., very low activity toward ethanol (Km beyond measurement) and high selectivity for formaldehyde/glutathione (S-hydroxymethylglutathione; kcat/Km = 160,000 min-1.mM-1). Between the present class III and the ethanol-active class I enzymes, however, patterns of variability differ greatly, highlighting fundamentally separate molecular properties of these two alcohol dehydrogenases, with class III resembling enzymes in general and class I showing high variation. The gene coding for the Drosophila class III enzyme produces an mRNA of about 1.36 kb that is present at all developmental stages of the fly, compatible with the constitutive nature of the vertebrate enzyme. Taken together, the results bridge a previously apparent gap in the distribution of medium-chain alcohol dehydrogenases and establish a strictly conserved class III enzyme, consistent with an important role for this enzyme in cellular metabolism. Images PMID:8197167

  19. Psoriatic therapeutics and glucose-6-phosphate dehydrogenase.

    PubMed

    Cotton, D W; van Rossum, E

    1975-01-01

    The inhibitory effects of various agents on the enzyme glucose-6-phosphate dehydrogenase have been studied in vitro. Stress is laid on the calculation of kinetic parameters such as true K-I values. The most active inhibitor was methotrexate, closely followed by cGMP. The increase in inhibitory activity after incubation of methotrexate with liver slices is discussed. PMID:167665

  20. Multiple retinoid dehydrogenases in testes cytosol from alcohol dehydrogenase negative or positive deermice.

    PubMed

    Posch, K C; Napoli, J L

    1992-05-28

    Retinoic acid syntheses from retinol by cytosol from testes of alcohol dehydrogenase negative or positive deermice were similar in specific activity and in their insensitivity to 1 M ethanol or 100 mM 4-methylpyrazole. Anion-exchange followed by size-exclusion chromatography revealed multiple and similarly migrating peaks in each cytosol that had both retinol and retinal dehydrogenase activities. Thus, the effects of ethanol on testes cannot be caused by direct inhibition of cytosolic retinoic acid synthesis because retinoid dehydrogenases distinct from mouse class A2 alcohol dehydrogenases, which corresponds to human class I, occurred in testes and they were not inhibited by ethanol. These data also demonstrate the occurrence of multiple cytosolic retinoic acid synthesis activities and indicate that the two reactions of cytosolic retinoic acid synthesis, retinol and retinal dehydrogenation, may be catalyzed by enzymes that occur as complexes. PMID:1599517

  1. Characterization of xylitol dehydrogenase from Debaryomyces hansenii

    SciTech Connect

    Girio, F.M.; Amaral-Collaco, M.T.; Pelica, F.

    1996-01-01

    The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells of Debaryomyces hansenii was partially purified in two chromatographic steps, and characterization studies were carried out in order to investigate the role of the xylitol dehydrogenase-catalyzed step in the regulation of D-xylose metabolism. The enzyme was most active at pH 9.0-9.5, and exhibited a broad polyol specificity. The Michaelis constants for xylitol and NAD{sup +} were 16.5 and 0.55 mM, respectively. Ca{sup 2+}, Mg{sup 2+}, and Mn{sup 2+} did not affect the enzyme activity. Conversely, Zn{sup 2+}, Cd{sup 2+}, and Co{sup 2+} strongly inhibited the enzyme activity. It was concluded that NAD{sup +}-xylitol dehydrogenase from D. hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and K{sub m} value for xylitol, and therefore should be named L-iditol:NAD{sup +}-5-oxidoreductase (EC 1.1.1.14). The reason D. hansenii is a good xylitol producer is not because of its value of K for xylitol, which is low enough to assure its fast oxidation by NAD{sup +}-xylitol dehydrogenase. However, a higher K{sub m} value of xylitol dehydrogenase for NAD{sup +} compared to the K{sub m} values of other xylose-fermenting yeasts may be responsible for the higher xylitol yields. 22 refs., 4 figs., 2 tabs.

  2. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase.

    PubMed

    Hurley, J H; Thorsness, P E; Ramalingam, V; Helmers, N H; Koshland, D E; Stroud, R M

    1989-11-01

    The structure of isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] from Escherichia coli has been solved and refined at 2.5 A resolution and is topologically different from that of any other dehydrogenase. This enzyme, a dimer of identical 416-residue subunits, is inactivated by phosphorylation at Ser-113, which lies at the edge of an interdomain pocket that also contains many residues conserved between isocitrate dehydrogenase and isopropylmalate dehydrogenase. Isocitrate dehydrogenase contains an unusual clasp-like domain in which both polypeptide chains in the dimer interlock. Based on the structure of isocitrate dehydrogenase and conservation with isopropylmalate dehydrogenase, we suggest that the active site lies in an interdomain pocket close to the phosphorylation site. PMID:2682654

  3. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

    Peafowl, like other vertebrates, contain multiple forms of lactate dehydrogenase. The electrophoretic properties of the peafowl isoenzymes are unusual in that the isoenzyme from heart tissue can be either more or less anodic than that of muscle, depending on the pH. This finding focuses attention on the problem of isoenzyme identification. It is suggested that isoenzymes be identified on the basis of properties that are chemically and biologically more significant than electrophoretic mobility. PMID:5917779

  4. Neuropathology in Succinic Semialdehyde Dehydrogenase Deficiency

    PubMed Central

    Knerr, Ina; Gibson, K. Michael; Murdoch, Geoffrey; Salomons, Gajja S.; Jakobs, Cornelis; Combs, Susan; Pearl, Phillip L.

    2010-01-01

    Reported here is the novel finding of neuropathology in a patient with succinic semialdehyde dehydrogenase deficiency, an inherited disorder of γ-aminobutyric acid metabolism characterized by intellectual deficiency, hypotonia, and epilepsy, with 4-hydroxybutyric aciduria and abnormalities of the globus pallidus on neuroimaging. A 19-year-old woman of European origin with a neurodevelopmental disorder and epilepsy died unexpectedly in 1998. A postmortem examination was performed, with a final diagnosis of sudden unexpected death in epilepsy patients. Eight years later, her sister with a neurodevelopmental disorder presented at 13 years of age with seizures and was diagnosed with succinic semialdehyde dehydrogenase deficiency. In the decedent, succinic semialdehyde dehydrogenase deficiency was established at the molecular level, 10 years after her death, using genomic DNA from brain tissue specimens. The neuropathologic findings revealed striking discoloration of the globi pallidi, leptomeningeal congestion, and a scar in the frontal cortex. After detection of the pathogenic homozygous mutation c.1226G>A, p.Gly409Asp in the living sister, it was confirmed in the decedent. An underlying metabolic disease may be an additional risk factor for sudden unexpected death in epilepsy patients. PMID:20304328

  5. Succinate dehydrogenase-deficient gastrointestinal stromal tumors

    PubMed Central

    Wang, Ya-Mei; Gu, Meng-Li; Ji, Feng

    2015-01-01

    Most gastrointestinal stromal tumors (GISTs) are characterized by KIT or platelet-derived growth factor alpha (PDGFRA) activating mutations. However, there are still 10%-15% of GISTs lacking KIT and PDGFRA mutations, called wild-type GISTs (WT GISTs). Among these so-called WT GISTs, a small subset is associated with succinate dehydrogenase (SDH) deficiency, known as SDH-deficient GISTs. In addition, GISTs that occur in Carney triad and Carney-Stratakis syndrome represent specific examples of SDH-deficient GISTs. SDH-deficient GISTs locate exclusively in the stomach, showing predilection for children and young adults with female preponderance. The tumor generally pursues an indolent course and exhibits primary resistance to imatinib therapy in most cases. Loss of succinate dehydrogenase subunit B expression and overexpression of insulin-like growth factor 1 receptor (IGF1R) are common features of SDH-deficient GISTs. In WT GISTs without succinate dehydrogenase activity, upregulation of hypoxia-inducible factor 1α may lead to increased growth signaling through IGF1R and vascular endothelial growth factor receptor (VEGFR). As a result, IGF1R and VEGFR are promising to be the novel therapeutic targets of GISTs. This review will update the current knowledge on characteristics of SDH-deficient GISTs and further discuss the possible mechanisms of tumorigenesis and clinical management of SDH-deficient GISTs. PMID:25741136

  6. Dihydrodiol dehydrogenase and polycyclic aromatic hydrocarbon metabolism

    SciTech Connect

    Smithgall, T.E.

    1986-01-01

    Carcinogenic activation of polycyclic aromatic hydrocarbons by microsomal monoxygenases proceeds through trans-dihydrodiol metabolites to diol-epoxide ultimate carcinogens. This thesis directly investigated the role of dihydrodiol dehydrogenase, a cytosolic NAD(P)-linked oxidoreductase, in the detoxification of polycyclic aromatic trans-dihydrodiols. A wide variety of non-K-region trans-dihydrodiols were synthesized and shown to be substrates for the homogeneous rat liver dehydrogenase, including several potent proximate carcinogens derived from 7,12-dimethylbenz(a)anthracene, 5-methylchrysene, and benzo(a)pyrene. Since microsomal activation of polycyclic aromatic hydrocarbons is highly stereospecific, the stereochemical course of enzymatic trans-dihydrodiol oxidation was monitored using circular dichroism spectropolarimetry. The major product formed from the dehydrogenase-catalyzed oxidation of the trans-1,2-dihydrodiol of naphthalene was characterized using UV, IR, NMR, and mass spectroscopy, and appears to be 4-hydroxy-1,2-naphthoquinone. Mass spectral analysis suggests that an analogous hydroxylated o-quinone is formed as the major product of benzo(a)pyrene-7,8-dihydrodiol oxidation. Enzymatic oxidation of trans-dihydrodiols was shown to be potently inhibited by all of the major classes of the nonsteroidal antiinflammatory drugs. Enhancement of trans-dihydrodiol proximate carcinogen oxidation may protect against possible adverse effects of the aspirin-like drugs, and help maintain the balance between activation and detoxification of polycyclic aromatic hydrocarbons.

  7. Isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma.

    PubMed

    Kipp, Benjamin R; Voss, Jesse S; Kerr, Sarah E; Barr Fritcher, Emily G; Graham, Rondell P; Zhang, Lizhi; Highsmith, W Edward; Zhang, Jun; Roberts, Lewis R; Gores, Gregory J; Halling, Kevin C

    2012-10-01

    Somatic mutations in isocitrate dehydrogenase 1 and 2 genes are common in gliomas and help stratify patients with brain cancer into histologic and molecular subtypes. However, these mutations are considered rare in other solid tumors. The aims of this study were to determine the frequency of isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma and to assess histopathologic differences between specimens with and without an isocitrate dehydrogenase mutation. We sequenced 94 formalin-fixed, paraffin-embedded cholangiocarcinoma (67 intrahepatic and 27 extrahepatic) assessing for isocitrate dehydrogenase 1 (codon 132) and isocitrate dehydrogenase 2 (codons 140 and 172) mutations. Multiple histopathologic characteristics were also evaluated and compared with isocitrate dehydrogenase 1/2 mutation status. Of the 94 evaluated specimens, 21 (22%) had a mutation including 14 isocitrate dehydrogenase 1 and 7 isocitrate dehydrogenase 2 mutations. Isocitrate dehydrogenase mutations were more frequently observed in intrahepatic cholangiocarcinoma than in extrahepatic cholangiocarcinoma (28% versus 7%, respectively; P = .030). The 14 isocitrate dehydrogenase 1 mutations were R132C (n = 9), R132S (n = 2), R132G (n = 2), and R132L (n = 1). The 7 isocitrate dehydrogenase 2 mutations were R172K (n = 5), R172M (n = 1), and R172G (n = 1). Isocitrate dehydrogenase mutations were more frequently observed in tumors with clear cell change (P < .001) and poorly differentiated histology (P = .012). The results of this study show for the first time that isocitrate dehydrogenase 1 and 2 genes are mutated in cholangiocarcinoma. The results of this study are encouraging because it identifies a new potential target for genotype-directed therapeutic trials and may represent a potential biomarker for earlier detection of cholangiocarcinoma in a subset of cases. PMID:22503487

  8. Lactate dehydrogenase X, malate dehydrogenase and total protein in rat spermatozoa during epididymal transit.

    PubMed

    Vermouth, N T; Carriazo, C S; Ponce, R H; Blanco, A

    1986-01-01

    Lactate dehydrogenase isozyme X (LDH X), malate dehydrogenase (MDH) and total soluble protein have been determined in lysates of spermatozoa isolated from caput, corpus and cauda of rat epididymis. Transit of spermatozoa through epididymis is accompanied by a reduction of LDH X, MDH and total protein per cell in sexually rested animals. The profiles of reduction along epididymal segments are different for the three variables studied. Mating with receptive females during the 5 days prior to determinations increases significantly the levels of MDH in spermatozoa from all sections of epididymis and produces increase of total soluble protein in the cells contained in cauda. PMID:3956158

  9. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  10. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the blood... conditions known to cause increased lactic dehydrogenase levels. (b) Classification. Class I...

  11. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the blood... conditions known to cause increased lactic dehydrogenase levels. (b) Classification. Class I...

  12. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the blood... conditions known to cause increased lactic dehydrogenase levels. (b) Classification. Class I...

  13. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... blood cells), myocardial infarction (heart disease), and some forms of leukemia (cancer of the blood... conditions known to cause increased lactic dehydrogenase levels. (b) Classification. Class I...

  14. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary...

  15. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary...

  16. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary...

  17. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary...

  18. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... and plasma. Isocitric dehydrogenase measurements are used in the diagnosis and treatment of liver disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary...

  19. Lactate dehydrogenase in sickle cell disease.

    PubMed

    Stankovic Stojanovic, Katia; Lionnet, François

    2016-07-01

    Lactate dehydrogenase (LDH) activity is elevated in many pathological states. Interest in LDH activity in sickle cell disease (SCD) has developed out of an increased comprehension of the pathophysiological process and the clinical course of the disease. Elevated LDH activity in SCD comes from various mechanisms, especially intravascular hemolysis, as well as ischemia-reperfusion damage and tissular necrosis. Intravascular hemolysis is associated with vasoconstriction, platelet activation, endothelial damage, and vascular complications. LDH has been used as a diagnostic and prognostic factor of acute and chronic complications. In this review we have evaluated the literature where LDH activity was examined during steady-state or acute conditions in SCD. PMID:27138446

  20. Substrate specificity of sheep liver sorbitol dehydrogenase.

    PubMed Central

    Lindstad, R I; Köll, P; McKinley-McKee, J S

    1998-01-01

    The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of

  1. Methylenetetrahydrofolate dehydrogenase from Clostridium formicoaceticum and methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase (combined) from Clostridium thermoaceticum

    SciTech Connect

    Ljungdahl, L.G.; O'Brien, W.E.; Moore, M.R.; Liu, M.T.

    1980-01-01

    Methylenetetrahydrofolate dehydrogenase is widely distributed and has been found in every cell type investigated. The NAD-specific enzyme has been purified to homogeneity from Clostridium formicoaceticum and the NADP-specific enzyme has been obtained from Clostridium thermoaceticum. Other sources of the NADP-specific enzyme are Streptococcus species, Escherichia coli, Clostridium cylindrosporum, Salmonella typhimurium, yeast, liver from various animals, calf thymus, and plants. The NAD-specific enzyme has been demonstrated in Acetobacterium woodii, some methane bacteria, and in Ehrlich ascites tumor cells. Of considerable interest are the observations that in porcine and ovine livers, as well as in yeast, methylenetetrahydrofolate dehydrogenase purified to homogeneity also contains methylenetetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase activities. Now it appears that the purified methylenetetrahydrofolate dehydrogenase from C. thermoaceticum also has cyclohydrolase but not synthetase activity. Methylenetetrahydrofolate dehydrogenase has been discussed previously in this series, as has methenyltetrahydrofolate cyclohydrolase. In C. formicoaceticum and C. thermoaceticum these tetrahydrofolate-dependent enzymes participate in a sequence of metabolic reactions by which carbon dioxide is reduced to the methyl group of 5-methyltetrahydrofolate which in turn is utilized for the synthesis of acetate. This pathway provides the mechanism for disposing of reducing equivalents generated in glycolysis.

  2. Catecholamine regulation of lactate dehydrogenase in rat brain cell culture

    SciTech Connect

    Kumar, S.; McGinnis, J.F.; de Vellis, J.

    1980-03-25

    The mechanism of catecholamine induction of the soluble cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27) was studied in the rat glial tumor cell line, C6. Lactate dehydrogenase was partially purified from extracts of (/sup 3/H)leucine-labeled cells by affinity gel chromatography and quantitatively immunoprecipitated with anti-lactate dehydrogenase-5 IgG and with antilactate dehydrogenase-1 IgG. The immunoprecipitates were dissociated and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Using this methodology, the increased enzyme activity of lactate dehydrogenase in norepinephrine-treated C6 cells was observed to be concomitant with the increased synthesis of enzyme molecules. Despite the continued presence of norepinephrine, the specific increase in the rate of synthesis of lactate dehydrogenase was transient. It was first detected at 4 h, was maximum at 9 h, and returned to basal levels by 24 h. The half-life of lactate dehydrogenase enzyme activity was 36 h during the induction and 40 h during deinduction. The half-life for decay of /sup 3/H-labeled lactate dehydrogenase was 41 h. These observations suggest that the increase in lactate dehydrogenase activity in norepinephrine-treated cells does not involve any change in the rate of degradation. Norepinephrine increased the specific rate of synthesis of both lactate dehydrogenase-5 (a tetramer of four M subunits) and lactate dehydrogenase-1 (a tetramer of four H subunits), although to different extents. Since these subunits are coded for by two separate genes on separate chromosomes, it suggests that the regulatory mechanism involves at least two separate sites of action.

  3. Aldehyde dehydrogenase protein superfamily in maize.

    PubMed

    Zhou, Mei-Liang; Zhang, Qian; Zhou, Ming; Qi, Lei-Peng; Yang, Xiong-Bang; Zhang, Kai-Xuan; Pang, Jun-Feng; Zhu, Xue-Mei; Shao, Ji-Rong; Tang, Yi-Xiong; Wu, Yan-Min

    2012-11-01

    Maize (Zea mays ssp. mays L.) is an important model organism for fundamental research in the agro-biotechnology field. Aldehydes were generated in response to a suite of environmental stresses that perturb metabolism including salinity, dehydration, desiccation, and cold and heat shock. Many biologically important aldehydes are metabolized by the superfamily of NAD(P)(+)-dependent aldehyde dehydrogenases. Here, starting from the database of Z. mays, we identified 28 aldehyde dehydrogenase (ALDH) genes and 48 transcripts by the in silico cloning method using the ALDH-conserved domain amino acid sequence of Arabidopsis and rice as a probe. Phylogenetic analysis shows that all 28 members of the ALDH gene families were classified to ten distinct subfamilies. Microarray data and quantitative real-time PCR analysis reveal that ZmALDH9, ZmALDH13, and ZmALDH17 genes involve the function of drought stress, acid tolerance, and pathogens infection. These results suggested that these three ZmALDH genes might be potentially useful in maize genetic improvement. PMID:22983498

  4. Alcohol dehydrogenases from olive (Olea europaea) fruit.

    PubMed

    Salas, J J; Sánchez, J

    1998-05-01

    Alcohol dehydrogenase activity was detected in extracts from the pericarp tissues of developing olive fruits using hexanal as the substrate. Total activity in the crude extract was 20-fold higher with NADPH than with NADH. Three discrete enzymes were resolved by means of a purification protocol involving ammonium sulfate fractionation followed by ion-exchange and affinity chromatography. One of the enzymes was NAD-dependent and displayed a high K(m) for hexanal (K(m) = 2.1 mM). Two NADP-dependent alcohol dehydrogenases were resolved, one showing a high K(m) for hexanal (K(m) = 1.9 mM) and the second with a lower K(m) for the same substrate (K(m) = 0.04 mM). The three enzymes have been partially purified and their kinetic parameters and specificities for various aldehydes determined. The involvement of these enzymes in the biogenesis of six carbon alcohols constituent of the aroma of olive oil is discussed. PMID:9621451

  5. Phosphorylation-dephosphorylation of yeast pyruvate dehydrogenase

    SciTech Connect

    Uhlinger, D.J.; Reed, L.J.

    1986-05-01

    Pyruvate dehydrogenase complex (PDC) was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase (PDH) kinase activity was detected at any stage of the purification. However, the purified PDC was phosphorylated and inactivated by purified PDH kinase from bovine kidney mitochondria, Mg/sup 2 +/, and (..gamma..-/sup 32/P)ATP. The protein-bound radioactivity was localized in the PDH ..cap alpha.. subunit. The phosphorylated, inactivated PDC was dephosphorylated and reactivated with purified bovine PDH phosphatase, Mg/sup 2 +/, and Ca/sup 2 +/. From a tryptic digest of phosphorylated yeast PDC a radioactive peptide was isolated by anion and reverse phase HPLC. The sequence of this tetradecapeptide is Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphopeptide derived from the ..cap alpha.. subunit of bovine kidney and heart PDH: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

  6. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Malic dehydrogenase test system. 862.1500 Section 862.1500 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1500 Malic dehydrogenase test system....

  7. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactate dehydrogenase test system. 862.1440 Section 862.1440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1440 Lactate dehydrogenase...

  8. 21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactic dehydrogenase immunological test system. 866.5560 Section 866.5560 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5560 Lactic dehydrogenase immunological...

  9. BACTERIAL EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF ARABIDOPSIS THALIANA PYRUVATE DEHYDROGENASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pyruvate dehydrogenase complex (PDC) is a very large multi-component structure that catalyzes decarboxylation of pyruvate, yielding CO2, NADH, and acetyl-CoA as products. The decarboxylation reaction is catalyzed by pyruvate dehydrogenase (E1). The PDC occupies a key position in intermediary met...

  10. GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

    EPA Science Inventory

    While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

  11. Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase

    PubMed Central

    Napora-Wijata, Kamila; Strohmeier, Gernot A.; Sonavane, Manoj N.; Avi, Manuela; Robins, Karen; Winkler, Margit

    2013-01-01

    Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases. PMID:24970175

  12. Antimicrobial Cellobiose Dehydrogenase-Chitosan Particles.

    PubMed

    Tegl, Gregor; Thallinger, Barbara; Beer, Bianca; Sygmund, Christoph; Ludwig, Roland; Rollett, Alexandra; Nyanhongo, Gibson S; Guebitz, Georg M

    2016-01-13

    Increasing prevalence of chronic wounds and microbial infection constitute a severe health challenge. The situation is further complicated by emerging multidrug resistance making the treatment of infections increasingly difficult. Here, a novel antimicrobial system based on in situ release of hydrogen peroxide (H2O2) by cellobiose dehydrogenase (CDH) immobilized on chitosan (CTS) particles is described. Covalent immobilization using carbodiimide coupling lead to a higher amount of protein immobilized on CTS (104 μg CDH/mg CTS) when compared to noncovalent immobilization, which, however, showed highest recovery of CDH activity (0.01 U/mg CTS). The CDH-CTS in situ generated H2O2 completely inhibited growth of Escherichia coli and Staphylococcus aureus over a period of 24 h. This resilient antimicrobial system represents a novel strategy for preventing infection with potential application in counteracting microbial colonization of chronic wounds. PMID:26672396

  13. Crystal structure of Arabidopsis thaliana cytokinin dehydrogenase

    SciTech Connect

    Bae, Euiyoung; Bingman, Craig A.; Bitto, Eduard; Aceti, David J.; Phillips, Jr., George N.

    2008-08-13

    Since first discovered in Zea mays, cytokinin dehydrogenase (CKX) genes have been identified in many plants including rice and Arabidopsis thaliana, which possesses CKX homologues (AtCKX1-AtCKX7). So far, the three-dimensional structure of only Z. mays CKX (ZmCKX1) has been determined. The crystal structures of ZmCKX1 have been solved in the native state and in complex with reaction products and a slowly reacting substrate. The structures revealed four glycosylated asparagine residues and a histidine residue covalently linked to FAD. Combined with the structural information, recent biochemical analyses of ZmCKX1 concluded that the final products of the reaction, adenine and a side chain aldehyde, are formed by nonenzymatic hydrolytic cleavage of cytokinin imine products resulting directly from CKX catalysis. Here, we report the crystal structure of AtCKX7 (gene locus At5g21482.1, UniProt code Q9FUJ1).

  14. Fast internal dynamics in alcohol dehydrogenase

    SciTech Connect

    Monkenbusch, M.; Stadler, A. Biehl, R.; Richter, D.; Ollivier, J.; Zamponi, M.

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D{sub 2}O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

  15. Stability of immobilized yeast alcohol dehydrogenase

    SciTech Connect

    Ooshima, H.; Genko, Y.; Harano, Y.

    1981-12-01

    The effects of substrate on stabilities of native (NA) and three kinds of immobilized yeast alcohol dehydrogenase (IMA), namely PGA (the carrier; porous glass), SEA (agarose gel) prepared covalently, and AMA (anion-exchange resin) prepared ionically, were studied. The following results were obtained. 1) The deactivations of NA and IMA free from the substrate or in the presence of ethanol obey the first-order kinetics, whereas, in the presence of butyraldehyde, their deactivation behaviors are explained on the basis of coexistence of two components of YADHs, namely the labile E1 and the comparatively stable E2, with different first-order deactivation constants. (2) A few attempts for stabilization of IMA were carried out from the viewpoint of the effects of crosslinkages among the subunits of YADH for PGA and the multibonding between the carrier and enzyme for SEA. The former is effective for the stabilization, whereas the latter is not. (Refs. 19).

  16. Betaine aldehyde dehydrogenase isozymes of spinach

    SciTech Connect

    Hanson, A.D.; Weretilnyk, E.A.; Weigel, P.

    1986-04-01

    Betaine is synthesized in spinach chloroplasts via the pathway Choline ..-->.. Betaine Aldehyde ..-->.. Betaine; the second step is catalyzed by betaine aldehyde dehydrogenase (BADH). The subcellular distribution of BADH was determined in leaf protoplast lysates; BADH isozymes were separated by 6-9% native PAGE. The chloroplast stromal fraction contains a single BADH isozyme (number1) that accounts for > 80% of the total protoplast activity; the extrachloroplastic fraction has a minor isozyme (number2) which migrates more slowly than number1. Both isozymes appear specific for betaine aldehyde, are more active with NAD than NADP, and show a ca. 3-fold activity increase in salinized leaves. The phenotype of a natural variant of isozyme number1 suggests that the enzyme is a dimer.

  17. Fast internal dynamics in alcohol dehydrogenase.

    PubMed

    Monkenbusch, M; Stadler, A; Biehl, R; Ollivier, J; Zamponi, M; Richter, D

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains. PMID:26298156

  18. Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

    PubMed

    Hoey, M E; Allison, N; Scott, A J; Fewson, C A

    1987-12-15

    L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus. PMID:3325042

  19. Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

    PubMed Central

    Hoey, M E; Allison, N; Scott, A J; Fewson, C A

    1987-01-01

    L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus. PMID:3325042

  20. Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci

    PubMed Central

    Pavlova, Sylvia I.; Jin, Ling; Gasparovich, Stephen R.

    2013-01-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci. PMID:23637459

  1. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. PMID:25542170

  2. Pyruvate Dehydrogenase Complex from Chloroplasts of Pisum sativum L 1

    PubMed Central

    Williams, Michael; Randall, Douglas D.

    1979-01-01

    Pyruvate dehydrogenase complex is associated with intact chloroplasts and mitochondria of 9-day-old Pisum sativum L. seedlings. The ratio of the mitochondrial complex to the chloroplast complex activities is about 3 to 1. Maximal rates observed for chloroplast pyruvate dehydrogenase complex activity ranged from 6 to 9 micromoles of NADH produced per milligram of chlorophyll per hour. Osmotic rupture of pea chloroplasts released 88% of the complex activity, indicating that chloroplast pyruvate dehydrogenase complex is a stromal complex. The pH optimum for chloroplast pyruvate dehydrogenase complex was between 7.8 and 8.2, whereas the mitochondrial pyruvate dehydrogenase complex had a pH optimum between 7.3 and 7.7. Chloroplast pyruvate dehydrogenase complex activity was specific for pyruvate, dependent upon coenzyme A and NAD and partially dependent upon Mg2+ and thiamine pyrophosphate. Chloroplast-associated pyruvate dehydrogenase complex provides a direct link between pyruvate metabolism and chloroplast fatty acid biosynthesis by providing the substrate, acetyl-CoA, necessary for membrane development in young plants. Images PMID:16661100

  3. ALDEHYDE DEHYDROGENASES EXPRESSION DURING POSTNATAL DEVELOPMENT: LIVER VS. LUNG

    EPA Science Inventory

    Aldehydes are highly reactive molecules present in the environment, and can be produced during biotransformation of xenobiotics. Although the lung can be a major target for aldehyde toxicity, development of aldehyde dehydrogenases (ALDHs), which detoxify aldehydes, in lung has be...

  4. Genetics Home Reference: 3-beta-hydroxysteroid dehydrogenase deficiency

    MedlinePlus

    ... not by hormone test. Clin Endocrinol (Oxf). 2003 Mar;58(3):323-31. Citation on PubMed Pang S, ... dehydrogenase deficiency. Endocrinol Metab Clin North Am. 2001 Mar;30(1):81-99, vi-vii. Review. Citation ...

  5. Quinoprotein alcohol dehydrogenase from ethanol-grown Pseudomonas aeruginosa.

    PubMed Central

    Groen, B; Frank, J; Duine, J A

    1984-01-01

    Cell-free extracts of Pseudomonas aeruginosa strains, grown on ethanol, showed dye-linked alcohol dehydrogenase activities. The enzyme responsible for this activity was purified to homogeneity. It appeared to contain two molecules of pyrroloquinoline quinone per enzyme molecule. In many respects, it resembled other quinoprotein alcohol dehydrogenases (EC 1.1.99.8), having a substrate specificity intermediate between that of methanol dehydrogenases and ethanol dehydrogenases in this group. On the other hand, it also showed dissimilarities: the enzyme was found to be a monomer (Mr 101 000), to need only one molecule of the suicide substrate cyclopropanol to become fully inactivated, and to have a different aromatic amino acid composition. PMID:6439190

  6. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  7. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  8. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  9. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  10. N-acylethanolamines as novel alcohol dehydrogenase 3 substrates.

    PubMed

    Ivkovic, Milena; Dempsey, Daniel R; Handa, Sumit; Hilton, Joshua H; Lowe, Edward W; Merkler, David J

    2011-02-15

    N-acylethanolamines (NAEs) are members of the fatty acid amide family. The NAEs have been proposed to serve as metabolic precursors to N-acylglycines (NAGs). The sequential oxidation of the NAEs by an alcohol dehydrogenase and an aldehyde dehydrogenase would yield the N-acylglycinals and/or the NAGs. Alcohol dehydrogenase 3 (ADH3) is one enzyme that might catalyze this reaction. To define a potential role for ADH3 in NAE catabolism, we synthesized a set of NAEs and evaluated these as ADH3 substrates. NAEs were oxidized by ADH3, yielding the N-acylglycinals as the product. The (V/K)(app) values for the NAEs included here were low relative to cinnamyl alcohol. Our data show that the NAEs can serve as alcohol dehydrogenase substrates. PMID:21144815

  11. Aldehyde dehydrogenase inhibitors from the mushroom Clitocybe clavipes.

    PubMed

    Kawagishi, Hirokazu; Miyazawa, Toshiyuki; Kume, Hiroko; Arimoto, Yasushi; Inakuma, Takahiro

    2002-11-01

    Five fatty acid derivatives including three novel compounds were isolated from the mushroom Clitocybe clavipe. Their structures were elucidated by spectral analyses. These compounds inhibited aldehyde dehydrogenase in vitro. PMID:12444711

  12. Purification and properties of carbon monoxide dehydrogenase from Methanococcus vannielii.

    PubMed Central

    DeMoll, E; Grahame, D A; Harnly, J M; Tsai, L; Stadtman, T C

    1987-01-01

    Carbon monoxide dehydrogenase was purified to homogeneity from Methanococcus vannielii grown with formate as the sole carbon source. The enzyme is composed of subunits with molecular weights of 89,000 and 21,000 in an alpha 2 beta 2 oligomeric structure. The native molecular weight of carbon monoxide dehydrogenase, determined by gel electrophoresis, is 220,000. The enzyme from M. vannielii contains 2 g-atoms of nickel per mol of enzyme. Except for its relatively high pH optimum of 10.5 and its slightly greater net positive charge, the enzyme from M. vannielii closely resembles carbon monoxide dehydrogenase isolated previously from acetate-grown Methanosarcina barkeri. Carbon monoxide dehydrogenase from M. vannielii constitutes 0.2% of the soluble protein of the cell. By comparison the enzyme comprises 5% of the soluble protein in acetate-grown cells of M. barkeri and approximately 1% in methanol-grown cells. Images PMID:3624199

  13. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  14. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  15. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  16. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  17. Mammalian class IV alcohol dehydrogenase (stomach alcohol dehydrogenase): structure, origin, and correlation with enzymology.

    PubMed Central

    Parés, X; Cederlund, E; Moreno, A; Hjelmqvist, L; Farrés, J; Jörnvall, H

    1994-01-01

    The structure of a mammalian class IV alcohol dehydrogenase has been determined by peptide analysis of the protein isolated from rat stomach. The structure indicates that the enzyme constitutes a separate alcohol dehydrogenase class, in agreement with the distinct enzymatic properties; the class IV enzyme is somewhat closer to class I (the "classical" liver alcohol dehydrogenase; approximately 68% residue identities) than to the other classes (II, III, and V; approximately 60% residue identities), suggesting that class IV might have originated through duplication of an early vertebrate class I gene. The activity of the class IV protein toward ethanol is even higher than that of the classical liver enzyme. Both Km and kcat values are high, the latter being the highest of any class characterized so far. Structurally, these properties are correlated with replacements at the active site, affecting both substrate and coenzyme binding. In particular, Ala-294 (instead of valine) results in increased space in the middle section of the substrate cleft, Gly-47 (instead of a basic residue) results in decreased charge interactions with the coenzyme pyrophosphate, and Tyr-363 (instead of a basic residue) may also affect coenzyme binding. In combination, these exchanges are compatible with a promotion of the off dissociation and an increased turnover rate. In contrast, residues at the inner part of the substrate cleft are bulky, accounting for low activity toward secondary alcohols and cyclohexanol. Exchanges at positions 259-261 involve minor shifts in glycine residues at a reverse turn in the coenzyme-binding fold. Clearly, class IV is distinct in structure, ethanol turnover, stomach expression, and possible emergence from class I. PMID:8127901

  18. Enzymic and structural studies on Drosophila alcohol dehydrogenase and other short-chain dehydrogenases/reductases.

    PubMed

    Smilda, T; Kamminga, A H; Reinders, P; Baron, W; van Hylckama Vlieg, J E; Beintema, J J

    2001-05-01

    Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several secondary alcohols were used to determine the kinetic parameters kcat and Km. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity for R(-) alcohols. At a high concentration of R(-) alcohols substrate activation occurs. The kcat and Km values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those at low substrate concentrations. Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic domains of these enzymes, but they do not have the structural features required for binding NAD+. Probably these lyases descend from an SDR, which has lost the capability to bind NAD+, but the enzyme reaction mechanisms may still be similar. PMID:11443349

  19. Human liver aldehyde dehydrogenase: coenzyme binding

    SciTech Connect

    Kosley, L.L.; Pietruszko, R.

    1987-05-01

    The binding of (U-/sup 14/C) NAD to mitochondrial (E2) and cytoplasmin(E1) aldehyde dehydrogenase was measured by gel filtration and sedimentation techniques. The binding data for NAD and (E1) yielded linear Scatchard plots giving a dissociation constant of 25 (+/- 8) uM and the stoichiometry of 2 mol of NAD bound per mol of E1. The binding data for NAD and (E2) gave nonlinear Scatchard plots. The binding of NADH to E2 was measured via fluorescence enhancement; this could not be done with E1 because there was no signal. The dissociation constant for E2 by this technique was 0.7 (+/- 0.4) uM and stoichiometry of 1.0 was obtained. The binding of (U-/sup 14/C) NADH to (E1) and (E2) was also measured by the sedimentation technique. The binding data for (E1) and NADH gave linear Scatchard plots giving a dissociation constant of 13 (+/- 6) uM and the stoichiometry of 2.0. The binding data for NADH to (E2) gave nonlinear Scatchard plots. With (E1), the dissociation constants for both NAD and NADH are similar to those determined kinetically, but the stoichiometry is only half of that found by stopped flow technique. With (E2) the dissociation constant by fluorometric procedure was 2 orders of magnitude less than that from catalytic reaction.

  20. Targeting isocitrate dehydrogenase (IDH) in cancer.

    PubMed

    Fujii, Takeo; Khawaja, Muhammad Rizwan; DiNardo, Courtney D; Atkins, Johnique T; Janku, Filip

    2016-05-01

    Isocitrate dehydrogenase (IDH) is an essential enzyme for cellular respiration in the tricarboxylic acid (TCA) cycle. Recurrent mutations in IDH1 or IDH2 are prevalent in several cancers including glioma, acute myeloid leukemia (AML), cholangiocarcinoma and chondrosarcoma. The mutated IDH1 and IDH2 proteins have a gain-of-function, neomorphic activity, catalyzing the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG) by NADPH. Cancer-associated IDH mutations block normal cellular differentiation and promote tumorigenesis via the abnormal production of the oncometabolite 2-HG. High levels of 2-HG have been shown to inhibit α-KG dependent dioxygenases, including histone and deoxyribonucleic acid (DNA) demethylases, which play a key role in regulating the epigenetic state of cells. Current targeted inhibitors of IDH1 (AG120, IDH305), IDH2 (AG221), and pan-IDH1/2 (AG881) selectively inhibit mutant IDH protein and induce cell differentiation in in vitro and in vivo models. Preliminary results from phase I clinical trials with IDH inhibitors in patients with advanced hematologic malignancies have demonstrated an objective response rate ranging from 31% to 40% with durable responses (>1 year) observed. Furthermore, the IDH inhibitors have demonstrated early signals of activity in solid tumors with IDH mutations, including cholangiocarcinomas and low grade gliomas. PMID:27355333

  1. SAXS fingerprints of aldehyde dehydrogenase oligomers.

    PubMed

    Tanner, John J

    2015-12-01

    Enzymes of the aldehyde dehydrogenase (ALDH) superfamily catalyze the nicotinamide adenine dinucleotide-dependent oxidation of aldehydes to carboxylic acids. ALDHs are important in detoxification of aldehydes, amino acid metabolism, embryogenesis and development, neurotransmission, oxidative stress, and cancer. Mutations in genes encoding ALDHs cause metabolic disorders, including alcohol flush reaction (ALDH2), Sjögren-Larsson syndrome (ALDH3A2), hyperprolinemia type II (ALDH4A1), γ-hydroxybutyric aciduria (ALDH5A1), methylmalonic aciduria (ALDH6A1), pyridoxine dependent epilepsy (ALDH7A1), and hyperammonemia (ALDH18A1). We previously reported crystal structures and small-angle X-ray scattering (SAXS) analyses of ALDHs exhibiting dimeric, tetrameric, and hexameric oligomeric states (Luo et al., Biochemistry 54 (2015) 5513-5522; Luo et al., J. Mol. Biol. 425 (2013) 3106-3120). Herein I provide the SAXS curves, radii of gyration, and distance distribution functions for the three types of ALDH oligomer. The SAXS curves and associated analysis provide diagnostic fingerprints that allow rapid identification of the type of ALDH oligomer that is present in solution. The data sets provided here serve as a benchmark for characterizing oligomerization of ALDHs. PMID:26693506

  2. Eucalypt NADP-Dependent Isocitrate Dehydrogenase1

    PubMed Central

    Boiffin, Vincent; Hodges, Michael; Gálvez, Susana; Balestrini, Raffaella; Bonfante, Paola; Gadal, Pierre; Martin, Francis

    1998-01-01

    NADP-dependent isocitrate dehydrogenase (NADP-ICDH) activity is increased in roots of Eucalyptus globulus subsp. bicostata ex Maiden Kirkp. during colonization by the ectomycorrhizal fungus Pisolithus tinctorius Coker and Couch. To investigate the regulation of the enzyme expression, a cDNA (EgIcdh) encoding the NADP-ICDH was isolated from a cDNA library of E. globulus-P. tinctorius ectomycorrhizae. The putative polypeptide sequence of EgIcdh showed a high amino acid similarity with plant NADP-ICDHs. Because the deduced EgICDH protein lacks an amino-terminal targeting sequence and shows highest similarity to plant cytosolic ICDHs, it probably represents a cytoplasmic isoform. RNA analysis showed that the steady-state level of EgIcdh transcripts was enhanced nearly 2-fold in ectomycorrhizal roots compared with nonmycorrhizal roots. Increased accumulation of NADP-ICDH transcripts occurred as early as 2 d after contact and likely led to the observed increased enzyme activity. Indirect immunofluorescence microscopy indicated that NADP-ICDH was preferentially accumulated in the epidermis and stele parenchyma of nonmycorrhizal and ectomycorrhizal lateral roots. The putative role of cytosolic NADP-ICDH in ectomycorrhizae is discussed. PMID:9662536

  3. Aggregation states of mitochondrial malate dehydrogenase.

    PubMed Central

    Sánchez, S. A.; Hazlett, T. L.; Brunet, J. E.; Jameson, D. M.

    1998-01-01

    The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 +/- 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH-fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer-monomer dissociation constant for mMDH is below 10(-9) M. In contrast to reports in the literature suggesting a pH-dependent dissociation of mMDH, the oligomeric state of this mMDH-fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH-fluorescein conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer-monomer equilibrium. PMID:9792106

  4. Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities

    PubMed Central

    Chen, Che-Hong; Ferreira, Julio Cesar Batista; Gross, Eric R.; Mochly-Rosen, Daria

    2014-01-01

    A family of detoxifying enzymes called aldehyde dehydrogenases (ALDHs) has been a subject of recent interest, as its role in detoxifying aldehydes that accumulate through metabolism and to which we are exposed from the environment has been elucidated. Although the human genome has 19 ALDH genes, one ALDH emerges as a particularly important enzyme in a variety of human pathologies. This ALDH, ALDH2, is located in the mitochondrial matrix with much known about its role in ethanol metabolism. Less known is a new body of research to be discussed in this review, suggesting that ALDH2 dysfunction may contribute to a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer. Recent studies suggest that ALDH2 dysfunction is also associated with Fanconi anemia, pain, osteoporosis, and the process of aging. Furthermore, an ALDH2 inactivating mutation (termed ALDH2*2) is the most common single point mutation in humans, and epidemiological studies suggest a correlation between this inactivating mutation and increased propensity for common human pathologies. These data together with studies in animal models and the use of new pharmacological tools that activate ALDH2 depict a new picture related to ALDH2 as a critical health-promoting enzyme. PMID:24382882

  5. Postischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity

    PubMed Central

    Richards, Erica M.; Rosenthal, Robert E.; Kristian, Tibor; Fiskum, Gary

    2008-01-01

    The pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme that catalyzes the oxidative decarboxylation of pyruvate and represents the sole bridge between anaerobic and aerobic cerebral energy metabolism. Previous studies demonstrating loss of PDHC enzyme activity and immunoreactivity during reperfusion after cerebral ischemia suggest that oxidative modifications are involved. This study tested the hypothesis that hyperoxic reperfusion exacerbates loss of PDHC enzyme activity, possibly due to tyrosine nitration or S-nitrosation. We used a clinically relevant canine ventricular fibrillation cardiac arrest model in which, after resuscitation and ventilation on either 100% O2 (hyperoxic) or 21–30% O2 (normoxic), animals were sacrificed at 2 h reperfusion and the brains removed for enzyme activity and immunoreactivity measurements. Animals resuscitated under hyperoxic conditions exhibited decreased PDHC activity and elevated 3-nitrotyrosine immunoreactivity in the hippocampus but not the cortex, compared to nonischemic controls. These measures were unchanged in normoxic animals. In vitro exposure of purified PDHC to peroxynitrite resulted in a dose-dependent loss of activity and increased nitrotyrosine immunoreactivity. These results support the hypothesis that oxidative stress contributes to loss of hippocampal PDHC activity during cerebral ischemia and reperfusion and suggest that PDHC is a target of peroxynitrite. PMID:16716897

  6. Iodination of glyceraldehyde 3-phosphate dehydrogenase

    PubMed Central

    Thomas, Jean O.; Harris, J. Ieuan

    1970-01-01

    1. A high degree of homology in the positions of tyrosine residues in glyceraldehyde 3-phosphate dehydrogenase from lobster and pig muscle, and from yeast, prompted an examination of the reactivity of tyrosine residues in the enzyme. 2. Iodination of the enzyme from lobster muscle with low concentrations of potassium tri-[125I]-iodide led to the identification of tyrosine residues of differing reactivity. Tyrosine-46 appeared to be the most reactive in the native enzyme. 3. When the monocarboxymethylated enzyme was briefly treated with small amounts of iodine, iodination could be confined almost entirely to tyrosine-46 in the lobster enzyme; tyrosine-39 or tyrosine-42, or both, were also beginning to react. 4. These three tyrosine residues were also those that reacted most readily in the carboxymethylated pig and yeast enzymes. 5. The difficulties in attaining specific reaction of the native enzyme are considered. 6. The differences between our results and those of other workers are discussed. ImagesPLATE 1PLATE 2 PMID:5530750

  7. [Pyruvate dehydrogenase deficiency and cerebral malformations].

    PubMed

    Eirís, J; Alvarez-Moreno, A; Briones, P; Alonso-Alonso, C; Castro-Gago, M

    1996-10-01

    Pyruvate dehydrogenase (PDH) deficiency is a major cause of primary lactic acidosis and severe global developmental delay. A deficiency of PDH E1 alpha, a subunit of the PDH complex is a prominent cause of congenital lactic acidosis. The E1 alpha cDNA and corresponding genomic DNA have been located in the short arm of the X-chromosome (Xp22-1). A isolated 'cerebral' lactic acidosis with cerebral dysgenesis is a recognized pattern of presentation of PDH deficiency. Here, we report clinical features, magnetic resonance, and biochemical studies of two females aged 6 months (case 1) and 26 months (case 2). Both had severe development delay, minor dysmorphic features, microcephaly, severe hypoplasia of the corpus callosum, cerebral atrophy, ventricular dilatation and increase in serum lactate levels without systemic acidosis. Urinary organic acid profile was compatible with PDH deficiency. Increased CSF lactate and pyruvate levels and reduced total PDH and PDH E1 activities in muscle and fibroblasts were observed in case 1. Otherwise, decreased total PDH activity in muscle but not in fibroblasts was seen in case 2. The PDH E1á gene was sequenced in the case 1 and a deletion in exon 7 was demonstrated. Dysmorphism with severe cerebral malformations in female patients merits a metabolic evaluation, including determination of lactate and pyruvate levels in CSF. PMID:8983728

  8. Inhibitors of 17beta-hydroxysteroid dehydrogenase type 1.

    PubMed

    Brozic, P; Lanisnik Risner, T; Gobec, S

    2008-01-01

    Carcinogenesis of hormone-related cancers involves hormone-stimulated cell proliferation, which increases the number of cell divisions and the opportunity for random genetic errors. In target tissues, steroid hormones are interconverted between their potent, high affinity forms for their respective receptors and their inactive, low affinity forms. One group of enzymes responsible for these interconversions are the hydroxysteroid dehydrogenases, which regulate ligand access to steroid receptors and thus act at a pre-receptor level. As part of this group, the 17beta-hydroxysteroid dehydrogenases catalyze either oxidation of hydroxyl groups or reduction of keto groups at steroid position C17. The thoroughly characterized 17beta-hydroxysteroid dehydrogenase type 1 activates the less active estrone to estradiol, a potent ligand for estrogen receptors. This isoform is expressed in gonads, where it affects circulating levels of estradiol, and in peripheral tissue, where it regulates ligand occupancy of estrogen receptors. Inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 are thus highly interesting potential therapeutic agents for the control of estrogen-dependent diseases such as endometriosis, as well as breast and ovarian cancers. Here, we present the review on the recent development of inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 published and patented since the previous review of 17beta-hydroxysteroid dehydrogenase inhibitors of Poirier (Curr. Med. Chem., 2003, 10, 453). These inhibitors are divided into two separate groups according to their chemical structures: steroidal and non-steroidal 17beta-hydroxysteroid dehydrogenase type 1 inhibitors. Their estrogenic/ proliferative activities and selectivities over other 17beta-hydroxysteroid dehydrogenases that are involved in local regulation of estrogen action (types 2, 7 and 12) are also presented. PMID:18220769

  9. Succinate Dehydrogenase Loss in Familial Paraganglioma: Biochemistry, Genetics, and Epigenetics

    PubMed Central

    Her, Yeng F.; Maher, L. James

    2015-01-01

    It is counterintuitive that metabolic defects reducing ATP production can cause, rather than protect from, cancer. Yet this is precisely the case for familial paraganglioma, a form of neuroendocrine malignancy caused by loss of succinate dehydrogenase in the tricarboxylic acid cycle. Here we review biochemical, genetic, and epigenetic considerations in succinate dehydrogenase loss and present leading models and mysteries associated with this fascinating and important tumor. PMID:26294907

  10. Role of threonine dehydrogenase in Escherichia coli threonine degradation.

    PubMed Central

    Potter, R; Kapoor, V; Newman, E B

    1977-01-01

    Threonine was used as nitrogen source by Escherichia coli K-12 through a pathway beginning with the enzyme threonine dehydrogenase. The 2-amino-3-ketobutyrate formed was converted to glycine, and the glycine was converted to serine, which acted as the actual nitrogen donor. The enzyme formed under anaerobic conditions and known as threonine deaminase (biodegradative) is less widespread than threonine dehydrogenase and may be involved in energy metabolism rather than in threonine degradation per se. PMID:334738

  11. Retinol dehydrogenase 10 but not retinol/sterol dehydrogenase(s) regulates the expression of retinoic acid-responsive genes in human transgenic skin raft culture.

    PubMed

    Lee, Seung-Ah; Belyaeva, Olga V; Wu, Lizhi; Kedishvili, Natalia Y

    2011-04-15

    Retinoic acid is essential for skin growth and differentiation, and its concentration in skin is controlled tightly. In humans, four different members of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins were proposed to catalyze the rate-limiting step in the biosynthesis of retinoic acid (the oxidation of retinol to retinaldehyde). Epidermis contains at least three of these enzymes, but their relative importance for retinoic acid biosynthesis and regulation of gene expression during growth and differentiation of epidermis is not known. Here, we investigated the effect of the four human SDRs on retinoic acid biosynthesis, and their impact on growth and differentiation of keratinocytes using organotypic skin raft culture model of human epidermis. The results of this study demonstrate that ectopic expression of retinol dehydrogenase 10 (RDH10, SDR16C4) in skin rafts dramatically increases proliferation and inhibits differentiation of keratinocytes, consistent with the increased steady-state levels of retinoic acid and activation of retinoic acid-inducible genes in RDH10 rafts. In contrast, SDRs with dual retinol/sterol substrate specificity, namely retinol dehydrogenase 4 (RoDH4, SDR9C8), RoDH-like 3α-hydroxysteroid dehydrogenase (RL-HSD, SDR9C6), and RDH-like SDR (RDHL, SDR9C4) do not affect the expression of retinoic acid-inducible genes but alter the expression levels of several components of extracellular matrix. These results reveal essential differences in the metabolic contribution of RDH10 versus retinol/sterol dehydrogenases to retinoic acid biosynthesis and provide the first evidence that non-retinoid metabolic products of retinol/sterol dehydrogenases affect gene expression in human epidermis. PMID:21345790

  12. Retinol Dehydrogenase 10 but Not Retinol/Sterol Dehydrogenase(s) Regulates the Expression of Retinoic Acid-responsive Genes in Human Transgenic Skin Raft Culture*

    PubMed Central

    Lee, Seung-Ah; Belyaeva, Olga V.; Wu, Lizhi; Kedishvili, Natalia Y.

    2011-01-01

    Retinoic acid is essential for skin growth and differentiation, and its concentration in skin is controlled tightly. In humans, four different members of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins were proposed to catalyze the rate-limiting step in the biosynthesis of retinoic acid (the oxidation of retinol to retinaldehyde). Epidermis contains at least three of these enzymes, but their relative importance for retinoic acid biosynthesis and regulation of gene expression during growth and differentiation of epidermis is not known. Here, we investigated the effect of the four human SDRs on retinoic acid biosynthesis, and their impact on growth and differentiation of keratinocytes using organotypic skin raft culture model of human epidermis. The results of this study demonstrate that ectopic expression of retinol dehydrogenase 10 (RDH10, SDR16C4) in skin rafts dramatically increases proliferation and inhibits differentiation of keratinocytes, consistent with the increased steady-state levels of retinoic acid and activation of retinoic acid-inducible genes in RDH10 rafts. In contrast, SDRs with dual retinol/sterol substrate specificity, namely retinol dehydrogenase 4 (RoDH4, SDR9C8), RoDH-like 3α-hydroxysteroid dehydrogenase (RL-HSD, SDR9C6), and RDH-like SDR (RDHL, SDR9C4) do not affect the expression of retinoic acid-inducible genes but alter the expression levels of several components of extracellular matrix. These results reveal essential differences in the metabolic contribution of RDH10 versus retinol/sterol dehydrogenases to retinoic acid biosynthesis and provide the first evidence that non-retinoid metabolic products of retinol/sterol dehydrogenases affect gene expression in human epidermis. PMID:21345790

  13. Light modulation of glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase by photosynthetic electron flow in pea chloroplasts

    SciTech Connect

    Akamba, L.M.; Anderson, L.E.

    1981-02-01

    Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) and light inactivation of glucose-6-P dehydrogenase (EC 1.1.1.49) appear to be modulated within pea leaf chloroplasts by mediators which are reduced by photosynthetic electron flow from the photosystem I reaction center. Dichlorophenyl-1,1-dimethylurea inhibition of this modulation can be completely reversed by ascorbate plus 2,6-dichlorophenolindophenol in broken chloroplasts, but not in intact chloroplasts. Intact chloroplasts are impermeable to 2,6-dichlorophenolindophenol at pH 7.5. Studies on the effect of light in reconstituted chloroplasts with photosystem I-enriched particles in the place of whole thylakoids revealed that photosystem I participants in the light modulation of NADP-linked glyceraldehyde-3-P dehydrogenase and of glucose-6-P dehydrogenase.

  14. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    SciTech Connect

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.; Kakar, S. N.; Stevens, F. J.; Donnelly, M. I.; Nebraska Wesleyan Univ.

    1995-09-10

    Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the native enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant

  15. Journal of Accelerative Learning and Teaching, 1995.

    ERIC Educational Resources Information Center

    Journal of Accelerative Learning and Teaching, 1995

    1995-01-01

    Issues 1 and 2 (combined) of the 1995 journal contain these articles: "Accelerated Learning in a Beginning College-Level French Class at the University of Houston" (Patrice Caux); "The Psychobiology of Learning and Memory" (Don Schuster); "Do the Seeds of Accelerated Language Learning and Teaching Lie in a Behavioral Carrier Wave?" (R. M. Dunham,…

  16. Structural Studies of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand S.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Human pyruvate dehydrogenase (E1) catalyzes the irreversible decarboxylation of pyruvate in the presence of Mg(2+) and thiamin pyrophosphate (TPP) followed by the rate-limiting reductive acetylation of the lipoyl moiety linked to dihydrolipoamide acetyltransferase. The three-dimensional structure of human E1 is elucidated using the methods of macromolecular X-ray crystallography. The structure is an alpha, alpha', beta and beta' tetramer with the protein units being in the tetrahedral arrangement. Each 361-residue alpha-subunit and 329-residue beta-subunit is composed of a beta-sheet core surrounded by alpha-helical domains. Each subunit is in extensive contact with all the three subunits involving TPP and magnesium cofactors, and potassium ions. The two binding sites for TPP are at the alpha-beta' and alpha'-beta interfaces, each involving a magnesium ion and Phe6l, His63, Tyr89, and Met200 from the alpha-subunit (or alpha'-subunit), and Met81 Phe85, His128 from the beta-subunit (or beta'-subunit). K+ ions are nestled between two beta-sheets and the end of an alpha-helix in each beta-subunit, where they are coordinated by four carbonyl oxygen groups from Ile12, Ala160, Asp163, and Asnl65, and a water molecule. The catalytic C2 carbon of thiazolium ring in this structure forms a 3.2 A contact with a water molecule involved in a series of H-bonds with other water molecules, and indirectly with amino acids including those involved in the catalysis and regulation of the enzyme.

  17. Succinate dehydrogenase gene mutations in cardiac paragangliomas.

    PubMed

    Martucci, Victoria L; Emaminia, Abbas; del Rivero, Jaydira; Lechan, Ronald M; Magoon, Bindiya T; Galia, Analyza; Fojo, Tito; Leung, Steve; Lorusso, Roberto; Jimenez, Camilo; Shulkin, Barry L; Audibert, Jennifer L; Adams, Karen T; Rosing, Douglas R; Vaidya, Anand; Dluhy, Robert G; Horvath, Keith A; Pacak, Karel

    2015-06-15

    Pheochromocytomas and paragangliomas are chromaffin cell tumors arising from neuroendocrine cells. At least 1/3 of paragangliomas are related to germline mutations in 1 of 17 genes. Although these tumors can occur throughout the body, cardiac paragangliomas are very rare, accounting for <0.3% of mediastinal tumors. The purpose of this study was to determine the clinical characteristics of patients with cardiac paragangliomas, particularly focusing on their genetic backgrounds. A retrospective chart analysis of 15 patients with cardiac paragangliomas was performed to determine clinical presentation, genetic background, diagnostic workup, and outcomes. The average age at diagnosis was 41.9 years. Typical symptoms of paraganglioma (e.g., hypertension, sweating, palpitations, headache) were reported at initial presentation in 13 patients (86.7%); the remaining 2, as well as 4 symptomatic patients, initially presented with cardiac-specific symptoms (e.g., chest pain, dyspnea). Genetic testing was done in 13 patients (86.7%); 10 (76.9%) were positive for mutations in succinate dehydrogenase (SDHx) subunits B, C, or D. Thirteen patients (86.7%) underwent surgery to remove the paraganglioma with no intraoperative morbidity or mortality; 1 additional patient underwent surgical resection but experienced intraoperative complications after removal of the tumor due to co-morbidities and did not survive. SDHx mutations are known to be associated with mediastinal locations and malignant behavior of paragangliomas. In this report, the investigators extend the locations of predominantly SDHx-related paragangliomas to cardiac tumors. In conclusion, cardiac paragangliomas are frequently associated with underlying SDHx germline mutations, suggesting a need for genetic testing of all patients with this rare tumor. PMID:25896150

  18. The Carbon Monoxide Dehydrogenase from Desulfovibrio vulgaris.

    PubMed

    Hadj-Saïd, Jessica; Pandelia, Maria-Eirini; Léger, Christophe; Fourmond, Vincent; Dementin, Sébastien

    2015-12-01

    Ni-containing Carbon Monoxide Dehydrogenases (CODHs) catalyze the reversible conversion between CO and CO₂and are involved in energy conservation and carbon fixation. These homodimeric enzymes house two NiFeS active sites (C-clusters) and three accessory [4Fe-4S] clusters. The Desulfovibrio vulgaris (Dv) genome contains a two-gene CODH operon coding for a CODH (cooS) and a maturation protein (cooC) involved in nickel insertion in the active site. According to the literature, the question of the precise function of CooC as a chaperone folding the C-cluster in a form which accommodates free nickel or as a mere nickel donor is not resolved. Here, we report the biochemical and spectroscopic characterization of two recombinant forms of the CODH, produced in the absence and in the presence of CooC, designated CooS and CooS(C), respectively. CooS contains no nickel and cannot be activated, supporting the idea that the role of CooC is to fold the C-cluster so that it can bind nickel. As expected, CooS(C) is Ni-loaded, reversibly converts CO and CO₂, displays the typical Cred1 and Cred2 EPR signatures of the C-cluster and activates in the presence of methyl viologen and CO in an autocatalytic process. However, Ni-loaded CooS(C) reaches maximum activity only upon reductive treatment in the presence of exogenous nickel, a phenomenon that had not been observed before. Surprisingly, the enzyme displays the Cred1 and Cred2 signatures whether it has been activated or not, showing that this activation process of the Ni-loaded Dv CODH is not associated with structural changes at the active site. PMID:26255854

  19. Assessment of toxicity using dehydrogenases activity and mathematical modeling.

    PubMed

    Matyja, Konrad; Małachowska-Jutsz, Anna; Mazur, Anna K; Grabas, Kazimierz

    2016-07-01

    Dehydrogenase activity is frequently used to assess the general condition of microorganisms in soil and activated sludge. Many studies have investigated the inhibition of dehydrogenase activity by various compounds, including heavy metal ions. However, the time after which the measurements are carried out is often chosen arbitrarily. Thus, it can be difficult to estimate how the toxic effects of compounds vary during the reaction and when the maximum of the effect would be reached. Hence, the aim of this study was to create simple and useful mathematical model describing changes in dehydrogenase activity during exposure to substances that inactivate enzymes. Our model is based on the Lagergrens pseudo-first-order equation, the rate of chemical reactions, enzyme activity, and inactivation and was created to describe short-term changes in dehydrogenase activity. The main assumption of our model is that toxic substances cause irreversible inactivation of enzyme units. The model is able to predict the maximum direct toxic effect (MDTE) and the time to reach this maximum (TMDTE). In order to validate our model, we present two examples: inactivation of dehydrogenase in microorganisms in soil and activated sludge. The model was applied successfully for cadmium and copper ions. Our results indicate that the predicted MDTE and TMDTE are more appropriate than EC50 and IC50 for toxicity assessments, except for long exposure times. PMID:27021434

  20. Characterization and purification of carbon monoxide dehydrogenase from Methanosarcina barkeri.

    PubMed Central

    Krzycki, J A; Zeikus, J G

    1984-01-01

    Carbon monoxide-dependent production of H2, CO2, and CH4 was detected in crude cell extracts of acetate-grown Methanosarcina barkeri. This metabolic transformation was associated with an active methyl viologen-linked CO dehydrogenase activity (5 to 10 U/mg of protein). Carbon monoxide dehydrogenase activity was inhibited 85% by 10 microM KCN and was rapidly inactivated by O2. The enzyme was nearly homogeneous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%). The native purified enzyme displayed a molecular weight of 232,000 and a two-subunit composition of 92,000 and 18,000 daltons. The enzyme was shown to contain nickel by isolation of radioactive CO dehydrogenase from cells grown in 63Ni. Analysis of enzyme kinetic properties revealed an apparent Km of 5 mM for CO and a Vmax of 1,300 U/mg of protein. The spectral properties of the enzyme were similar to those published for CO dehydrogenase from acetogenic anaerobes. The physiological functions of the enzyme are discussed. Images PMID:6425262

  1. Dehydrogenase activity of forest soils depends on the assay used

    NASA Astrophysics Data System (ADS)

    Januszek, Kazimierz; Długa, Joanna; Socha, Jarosław

    2015-01-01

    Dehydrogenases are exclusively intracellular enzymes, which play an important role in the initial stages of oxidation of soil organic matter. One of the most frequently used methods to estimate dehydrogenase activity in soil is based on the use of triphenyltetrazolium chloride as an artificial electron acceptor. The purpose of this study was to compare the activity of dehydrogenases of forest soils with varied physicochemical properties using different triphenyltetrazolium chloride assays. The determination was carried out using the original procedure by Casida et al., a modification of the procedure which involves the use of Ca(OH)2 instead of CaCO3, the Thalmann method, and the assay by Casida et al. without addition of buffer or any salt. Soil dehydrogenase activity depended on the assay used. Dehydrogenase determined by the Casida et al. method without addition of buffer or any salt correlated with the pH values of soils. The autoclaved strongly acidic samples of control soils showed high concentrations of triphenylformazan, probably due to chemical reduction of triphenyltetrazolium chloride. There is, therefore, a need for a sterilization method other than autoclaving, ie a process that results in significant changes in soil properties, thus helping to increase the chemical reduction of triphenyltetrazolium chloride.

  2. Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex

    SciTech Connect

    Park, Yun-Hee; Patel, Mulchand S.

    2010-05-07

    Unlike pyruvate dehydrogenase complexes (PDCs) from prokaryotes, PDCs from higher eukaryotes have an additional structural component, E3-binding protein (BP), for binding of dihydrolipoamide dehydrogenase (E3) in the complex. Based on the 3D structure of the subcomplex of human (h) E3 with the di-domain (L3S1) of hBP, the amino acid residues (H348, D413, Y438, and R447) of hE3 for binding to hBP were substituted singly by alanine or other residues. These substitutions did not have large effects on hE3 activity when measured in its free form. However, when these hE3 mutants were reconstituted in the complex, the PDC activity was significantly reduced to 9% for Y438A, 20% for Y438H, and 18% for D413A. The binding of hE3 mutants with L3S1 determined by isothermal titration calorimetry revealed that the binding affinities of the Y438A, Y438H, and D413A mutants to L3S1 were severely reduced (1019-, 607-, and 402-fold, respectively). Unlike wild-type hE3 the binding of the Y438A mutant to L3S1 was accompanied by an unfavorable enthalpy change and a large positive entropy change. These results indicate that hE3-Y438 and hE3-D413 play important roles in binding of hE3 to hBP.

  3. Evaluation of NAD(P)-Dependent Dehydrogenase Activities in Neutrophilic Granulocytes by the Bioluminescent Method.

    PubMed

    Savchenko, A A

    2015-09-01

    Bioluminescent method for measurements of the neutrophilic NAD(P)-dependent dehydrogenases (lactate dehydrogenase, NAD-dependent malate dehydrogenase, NADP-dependent decarboxylating malate dehydrogenase, NAD-dependent isocitrate dehydrogenase, and glucose- 6-phosphate dehydrogenase) is developed. The sensitivity of the method allows minimization of the volume of biological material for measurements to 104 neutrophils per analysis. The method is tried in patients with diffuse purulent peritonitis. Low levels of NADPH synthesis enzymes and high levels of enzymes determining the substrate flow by the Krebs cycle found in these patients can lead to attenuation of functional activity of cells. PMID:26468025

  4. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci.

    PubMed

    Peralta, Guillermo Hugo; Bergamini, Carina Viviana; Hynes, Erica Rut

    2016-01-01

    Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor. PMID:27266631

  5. Elevated plasma citrulline: look for dihydrolipoamide dehydrogenase deficiency.

    PubMed

    Haviv, Ruby; Zeharia, Avraham; Belaiche, Corinne; Haimi Cohen, Yishai; Saada, Ann

    2014-02-01

    The E3 subunit of the pyruvate dehydrogenase complex (dihydrolipoamide dehydrogenase/dihydrolipoyl dehydrogenase/DLD/lipoamide dehydrogenase/LAD), is a mitochondrial matrix enzyme and also a part of the branched-chain ketoacid dehydrogenase and alpha-ketoglutarate dehydrogenase complexes. DLD deficiency (MIM #246900), is relatively frequent in the Ashkenazi Jewish population but occurs in other populations as well. Early diagnosis is important to prevent episodes of metabolic decompensation, liver failure, and encephalopathy. The clinical presentations are varied and may include Reye-like syndrome, hepatic failure, myopathy, and myoglobinuria. Laboratory markers, such as elevated urinary alpha-ketoglutarate, blood pyruvate, lactate, and ammonia, are mostly nonspecific and not always present, making the diagnosis difficult. Since we observed elevated plasma citrulline levels in a number of confirmed cases, we retrospectively examined the value of citrulline as a biochemical marker for DLD deficiency. Data was gathered from the files of 17 pediatric patients with DLD deficiency, confirmed by enzymatic and genetic analysis. The control group included 19 patients in whom urea cycle defects were ruled out but DLD deficiency was suspected. Seven of the DLD-deficient patients presented with elevated plasma citrulline levels (median value 205 μM, range 59-282 μM) (normal range 1-45 μM) while none in the control patient group. In five patients, elevated citrulline was associated with elevated plasma glutamine and metabolic acidosis. Interestingly, elevated plasma citrulline was associated with the common G229C mutation. In conclusion, we suggest that elevated plasma citrulline in the absence of urea cycle defects warrants an investigation for DLD deficiency. PMID:23995961

  6. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

    SciTech Connect

    Bulfer, Stacie L.; Hendershot, Jenna M.; Trievel, Raymond C.

    2013-09-18

    Lysine biosynthesis in fungi, euglena, and certain archaebacteria occurs through the {alpha}-aminoadipate pathway. Enzymes in the first steps of this pathway have been proposed as potential targets for the development of antifungal therapies, as they are absent in animals but are conserved in several pathogenic fungi species, including Candida, Cryptococcus, and Aspergillus. One potential antifungal target in the {alpha}-aminoadipate pathway is the third enzyme in the pathway, homoisocitrate dehydrogenase (HICDH), which catalyzes the divalent metal-dependent conversion of homoisocitrate to 2-oxoadipate (2-OA) using nicotinamide adenine dinucleotide (NAD{sup +}) as a cofactor. HICDH belogns to a family of {beta}-hydroxyacid oxidative decarboxylases that includes malate dehydrogenase, tartrate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase (ICDH), and 3-isopropylmalte dehydrogenase (IPMDH). ICDH and IPMDH are well-characterized enzymes that catalyze the decarboxylation of isocitrate to yield 2-oxoglutarate (2-OG) in the citric acid cycle and the conversion of 3-isopropylmalate to 2-oxoisovalerate in the leucine biosynthetic pathway, respectively. Recent structural and biochemical studies of HICDH reveal that this enzyme shares sequence, structural, and mechanistic homology with ICDH and IPMDH. To date, the only published structures of HICDH are from the archaebacteria Thermus thermophilus (TtHICDH). Fungal HICDHs diverge from TtHICDH in several aspects, including their thermal stability, oligomerization state, and substrate specificity, thus warranting further characterization. To gain insights into these differences, they determined crystal structures of a fungal Schizosaccharomyces pombe HICDH (SpHICDH) as an apoenzyme and as a binary complex with additive tripeptide glycyl-glycyl-glycine (GGG) to 1.55 {angstrom} and 1.85 {angstrom} resolution, respectively. Finally, a comparison of the SpHICDH and TtHICDH structures reveal differences in

  7. Reversible inactivation of CO dehydrogenase with thiol compounds

    SciTech Connect

    Kreß, Oliver; Gnida, Manuel; Pelzmann, Astrid M.; Marx, Christian; Meyer-Klaucke, Wolfram; Meyer, Ortwin

    2014-05-09

    Highlights: • Rather large thiols (e.g. coenzyme A) can reach the active site of CO dehydrogenase. • CO- and H{sub 2}-oxidizing activity of CO dehydrogenase is inhibited by thiols. • Inhibition by thiols was reversed by CO or upon lowering the thiol concentration. • Thiols coordinate the Cu ion in the [CuSMo(=O)OH] active site as a third ligand. - Abstract: Carbon monoxide dehydrogenase (CO dehydrogenase) from Oligotropha carboxidovorans is a structurally characterized member of the molybdenum hydroxylase enzyme family. It catalyzes the oxidation of CO (CO + H{sub 2}O → CO{sub 2} + 2e{sup −} + 2H{sup +}) which proceeds at a unique [CuSMo(=O)OH] metal cluster. Because of changing activities of CO dehydrogenase, particularly in subcellular fractions, we speculated whether the enzyme would be subject to regulation by thiols (RSH). Here we establish inhibition of CO dehydrogenase by thiols and report the corresponding K{sub i}-values (mM): L-cysteine (5.2), D-cysteine (9.7), N-acetyl-L-cysteine (8.2), D,L-homocysteine (25.8), L-cysteine–glycine (2.0), dithiothreitol (4.1), coenzyme A (8.3), and 2-mercaptoethanol (9.3). Inhibition of the enzyme was reversed by CO or upon lowering the thiol concentration. Electron paramagnetic resonance spectroscopy (EPR) and X-ray absorption spectroscopy (XAS) of thiol-inhibited CO dehydrogenase revealed a bimetallic site in which the RSH coordinates to the Cu-ion as a third ligand ([Mo{sup VI}(=O)OH{sub (2)}SCu{sup I}(SR)S-Cys]) leaving the redox state of the Cu(I) and the Mo(VI) unchanged. Collectively, our findings establish a regulation of CO dehydrogenase activity by thiols in vitro. They also corroborate the hypothesis that CO interacts with the Cu-ion first. The result that thiol compounds much larger than CO can freely travel through the substrate channel leading to the bimetallic cluster challenges previous concepts involving chaperone function and is of importance for an understanding how the sulfuration step in

  8. Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

    PubMed Central

    Kolk, A H; van Kuyk, L; Boettcher, B

    1978-01-01

    Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein. Images Fig. 2. Fig. 3. PMID:213050

  9. Prostaglandin dehydrogenase and the initiation of labor.

    PubMed

    Challis, J R; Patel, F A; Pomini, F

    1999-01-01

    In summary, these studies have suggested that prostaglandin dehydrogenase may have a central role to play in the mechanisms which determine biologically active prostaglandin concentrations within human fetal membranes and placenta at the time of labor, at term or preterm. Moreover, our studies indicate that the regulation of PGDH may by multifactorial (figure 3). In certain regions of the membranes, we suggest that PGDH expression may be influenced by levels of anti-inflammatory and pro-inflammatory cytokines. In other regions of the membranes, we suggest that PGDH may be regulated at a transcriptional level by competing activities of progesterone and cortisol. The action of progesterone could be effected through systemically-derived steroid, or by locally synthesized steroid, acting in a paracrine and/or autocrine fashion. The effects of cortisol in placenta must be due to glucocorticoid derived from the maternal or fetal compartment, since the placenta lacks the hydroxylases required for endogenous cortisol production. However, metabolism of cortisol by 11 beta-HSD-2 reduces the potency of this glucocorticoid in placental tissue. In chorion however, cortisol may be formed locally, from cortisone, in addition to its being derived from the maternal circulation and/or from the amniotic fluid. Our current studies do not allow us to delineate whether the effects of progesterone and cortisol on PGDH are exerted through the glucocorticoid receptor (GR) or progesterone receptor (PR) or both. It is possible that through pregnancy, PGDH activity is maintained by progesterone acting either through low levels of PR in membranes, or, more likely, acting through GR. At term, elevated levels of cortisol compete with and displace progesterone from GR, resulting in inhibition of PGDH transcription and activity. In this way, local withdrawal of progesterone action would be effected within human intrauterine tissues, without requiring changes in systemic, circulating progesterone

  10. NADP+-Preferring d-Lactate Dehydrogenase from Sporolactobacillus inulinus

    PubMed Central

    Zhu, Lingfeng; Xu, Xiaoling; Wang, Limin; Ma, Yanhe

    2015-01-01

    Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD+ as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn174 was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases. PMID:26150461

  11. KINETIC PROPERTIES OF MALIC DEHYDROGENASE FROM THREE CULTIVARS OF RICE

    EPA Science Inventory

    Temperature induced changes in the kinetics of the enzyme malic dehydrogenase (MON) were investigated in three cultivars of rice(Oryza sativa L.). Cultivars, included IR74, SWAT2, and N22. Plants were grown in a controlled environment chamber for 29 days, at 31 degrees C day/25 d...

  12. Efficiency of superoxide anions in the inactivation of selected dehydrogenases

    NASA Astrophysics Data System (ADS)

    Rodacka, Aleksandra; Serafin, Eligiusz; Puchala, Mieczyslaw

    2010-09-01

    The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as rad OH and ONOO -. In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion. Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.

  13. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Hydroxybutyric dehydrogenase test system. 862.1380 Section 862.1380 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  14. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase.

    PubMed Central

    Mat-Jan, F; Alam, K Y; Clark, D P

    1989-01-01

    Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion. PMID:2644194

  15. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  16. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  17. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1445 Lactate...

  18. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1445 Lactate...

  19. Genetics Home Reference: 3-hydroxyacyl-CoA dehydrogenase deficiency

    MedlinePlus

    ... step that metabolizes groups of fats called medium-chain fatty acids and short-chain fatty acids. Mutations in the HADH gene lead ... a shortage of 3-hydroxyacyl-CoA dehydrogenase. Medium-chain and short-chain fatty acids cannot be metabolized ...

  20. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1445 Lactate...

  1. Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene

    PubMed Central

    Sadeghi, H. Mir Mohammad; Ahmadi, R.; Aghaabdollahian, S.; Mofid, M.R.; Ghaemi, Y.; Abedi, D.

    2011-01-01

    Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. coli XL1-blue competent cells. Following plasmid preparation, the cloned gene was digested out and ligated into the expression vector pET-22b(+). Electrophoresis of PCR product showed a 789 bp band. Recombinant plasmid (rpTZ57R) was then constructed. This plasmid was double digested with XhoI and EcoRI resulting in 800 bp and 2900 bp bands. The obtained insert was ligated into pET-22b(+) vector and its orientation was confirmed with XhoI and BamHI restriction enzymes. In conclusion, in the present study the recombinant expression vector containing xylitol dehydrogenase gene has been constructed and can be used for the production of this enzyme in high quantities. PMID:22110522

  2. Succinate dehydrogenase is the regulator of respiration in Mycobacterium tuberculosis.

    PubMed

    Hartman, Travis; Weinrick, Brian; Vilchèze, Catherine; Berney, Michael; Tufariello, Joanne; Cook, Gregory M; Jacobs, William R

    2014-11-01

    In chronic infection, Mycobacterium tuberculosis bacilli are thought to enter a metabolic program that provides sufficient energy for maintenance of the protonmotive force, but is insufficient to meet the demands of cellular growth. We sought to understand this metabolic downshift genetically by targeting succinate dehydrogenase, the enzyme which couples the growth processes controlled by the TCA cycle with the energy production resulting from the electron transport chain. M. tuberculosis contains two operons which are predicted to encode succinate dehydrogenase enzymes (sdh-1 and sdh-2); we found that deletion of Sdh1 contributes to an inability to survive long term stationary phase. Stable isotope labeling and mass spectrometry revealed that Sdh1 functions as a succinate dehydrogenase during aerobic growth, and that Sdh2 is dispensable for this catalysis, but partially overlapping activities ensure that the loss of one enzyme can incompletely compensate for loss of the other. Deletion of Sdh1 disturbs the rate of respiration via the mycobacterial electron transport chain, resulting in an increased proportion of reduced electron carrier (menaquinol) which leads to increased oxygen consumption. The loss of respiratory control leads to an inability to recover from stationary phase. We propose a model in which succinate dehydrogenase is a governor of cellular respiration in the adaptation to low oxygen environments. PMID:25412183

  3. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  4. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  5. Spatial variability of the dehydrogenase activity in forest soils

    NASA Astrophysics Data System (ADS)

    Błońska, Ewa; Lasota, Jarosław

    2014-05-01

    The aim of this study was to assess the spatial variability of the dehydrogenase activity (DH) in forest soils using geostatistics. We have studied variability soil dehydrogenase and their relationship with variability of some physic-chemical properties. Two study areas (A and B) were set up in southern Poland in the Zlotoryja Forest District. Study areas were covered by different types of vegetation (A- broadleaf forest with beech, ash and sycamore), B- coniferous forest with Norway spruce). The soils were classified as Dystric Cambisols (WRB 2006). The samples for laboratory testing were collected from 49 places on each areas. 15 cm of surface horizon of soil were taken (with previously removed litter). Dehydrogenase activity was marked with Lenhard's method according to the Casida procedure. Soil pH, nitrogen (N) and soil organic carbon (C) content (by LECO CNS 2000 carbon analyzer) was marked. C/N ratio was calculated. Particle size composition was determined using laser diffraction. Statistical analysis were performed using STATISTICA 10 software. Geostatistical analysis and mapping were done by application of GS 9+ (Gamma Design) and Surfer 11 (Golden Software). The activity of DH ranged between 5,02 and 71,20 mg TPP• kg-1 •24 h-1 on the A area and between 0,94 and 16,47 mg TPP• kg-1 •24 h-1. Differences in spatial variability of the analised features were noted. The variability of dehydrogenase activity on the A study area was described by an exponential model, whereas on the B study area the spatial correlation has not been noted. The relationship of dehydrogenase activity with the remaining parameters of soil was noted only in the case of A study area. The variability of organic carbon content on the A and B study areas were described by an exponential model. The variability of nitrogen content on both areas were described by an spherical model.

  6. The alcohol dehydrogenase isoenzyme alcohol dehydrogenase IV as a candidate marker of Helicobacter pylori infection

    PubMed Central

    Laniewska-Dunaj, Magdalena; Strumnik, Anna; Szmitkowski, Maciej

    2014-01-01

    Introduction Helicobacter pylori infection is associated with decreased alcohol dehydrogenase (ADH) activity in the gastric mucosa. The decrease in gastric ADH activity depends on the severity of inflammation and mucosal injury. This damage can be a reason of the release of enzyme from gastric mucosa and leads to the increase of the ADH activity in the sera of patients with H. pylori infection. Material and methods Serum samples were taken from 140 patients with H. pylori infection. Total ADH activity was measured by photometric method with p-nitrosodimethylaniline as a substrate and ALDH activity by the fluorometric method with 6-methoxy-2-naphtaldehyde. For the measurement of the activity of class I and II isoenzymes we employed the fluorometric methods, with class-specific fluorogenic substrates. The activity of class III ADH was measured by the photometric method with n-octanol and class IV with m-nitrobenzaldehyde as a substrate. Results The activity of ADH IV in the serum of patients with H. pylori infection increased about 42% (7.86 mU/l) in the comparison to the control level (4.52 mU/l). Total activity of ADH was 1105 mU/l in patients group and 682 mU/l in control. The diagnostic sensitivity for ADH IV was 88%, specificity 90%, positive and negative predictive values were 91% and 84% respectively. Area under ROC curve for ADH IV was 0.84. Conclusions Helicobacter pylori infection of gastric mucosa is reflected in the serum by significant increase of class IV and total ADH activity. The results suggest a potential role for ADH IV as a marker of H. pylori infection. PMID:25395946

  7. Succinate dehydrogenase subunit D and succinate dehydrogenase subunit B mutation analysis in canine phaeochromocytoma and paraganglioma.

    PubMed

    Holt, D E; Henthorn, P; Howell, V M; Robinson, B G; Benn, D E

    2014-07-01

    Phaeochromocytomas (PCs) are tumours of the adrenal medulla chromaffin cells. Paragangliomas (PGLs) arise in sympathetic ganglia (previously called extra-adrenal PCs) or in non-chromaffin parasympathetic ganglia cells that are usually non-secretory. Parenchymal cells from these tumours have a common embryological origin from neural crest ectoderm. Several case series of canine PCs and PGLs have been published and a link between the increased incidence of chemoreceptor neoplasia in brachycephalic dog breeds and chronic hypoxia has been postulated. A similar link to hypoxia in man led to the identification of germline heterozygous mutations in the gene encoding succinate dehydrogenase subunit D (SDHD) and subsequently SDHA, SDHB and SDHC in similar tumours. We investigated canine PCs (n = 6) and PGLs (n = 2) for SDHD and SDHB mutations and in one PGL found a somatic SDHD mutation c.365A>G (p.Lys122Arg) in exon 4, which was not present in normal tissue from this brachycephalic dog. Two PCs were heterozygous for both c.365A>G (p.Lys122Arg) mutation and an exon 3 silent variant c.291G>A. We also identified the heterozygous SDHB exon 2 mutation c.113G>A (p.Arg38Gln) in a PC. These results illustrate that genetic mutations may underlie tumourigenesis in canine PCs and PGLs. The spontaneous nature of these canine diseases and possible association of PGLs with hypoxia in brachycephalic breeds may make them an attractive model for studying the corresponding human tumours. PMID:24813157

  8. Regulation of human dihydrodiol dehydrogenase by Michael acceptor xenobiotics.

    PubMed

    Ciaccio, P J; Jaiswal, A K; Tew, K D

    1994-06-01

    A human oxidoreductase (H-37) that is overexpressed in ethacrynic acid-resistant HT29 colon cells (Ciaccio, P. J., Stuart, J.E., and Tew, K.D. (1993) Mol. Pharmacol. 43, 845-853) has been identified as a dihydrodiol dehydrogenase. Translated protein from a dihydrodiol dehydrogenase cDNA isolated from a library prepared from ethacrynic acid-resistant HT29 cell poly(A+) RNA was recognized by anti-H-37 IgG and was identical in molecular weight with H-37. The isolated cDNA was identical in both nucleotide and amino acid sequences with the recently cloned liver dihydrodiol dehydrogenase (Stolz, A., Hammond, L., Lou, H., Takikawa, H., Ronk, M., and Shively, J.E. (1993) J. Biol. Chem. 268, 10448-10457). Using this cDNA as probe, we have examined its induction by Michael acceptors. The steady state dihydrodiol dehydrogenase mRNA level in the ethacrynic acid-resistant line was increased 30-fold relative to that of wild-type cells. Twenty-four hour treatment of wild-type cells with ethacrynic acid or dimethyl maleate increased mRNA 10-fold and 5-fold, respectively. These changes are accompanied by both increased protein expression and increased NADP-dependent 1-acenaphthenol oxidative activity in cell cytosol. In gel shift assays, compared to wild type controls, increased binding of NAD(P)H quinone oxidoreductase human antioxidant response element (hARE) DNA to redox labile protein complexes present in treated and resistant cell nuclear extract was observed. Ethacrynic acid induced CAT activity 2-fold in Hepa1 cells stably transfected with NAD(P)H quinone oxidoreductase hARE-tk-CAT chimeric gene construct. Thus, dihydrodiol dehydrogenase protein is inducible by de novo synthesis from mRNA by structurally related monofunctional inducer Michael acceptors. Altered in vitro binding of nuclear protein to the hARE is indirect evidence for the involvement of an element similar to hARE in the regulation of dihydrodiol dehydrogenase by these agents. PMID:7515059

  9. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

    NASA Astrophysics Data System (ADS)

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

    2015-07-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

  10. [Cooperative properties of D-glyceraldehyde-3-phosphate dehydrogenase].

    PubMed

    Nagradova, N K

    1977-03-01

    The structure of the active center of glyceraldehyde-3-phosphate dehydrogenase and the arrangement of subunits in the tetrameric molecule is delineated. The mechanism of cooperative effects in the oligomer is considered, and the involvement of various regions of the active center and of different-subunit contact area in the realization of the cooperative phenomena is discussed. A special attention is paid to the effect of NAD+ bound to one of the subunits of the tetramer on the structure of an adjacent subunit and to the problem of the participation of the coenzyme in the creation of anion-binding sites of the enzyme. The conditions of reversible dissociation of the tetrameric apoenzyme molecule into dimers are depicted, and the role of NAD+ in the organization of the quaternary structure of the dehydrogenase is discussed. The problem of catalytic activity of the dimeric form of the enzyme is argued. PMID:193581

  11. Hydroxysteroid dehydrogenases (HSDs) in bacteria: a bioinformatic perspective.

    PubMed

    Kisiela, Michael; Skarka, Adam; Ebert, Bettina; Maser, Edmund

    2012-03-01

    Steroidal compounds including cholesterol, bile acids and steroid hormones play a central role in various physiological processes such as cell signaling, growth, reproduction, and energy homeostasis. Hydroxysteroid dehydrogenases (HSDs), which belong to the superfamily of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR), are important enzymes involved in the steroid hormone metabolism. HSDs function as an enzymatic switch that controls the access of receptor-active steroids to nuclear hormone receptors and thereby mediate a fine-tuning of the steroid response. The aim of this study was the identification of classified functional HSDs and the bioinformatic annotation of these proteins in all complete sequenced bacterial genomes followed by a phylogenetic analysis. For the bioinformatic annotation we constructed specific hidden Markov models in an iterative approach to provide a reliable identification for the specific catalytic groups of HSDs. Here, we show a detailed phylogenetic analysis of 3α-, 7α-, 12α-HSDs and two further functional related enzymes (3-ketosteroid-Δ(1)-dehydrogenase, 3-ketosteroid-Δ(4)(5α)-dehydrogenase) from the superfamily of SDRs. For some bacteria that have been previously reported to posses a specific HSD activity, we could annotate the corresponding HSD protein. The dominating phyla that were identified to express HSDs were that of Actinobacteria, Proteobacteria, and Firmicutes. Moreover, some evolutionarily more ancient microorganisms (e.g., Cyanobacteria and Euryachaeota) were found as well. A large number of HSD-expressing bacteria constitute the normal human gastro-intestinal flora. Another group of bacteria were originally isolated from natural habitats like seawater, soil, marine and permafrost sediments. These bacteria include polycyclic aromatic hydrocarbons-degrading species such as Pseudomonas, Burkholderia and Rhodococcus. In conclusion, HSDs are found in a wide variety of microorganisms including

  12. Novel Xylose Dehydrogenase in the Halophilic Archaeon Haloarcula marismortui†

    PubMed Central

    Johnsen, Ulrike; Schönheit, Peter

    2004-01-01

    During growth of the halophilic archaeon Haloarcula marismortui on d-xylose, a specific d-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP+ and NAD+ as cosubstrates with 10-fold higher affinity for NADP+. In addition to d-xylose, d-ribose was oxidized at similar kinetic constants, whereas d-glucose was used with about 70-fold lower catalytic efficiency (kcat/Km). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)—coding for a 39.9-kDA protein—was identified in the partially sequenced genome of H. marismortui. The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H. marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H. marismortui. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H. marismortui. PMID:15342590

  13. Reappraisal of the regulation of lactococcal L-lactate dehydrogenase.

    PubMed

    van Niel, Ed W J; Palmfeldt, Johan; Martin, Rani; Paese, Marco; Hahn-Hägerdal, Bärbel

    2004-03-01

    Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio. Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118). A more complete model of the kinetics of the regulation of lactococcal LDH is discussed. PMID:15006814

  14. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria

    PubMed Central

    Watanabe, Seiya; Sueda, Rui; Fukumori, Fumiyasu; Watanabe, Yasuo

    2015-01-01

    Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase. PMID:26382958

  15. A guide to 17beta-hydroxysteroid dehydrogenases.

    PubMed

    Adamski, J; Jakob, F J

    2001-01-22

    17beta-Hydroxysteroid dehydrogenases (17beta-HSD) are pivotal in controlling the biological potency of steroid hormones by catalyzing oxidation or reduction at position 17. Several 17beta-HSDs may as well metabolize further substrates including alcohols, bile acids, fatty acids and retinols. This review summarizes recent progress in the field of 17beta-HSD research provides an update of nomenclature. PMID:11165003

  16. Drosophila alcohol dehydrogenase: developmental studies on cryptic variant lines.

    PubMed

    Miglani, G S; Ampy, F R

    1981-10-01

    Thirty-five cryptic variant lines were used to examine the mechanisms involved in genetic modulation of alcohol metabolism in Drosophila. Late third-instar larval, preemergence pupal, and adult stages cultured at 18 and 28 C were examined. Spectrophotometric analyses for native alcohol dehydrogenase (ADH) activity and residual ADH activity after treatment with guanidine hydrochloride and heat were performed. Differential response of cryptic variants to treatment with the denaturants during development suggested that this variation may have an adaptive significance. PMID:6800354

  17. Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

    SciTech Connect

    White, Tommi A.; Tanner, John J.

    2005-08-01

    Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ{sup 1}-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  18. Biochemical mechanisms of glucose-6-phosphate dehydrogenase deficiency.

    PubMed Central

    Morelli, A; Benatti, U; Gaetani, G F; De Flora, A

    1978-01-01

    A solid-phase radioimmunoassay for human glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase; EC 1.1.1.49) was developed that allowed the specific activity of this enzyme protein to be measured in lysates from whole erythrocyte populations, in lysates from erythrocytes of different ages, and in purified samples. The enzyme was highly purified from erythrocytes of single donors by a simple procedure of affinity chromatography with insolubilized adenosine 2',5'-bisphosphate. These techniques were used in an attempt to elucidate the molecular mechanisms leading to deficiency of glucose-6-phosphate dehydrogenase activity in two genetic variants of the enzyme, i.e., the Mediterranean and the Seattle-like variants. The results indicate that the lowered activity of erythrocytes containing the Mediterranean variant of glucose-6-phosphate dehydrogenase is related to an enhanced rate of degradation of a catalytically defective protein synthesized at a nearly normal rate. Synthesis of a normally functioning protein and an increased breakdown of it are involved in the Seattle-like variant of the enzyme. Images PMID:273924

  19. Pyruvate dehydrogenase deficiency: molecular basis for intrafamilial heterogeneity.

    PubMed

    Fujii, T; Van Coster, R N; Old, S E; Medori, R; Winter, S; Gubits, R M; Matthews, P M; Brown, R M; Brown, G K; Dahl, H H

    1994-07-01

    Two half-brothers and their mother had symptomatic pyruvate dehydrogenase complex deficiency. The infants had severe congenital lactic acidosis, seizures, and apneic spells and died at the ages 3 and 4 months. The mother was less symptomatic with mental retardation, truncal ataxia, and dysarthria. The residual pyruvate dehydrogenase activities in cultured skin fibroblasts from the 2 infants and their mother were 7, 15, and 10% of control values. Immunoblot analysis showed negligible amounts of E1 alpha and E1 beta subunits of the complex. Northern blot analysis for the E1 alpha subunit showed normal results. In the 2 sons, complementary DNA sequence analysis revealed a cytosine to thymine mutation in exon 4, resulting in a change of arginine 127 to tryptophan in the E1 alpha subunit. Restriction enzyme analysis of the polymerase chain reaction product representing exon 4 of the E1 alpha gene revealed that the mother was a heterozygotes. Complementary DNA restriction analysis and methylation analysis of the X chromosome DXS255 loci revealed skewed activation of the mutant allele, consistent with the deficient pyruvate dehydrogenase activity in the mother's fibroblasts. The milder maternal phenotype is consistent with variable X-inactivation patterns in different organs of female heterozygotes. PMID:8024267

  20. A glycolate dehydrogenase in the mitochondria of Arabidopsis thaliana.

    PubMed

    Bari, Rafijul; Kebeish, Rashad; Kalamajka, Rainer; Rademacher, Thomas; Peterhänsel, Christoph

    2004-03-01

    The fixation of molecular O2 by the oxygenase activity of Rubisco leads to the formation of phosphoglycolate in the chloroplast that is further metabolized in the process of photorespiration. The initial step of this pathway is the oxidation of glycolate to glyoxylate. Whereas in higher plants this reaction takes place in peroxisomes and is dependent on oxygen as a co-factor, most algae oxidize glycolate in the mitochondria using organic co-factors. The identification and characterization of a novel glycolate dehydrogenase in Arabidopsis thaliana is reported here. The enzyme is dependent on organic co-factors and resembles algal glycolate dehydrogenases in its enzymatic properties. Mutants of E. coli incapable of glycolate oxidation can be complemented by overexpression of the Arabidopsis open reading frame. The corresponding RNA accumulates preferentially in illuminated leaves, but was also found in other tissues investigated. A fusion of the N-terminal part of the Arabidopsis glycolate dehydrogenase to red fluorescent protein accumulates in mitochondria when overexpressed in the homologous system. Based on these results it is proposed that the basic photorespiratory system of algae is conserved in higher plants. PMID:14966218

  1. Functional Analysis of a Mosquito Short Chain Dehydrogenase Cluster

    PubMed Central

    Mayoral, Jaime G.; Leonard, Kate T.; Defelipe, Lucas A.; Turjansksi, Adrian G.; Nouzova, Marcela; Noriegal, Fernando G.

    2013-01-01

    The short chain dehydrogenases (SDR) constitute one the oldest and largest families of enzymes with over 46,000 members in sequence databases. About 25% of all known dehydrogenases belong to the SDR family. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. This family is present in archaea, bacteria, and eukaryota, emphasizing their versatility and fundamental importance for metabolic processes. We identified a cluster of eight SDRs in the mosquito Aedes aegypti (AaSDRs). Members of the cluster differ in tissue specificity and developmental expression. Heterologous expression produced recombinant proteins that had diverse substrate specificities, but distinct from the conventional insect alcohol (ethanol) dehydrogenases. They are all NADP+-dependent and they have S-enantioselectivity and preference for secondary alcohols with 8–15 carbons. Homology modeling was used to build the structure of AaSDR1 and two additional cluster members. The computational study helped explain the selectivity towards the (10S)-isomers as well as the reduced activity of AaSDR4 and AaSDR9 for longer isoprenoid substrates. Similar clusters of SDRs are present in other species of insects, suggesting similar selection mechanisms causing duplication and diversification of this family of enzymes. PMID:23238893

  2. Novel yeast cell dehydrogenase activity assay in situ.

    PubMed

    Berłowska, Joanna; Kregiel, Dorota; Klimek, Leszek; Orzeszyna, Bartosz; Ambroziak, Wojciech

    2006-01-01

    The aim of this research was to develop a suitable method of succinate dehydrogenase activity assay in situ for different industrial yeast strains. For this purpose different compounds: EDTA, Triton X-100, sodium deoxycholate, digitonin, nystatin and beta-mercaptoethanol were used. The permeabilization process was controlled microscopically by primuline staining. Enzyme assay was conducted in whole yeast cells with Na-succinate as substrate, phenazine methosulfate (PMS) as electron carrier and in the presence one of two different tetrazolium salts: tetrazolium blue chloride (BT) or cyanoditolyl tetrazolium chloride (CTC) reduced during the assay. In comparabile studies of yeast vitality the amount of intracellular ATP was determined according to luciferin/luciferase method. During the succinate dehydrogenase assay in intact yeast cells without permeabilization, BT formazans were partially visualized in the cells, but CTC formazans appeared to be totally extracellular or associated with the plasma membrane. Under these conditions there was no linear relationship between formazan color intensity signal and yeast cell density. From all chemical compounds tested, only digitonin was effective in membrane permeabilization without negative influence on cell morphology. Furthermore, with digitonin-treated cells a linear relationship between formazan color intensity signal and yeast cell number was noticed. Significant decreasing of succinate dehydrogenase activity and ATP content were observed during aging of the tested yeast strains. PMID:17419290

  3. Isocitrate Dehydrogenase and Glutamate Synthesis in Acetobacter suboxydans1

    PubMed Central

    Greenfield, Seymour; Claus, G. W.

    1969-01-01

    Acetobacter suboxydans is an obligate aerobe for which an operative tricarboxylic acid cycle has not been demonstrated. Glutamate synthesis has been reported to occur by mechanisms other than those utilizing isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme not previously detected in this organism. We have recovered α-ketoglutarate and glutamate from a system containing citrate, nicotinamide adenine dinucleotide (NAD), a divalent cation, pyridoxal phosphate, an amino donor, and dialyzed, cell-free extract. Aconitase activity was readily detected in these extracts, but isocitrate dehydrogenase activity, measured by NAD reduction, was masked by a cyanide-resistant, particulate, reduced NAD oxidase. Isocitrate dehydrogenase activity could be demonstrated after centrifuging the extracts at 150,000 × g for 3 hr and treating the supernatant fluid with 2-heptyl-4-hydroxyquinoline N-oxide. It is concluded that A. suboxydans can utilize the conventional tricarboxylic acid cycle enzymes to convert citrate to α-ketoglutarate which can then undergo a transamination to glutamate. Images PMID:5361215

  4. Asp295 stabilizes the active-site loop structure of pyruvate dehydrogenase, facilitating phosphorylation of Ser292 by pyruvate dehydrogenase-kinase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed an invitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thaliana a2b2-hetero tetrameric pyruvate dehydrogenase (E1) plus A.thaliana E1-kinase (AtPDK). Upon addition of MgATP...

  5. X-linked glucose-6-phosphate dehydrogenase (G6PD) and autosomal 6-phosphogluconate dehydrogenase (6PGD) polymorphisms in baboons

    SciTech Connect

    VandeBerg, J.L.; Aivaliotis, M.J.; Samollow, P.B. )

    1992-12-01

    Electrophoretic polymorphisms of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were examined in captive colonies of five subspecies of baboons (Papio hamadryas). Phenotype frequencies and family data verified the X-linked inheritance of the G6PD polymorphism. Insufficient family data were available to confirm autosomal inheritance of the 6PGD polymorphism, but the electrophoretic patterns of variant types (putative heterozygotes) suggested the codominant expression of alleles at an autosomal locus. Implications of the G6PD polymorphism are discussed with regard to its utility as a marker system for research on X-chromosome inactivation during baboon development and for studies of clonal cell proliferation and/or cell selection during the development of atherosclerotic lesions in the baboon model. 61 refs., 1 fig., 4 tabs.

  6. Human dehydrogenase/reductase (SDR family) member 11 is a novel type of 17β-hydroxysteroid dehydrogenase.

    PubMed

    Endo, Satoshi; Miyagi, Namiki; Matsunaga, Toshiyuki; Hara, Akira; Ikari, Akira

    2016-03-25

    We report characterization of a member of the short-chain dehydrogenase/reductase superfamily encoded in a human gene, DHRS11. The recombinant protein (DHRS11) efficiently catalyzed the conversion of the 17-keto group of estrone, 4- and 5-androstenes and 5α-androstanes into their 17β-hydroxyl metabolites with NADPH as a coenzyme. In contrast, it exhibited reductive 3β-hydroxysteroid dehydrogenase activity toward 5β-androstanes, 5β-pregnanes, 4-pregnenes and bile acids. Additionally, DHRS11 reduced α-dicarbonyls (such as diacetyl and methylglyoxal) and alicyclic ketones (such as 1-indanone and loxoprofen). The enzyme activity was inhibited in a mixed-type manner by flavonoids, and competitively by carbenoxolone, glycyrrhetinic acid, zearalenone, curcumin and flufenamic acid. The expression of DHRS11 mRNA was observed widely in human tissues, most abundantly in testis, small intestine, colon, kidney and cancer cell lines. Thus, DHRS11 represents a novel type of 17β-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. PMID:26920053

  7. Evidence for distinct dehydrogenase and isomerase sites within a single 3. beta. -hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein

    SciTech Connect

    Luu-The, V.; Takahashi, Masakazu; de Launoit, Y.; Dumont, M.; Lachance, Y.; Labrie, F. )

    1991-09-10

    Complementary DNA encoding human 3{beta}-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3-{beta}-HSD) has been expressed in transfected GH{sub 4}C{sub 1} with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of ({sup 3}H)-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3{beta}-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5{alpha}-androstane-17{beta}-carboxamide) and its analogues of 5-androstenedione to 4-androstenedione with an approximately 1,000-fold higher K{sub i} value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3-{beta}-HSD protein. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.

  8. Pyruvate Dehydrogenase and Pyruvate Dehydrogenase Kinase Expression in Non Small Cell Lung Cancer and Tumor-Associated Stroma1

    PubMed Central

    Koukourakis, Michael I; Giatromanolaki, Alexandra; Sivridis, Efthimios; Gatter, Kevin C; Harris, Adrian L; “Tumor and Angiogenesis Research Group”

    2005-01-01

    Abstract Pyruvate dehydrogenase (PDH) catalyzes the conversion of pyruvate to acetyl-coenzyme A, which enters into the Krebs cycle, providing adenosine triphosphate (ATP) to the cell. PDH activity is under the control of pyruvate dehydrogenase kinases (PDKs). Under hypoxic conditions, conversion of pyruvate to lactate occurs, a reaction catalyzed by lactate dehydrogenase 5 (LDH5). In cancer cells, however, pyruvate is transformed to lactate occurs, regardless of the presence of oxygen (aerobic glycolysis/Warburg effect). Although hypoxic intratumoral conditions account for HIF1α stabilization and induction of anaerobic metabolism, recent data suggest that high pyruvate concentrations also result in HIF1α stabilization independently of hypoxia. In the present immunohistochemical study, we provide evidence that the PDH/PDK pathway is repressed in 73% of non small cell lung carcinomas, which may be a key reason for HIF1α stabilization and “aerobic glycolysis.” However, about half of PDH-deficient carcinomas are not able to switch on the HIF pathway, and patients harboring these tumors have an excellent postoperative outcome. A small subgroup of clinically aggressive tumors maintains a coherent PDH and HIF/LDH5 expression. In contrast to cancer cells, fibroblasts in the tumor-supporting stroma exhibit an intense PDH but reduced PDK1 expression favoring maximum PDH activity. This means that stroma may use lactic acid produced by tumor cells, preventing the creation of an intolerable intratumoral acidic environment at the same time. PMID:15736311

  9. The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

    PubMed Central

    Zammit, V A; Newsholme, E A

    1976-01-01

    Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is

  10. Dihydrolipoamide dehydrogenase from halophilic archaebacteria: purification and properties of the enzyme from halobacterium halobium

    SciTech Connect

    Danson, J.J.; McQuattie, A.; Stevenson, K.J.

    1986-07-01

    Halophilic archaebacteria possess dihydrolipoamide dehydrogenase activity but apparently lack the 2-oxoacid dehydrogenase multienzyme complexes of which it is usually an integral component. In this paper, the purification of dihydrolipoamide dehydrogenase from Halobacterium halobium is reported. The enzyme is a dimer with a polypeptide chain M/sub r/ of 58,000 (+/-3000). The amino acid composition of the enzyme is compared with those of the eubacterial and eukaryotic dihydrolipoamide dehydrogenases, and evidence is presented to suggest that the N-terminal amino acid of the H. halobium enzyme is blocked. Chemical modification with the trivalent arsenical reagent (p-aminophenyl)dichloroarsine indicates the involvement of a reversibly reducible disulfide bond in the enzyme's catalytic mechanism. The possible metabolic role of this dihydrolipoamide dehydrogenase in the absence of 2-oxoacid dehydrogenase complexes is discussed.

  11. Molecular structure of the pyruvate dehydrogenase complex from Escherichia coli K-12.

    PubMed

    Vogel, O; Hoehn, B; Henning, U

    1972-06-01

    The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 x 10(6). All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This "excess" component is bound differently than are the eight dimers in the core complex. PMID:4556465

  12. Molecular Structure of the Pyruvate Dehydrogenase Complex from Escherichia coli K-12

    PubMed Central

    Vogel, Otto; Hoehn, Barbara; Henning, Ulf

    1972-01-01

    The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 × 106. All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This “excess” component is bound differently than are the eight dimers in the core complex. Images PMID:4556465

  13. Localization of the major dehydrogenases in two methylotrophs by radiochemical labeling.

    PubMed Central

    Kasprzak, A A; Steenkamp, D J

    1983-01-01

    The localization of prominent proteins in intact cells of two methylotrophic bacteria, Hyphomicrobium sp. strain X and bacterium W3A1, was investigated by radiochemical labeling with [14C]isethionyl acetimidate. In bacterium W3A1, trimethylamine dehydrogenase was not labeled by the reagent and is, therefore, an intracellular protein, whereas the periplasmic location of the methylamine and methanol dehydrogenases was evidenced by being readily labeled in intact cells. Similarly, an intracellular location of the trimethylamine and dimethylamine dehydrogenases in Hyphomicrobium sp. strain X was indicated, whereas methanol dehydrogenase was periplasmic. Images PMID:6311799

  14. γ-Guanidinobutyraldehyde Dehydrogenase of Vicia faba Leaves

    PubMed Central

    Matsuda, Hitoshi; Suzuki, Yonezo

    1984-01-01

    γ-Guanidinobutyraldehyde dehydrogenase was purified 27-fold in 40% yield from extracts of Vicia faba leaves. High specificity exist only for γ-guanidinobutyraldehyde and γ-aminobutyraldehyde; the Km value was 3.4 micromolar for γ-guanidinobutyraldehyde, 25 micromolar for γ-aminobutyraldehyde, and 84 micromolar (case of γ-guanidinobutyraldehyde) for NAD, respectively. The enzyme had a molecular weight of approximately 83,000. Optimal pH and temperature for activity were 9.5 and 45°C, respectively. The enzyme was inhibited strongly by p-chloromercuribenzoate, N-ethylmaleimide, and zincon (2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene). PMID:16663901

  15. Direct Observation of Correlated Interdomain Motion in Alcohol Dehydrogenase

    SciTech Connect

    Biehl, Ralf; Monkenbusch, Michael; Richter, Dieter; Hoffmann, Bernd; Merkel, Rudolf; Falus, Peter; Preost, Sylvain

    2008-09-26

    Interdomain motions in proteins are essential to enable or promote biochemical function. Neutron spin-echo spectroscopy is used to directly observe the domain dynamics of the protein alcohol dehydrogenase. The collective motion of domains as revealed by their coherent form factor relates to the cleft opening dynamics between the binding and the catalytic domains enabling binding and release of the functional important cofactor. The cleft opening mode hardens as a result of an overall stiffening of the domain complex due to the binding of the cofactor.

  16. Some properties of aldehyde dehydrogenase from sheep liver mitochondria.

    PubMed Central

    Hart, G J; Dickinson, F M

    1977-01-01

    Aldehyde dehydrogenase from sheep liver mitochondria was purified to homogeneity as judged by electrophoresis on polyacrylamide gels, and by sedimentation-equilibrium experiments in the analytical ultracentrifuge. The enzyme has a molecular weight of 198000 and a subunit size of 48000, indicating that the molecule is a tetramer. Fluorescence and spectrophotometric titrations indicate that each subunit can bind 1 molecule of NADH. Enzymic activity is completely blocked by reaction of 4mol of 5,5'-dithiobis-(2-nitrobenzoate)/mol of enzyme. Excess of disulfiram or iodoacetamide decreases activity to only 50% of the control value, and only two thiol groups per molecule are apparently modified by these reagents. PMID:194582

  17. Malaria, favism and glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    Huheey, J E; Martin, D L

    1975-10-15

    Although glucose-6-phosphate dehydrogenase deficient individuals may suffer (sometimes fatally) from favism, a high incidence of this trait occurs in many Mediterranean populations. This apparent paradox is explained on the basis of a synergistic interaction between favism and G-6-PD deficiency that provides increased protection against malaria compared to that of the G-6-PD deficiency alone. This relationship is analogous to that between various hemoglobins and malaria in that there is selection for a more severe trait if it provides more protection against malaria. PMID:1107056

  18. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    SciTech Connect

    Woodward, J.

    1996-10-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based upon the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with continuous cofactor recycle. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value commodity chemical.

  19. Methylmalonic semialdehyde dehydrogenase deficiency: demonstration of defective valine and beta-alanine metabolism and reduced malonic semialdehyde dehydrogenase activity in cultured fibroblasts

    SciTech Connect

    Gray, R.G.; Pollitt, R.J.; Webley, J.

    1987-08-01

    Intact cultured fibroblasts from a child with a new metabolic disorder, thought to be due to a deficiency of methylmalonic semialdehyde dehydrogenase, produced labeled CO/sub 2/ normally from (1-/sup 14/C)valine but not from (2-/sup 14/C)valine. CO/sub 2/ production from labeled beta-alanine was also much reduced, confirming the suspicion that malonic semialdehyde dehydrogenase is also deficient in this condition. An assay for malonic semialdehyde dehydrogenase in cell homogenates showed low activity but it was impossible to assess the degree of reduction.

  20. Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.

    PubMed

    Wang, Qingzhao; Ingram, Lonnie O; Shanmugam, K T

    2011-11-22

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates. PMID:22065761

  1. Proline dehydrogenase 2 (PRODH2) is a hydroxyproline dehydrogenase (HYPDH) and molecular target for treating primary hyperoxaluria.

    PubMed

    Summitt, Candice B; Johnson, Lynnette C; Jönsson, Thomas J; Parsonage, Derek; Holmes, Ross P; Lowther, W Todd

    2015-03-01

    The primary hyperoxalurias (PH), types 1-3, are disorders of glyoxylate metabolism that result in increased oxalate production and calcium oxalate stone formation. The breakdown of trans-4-hydroxy-L-proline (Hyp) from endogenous and dietary sources of collagen makes a significant contribution to the cellular glyoxylate pool. Proline dehydrogenase 2 (PRODH2), historically known as hydroxyproline oxidase, is the first step in the hydroxyproline catabolic pathway and represents a drug target to reduce the glyoxylate and oxalate burden of PH patients. This study is the first report of the expression, purification, and biochemical characterization of human PRODH2. Evaluation of a panel of N-terminal and C-terminal truncation variants indicated that residues 157-515 contain the catalytic core with one FAD molecule. The 12-fold higher k(cat)/K(m) value of 0.93 M⁻¹·s⁻¹ for Hyp over Pro demonstrates the preference for Hyp as substrate. Moreover, an anaerobic titration determined a K(d) value of 125 μM for Hyp, a value ~1600-fold lower than the K(m) value. A survey of ubiquinone analogues revealed that menadione, duroquinone, and CoQ₁ reacted more efficiently than oxygen as the terminal electron acceptor during catalysis. Taken together, these data and the slow reactivity with sodium sulfite support that PRODH2 functions as a dehydrogenase and most likely utilizes CoQ₁₀ as the terminal electron acceptor in vivo. Thus, we propose that the name of PRODH2 be changed to hydroxyproline dehydrogenase (HYPDH). Three Hyp analogues were also identified to inhibit the activity of HYPDH, representing the first steps toward the development of a novel approach to treat all forms of PH. PMID:25697095

  2. Proline dehydrogenase 2 (PRODH2) is a hydroxyproline dehydrogenase (HYPDH) and molecular target for treating primary hyperoxaluria

    PubMed Central

    Summitt, Candice B.; Johnson, Lynnette C.; Jönsson, Thomas J.; Parsonage, Derek; Holmes, Ross P.; Lowther, W. Todd

    2015-01-01

    The primary hyperoxalurias (PH), types 1–3, are disorders of glyoxylate metabolism that result in increased oxalate production and calcium oxalate stone formation. The breakdown of trans-4-hydroxy-L-proline (Hyp) from endogenous and dietary sources of collagen makes a significant contribution to the cellular glyoxylate pool. Proline dehydrogenase 2 (PRODH2), historically known as hydroxyproline oxidase, is the first step in the hydroxyproline catabolic pathway and represents a drug target to reduce the glyoxylate and oxalate burden of PH patients. This study is the first report of the expression, purification, and biochemical characterization of human PRODH2. Evaluation of a panel of N-terminal and C-terminal truncation variants indicated that residues 157–515 contain the catalytic core with one FAD molecule. The 12-fold higher kcat/Km value of 0.93 M−1·s−1 for Hyp over Pro demonstrates the preference for Hyp as substrate. Moreover, an anaerobic titration determined a Kd value of 125 μM for Hyp, a value ~1600-fold lower than the Km value. A survey of ubiquinone analogues revealed that menadione, duroquinone, and CoQ1 reacted more efficiently than oxygen as the terminal electron acceptor during catalysis. Taken together, these data and the slow reactivity with sodium sulfite support that PRODH2 functions as a dehydrogenase and most likely utilizes CoQ10 as the terminal electron acceptor in vivo. Thus, we propose that the name of PRODH2 be changed to hydroxyproline dehydrogenase (HYPDH). Three Hyp analogues were also identified to inhibit the activity of HYPDH, representing the first steps toward the development of a novel approach to treat all forms of PH. PMID:25697095

  3. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

    PubMed Central

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

    2015-01-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization. PMID:26151670

  4. Characterization of the iron-sulfur centers in succinate dehydrogenase.

    PubMed Central

    Coles, C J; Holm, R H; Kurtz, D M; Orme-Johnson, W H; Rawlings, J; Singer, T P; Wong, G B

    1979-01-01

    Two techniques have been applied to the determination of the number and type (2-Fe, 4-Fe) of iron-sulfur centers in the iron-sulfur flavoprotein succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1]. One procedure uses p-CF3C6H4SH as an extrusion reagent and Fourier transform 19F nuclear magentic resonance as the method of detection and quantitation of extruded cores of these centers in the form of [Fe2S2(SRF)4]2- and [Fe4S4(SRF)4]2- (RF = p-C6H4CF3). The second procedure, interprotein core transfer, involves thiol displacement of iron-sulfur cores followed by specific core transfer to the apoproteins of Bacillus polymyxa ferredoxin and adrenodoxin. Detection and quantitation are accomplished by electron paramagnetic resonance of reduced proteins at low temperatures. Both procedures clearly show that succinate dehydrogenase contains two dimeric (Fe2S2) and one tetrameric (Fe4S4) centers per mole of histidyl flavin, accounting for all eight nonheme iron and eight labile sulfur atoms found by chemical analysis. These results remove uncertainties created by the less than stoichiometric amounts of binuclear centers detected by electron paramagnetic resonance after dithionite reduction and provide secure characterization of the iron-sulfur centers in this enzyme. PMID:226982

  5. Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals

    PubMed Central

    Schueren, Fabian; Lingner, Thomas; George, Rosemol; Hofhuis, Julia; Dickel, Corinna; Gärtner, Jutta; Thoms, Sven

    2014-01-01

    Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes. DOI: http://dx.doi.org/10.7554/eLife.03640.001 PMID:25247702

  6. High-pressure-induced water penetration into 3-isopropylmalate dehydrogenase

    SciTech Connect

    Nagae, Takayuki; Kawamura, Takashi; Chavas, Leonard M. G.; Niwa, Ken; Hasegawa, Masashi; Kato, Chiaki; Watanabe, Nobuhisa

    2012-03-01

    Structures of 3-isopropylmalate dehydrogenase were determined at pressures ranging from 0.1 to 650 MPa. Comparison of these structures gives a detailed picture of the swelling of a cavity at the dimer interface and the generation of a new cleft on the molecular surface, which are accompanied by water penetration. Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH.

  7. Structural analysis of fungus-derived FAD glucose dehydrogenase

    PubMed Central

    Yoshida, Hiromi; Sakai, Genki; Mori, Kazushige; Kojima, Katsuhiro; Kamitori, Shigehiro; Sode, Koji

    2015-01-01

    We report the first three-dimensional structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as the cofactor. This is currently the most advanced and popular enzyme used in glucose sensor strips manufactured for glycemic control by diabetic patients. We prepared recombinant nonglycosylated FAD-dependent glucose dehydrogenase (FADGDH) derived from Aspergillus flavus (AfGDH) and obtained the X-ray structures of the binary complex of enzyme and reduced FAD at a resolution of 1.78 Å and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. The overall structure is similar to that of fungal glucose oxidases (GOxs) reported till date. The ternary complex with reduced FAD and LGC revealed the residues recognizing the substrate. His505 and His548 were subjected for site-directed mutagenesis studies, and these two residues were revealed to form the catalytic pair, as those conserved in GOxs. The absence of residues that recognize the sixth hydroxyl group of the glucose of AfGDH, and the presence of significant cavity around the active site may account for this enzyme activity toward xylose. The structural information will contribute to the further engineering of FADGDH for use in more reliable and economical biosensing technology for diabetes management. PMID:26311535

  8. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation.

    PubMed

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B Martin; Ludwig, Roland; Divne, Christina

    2015-01-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization. PMID:26151670

  9. In Silico Analysis of Arabidopsis thaliana Peroxisomal 6-Phosphogluconate Dehydrogenase

    PubMed Central

    Fernández-Fernández, Álvaro D.; Corpas, Francisco J.

    2016-01-01

    NADPH, whose regeneration is critical for reductive biosynthesis and detoxification pathways, is an essential component in cell redox homeostasis. Peroxisomes are subcellular organelles with a complex biochemical machinery involved in signaling and stress processes by molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). NADPH is required by several peroxisomal enzymes involved in β-oxidation, NO, and glutathione (GSH) generation. Plants have various NADPH-generating dehydrogenases, one of which is 6-phosphogluconate dehydrogenase (6PGDH). Arabidopsis contains three 6PGDH genes that probably are encoded for cytosolic, chloroplastic/mitochondrial, and peroxisomal isozymes, although their specific functions remain largely unknown. This study focuses on the in silico analysis of the biochemical characteristics and gene expression of peroxisomal 6PGDH (p6PGDH) with the aim of understanding its potential function in the peroxisomal NADPH-recycling system. The data show that a group of plant 6PGDHs contains an archetypal type 1 peroxisomal targeting signal (PTS), while in silico gene expression analysis using affymetrix microarray data suggests that Arabidopsis p6PGDH appears to be mainly involved in xenobiotic response, growth, and developmental processes. PMID:27034898

  10. Phenylbutyrate Therapy for Pyruvate Dehydrogenase Complex Deficiency and Lactic Acidosis

    PubMed Central

    Ferriero, Rosa; Manco, Giuseppe; Lamantea, Eleonora; Nusco, Edoardo; Ferrante, Mariella I.; Sordino, Paolo; Stacpoole, Peter W.; Lee, Brendan; Zeviani, Massimo; Brunetti-Pierri, Nicola

    2014-01-01

    Lactic acidosis is a build-up of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1α subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57B6/L wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1α in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1α through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noam631 zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis. PMID:23467562

  11. Alcohol and aldehyde dehydrogenase polymorphisms in Chinese and Indian populations.

    PubMed

    Tan, Ene-Choo; Lim, Leslie; Leong, Jern-Yi; Lim, Jing-Yan; Lee, Arthur; Yang, Jun; Tan, Chay-Hoon; Winslow, Munidasa

    2010-01-01

    The association between two functional polymorphisms in alcohol dehydrogenase (ADH2/ADH1B) and aldehyde dehydrogenase (ALDH2) genes and alcohol dependence was examined in 182 Chinese and Indian patients undergoing treatment for alcohol dependence and 184 screened control subjects from Singapore. All subjects were screened by the Alcohol Use Disorders Identification Test (AUDIT). Patients were also administered the Severity of Alcohol Dependence Questionnaire (SADQ). Polymorphisms were genotyped by allele-specific polymerase chain reaction and selected genotypes confirmed by DNA sequencing or restriction fragment length polymorphism. Our results showed that frequencies of ADH1B*2 and ALDH2*2 were higher in controls compared to alcohol-dependent subjects for both Chinese and Indians. Frequencies of these two alleles were also higher in the 104 Chinese controls compared to the 80 Indian controls. None of the eight Chinese who were homozygous for both protective alleles was alcohol dependent. The higher frequencies of the protective alleles could explain the lower rate of alcohol dependence in Chinese. PMID:20025435

  12. Engineering of Pyranose Dehydrogenase for Increased Oxygen Reactivity

    PubMed Central

    Krondorfer, Iris; Lipp, Katharina; Brugger, Dagmar; Staudigl, Petra; Sygmund, Christoph; Haltrich, Dietmar; Peterbauer, Clemens K.

    2014-01-01

    Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity. PMID:24614932

  13. Crystal structure of a chimaeric bacterial glutamate dehydrogenase.

    PubMed

    Oliveira, Tânia; Sharkey, Michael A; Engel, Paul C; Khan, Amir R

    2016-06-01

    Glutamate dehydrogenases (EC 1.4.1.2-4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)(+) as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD(+) versus NADP(+), but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase from Clostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia coli enzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP(+) cofactor from the parent E. coli domain II, although there are subtle differences in catalytic activity. PMID:27303899

  14. A straightforward radiometric technique for measuring IMP dehydrogenase.

    PubMed

    Cooney, D A; Wilson, Y; McGee, E

    1983-04-15

    [2-3H]Inosinic acid ([2-3H]IMP) has been biosynthesized in good yield from [2-3H]hypoxanthine and PRPP via the action of a partially purified preparation of hypoxanthine/guanine phosphoribosyl transferase from mouse brain. The product was purified in one step by ascending paper chromatography, and used to assess the activity of IMP dehydrogenase. To conduct the assay, tritiated substrate is admixed with enzyme in a final volume of 10 microliters; NAD is present to serve as cofactor for the reaction, and allopurinol to inhibit the oxidation of any hypoxanthine generated as a consequence of side reactions. After an appropriate period of incubation, the 3H2O arising from the oxidation of tritiated IMP via [3H]NAD is isolated by quantitative microdistillation. Performed as described, the assay is facile, sensitive, and accurate, with the capability of detecting the dehydrogenation of as little as 1 pmol of [3H]IMP. Using it, measurements have been made of IMP dehydrogenase in a comprehensive array of mouse organs. Of these, pancreas contained the enzyme at the highest specific activity. PMID:6135372

  15. d-Xylose Metabolism in Hypocrea jecorina: Loss of the Xylitol Dehydrogenase Step Can Be Partially Compensated for by lad1-Encoded l-Arabinitol-4-Dehydrogenase

    PubMed Central

    Seiboth, Bernhard; Hartl, Lukas; Pail, Manuela; Kubicek, Christian P.

    2003-01-01

    With the goal of the genetic characterization of the d-xylose pathway in Hypocrea jecorina (anamorph: Trichoderma reesei), we cloned the xdh1 gene, encoding NAD-xylitol dehydrogenase, which catalyzes the second step of fungal d-xylose catabolism. This gene encodes a 363-amino-acid protein which has a mass of 38 kDa, belongs to the zinc-containing alcohol dehydrogenase family, exhibits high sequence identity to the published sequences of xylitol dehydrogenases from yeast origins, but contains a second, additional binding site for Zn2+. The enzyme catalyzed the NAD-dependent oxidation of xylitol and d-sorbitol and the NADH-dependent reduction of d-xylulose and d-fructose. No activity was observed with NADP, l-arabinose, or l-arabinitol. A single 1.4-kb transcript was formed during growth on xylan, d-xylose, l-arabinose, l-arabinitol and, at a lower abundance, xylitol, d-galactose, galactitol, and lactose but not on d-glucose and glycerol. xdh1 deletion mutants exhibited 50% reduced growth rates on d-xylose, whereas growth rates on xylitol remained unaltered. These mutants contained 30% of the xylitol dehydrogenase activity of the parent strain, indicating the presence of a second xylitol dehydrogenase. This activity was shown to be due to lad1-encoded l-arabinitol-4-dehydrogenase, because H. jecorina xdh1 lad1 double-deletion strains failed to grow on d-xylose or xylitol. In contrast, lad1 deletion strains of H. jecorina grew normally on these carbon sources. These results show that H. jecorina contains a single xylitol dehydrogenase which is encoded by xdh1 and is involved in the metabolism of d-xylose and that lad1-encoded l-arabinitol-4-dehydrogenase can compensate for it partially in mutants with a loss of xdh1 function. PMID:14555469

  16. Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.

    PubMed Central

    Wexler, I D; Kerr, D S; Ho, L; Lusk, M M; Pepin, R A; Javed, A A; Mole, J E; Jesse, B W; Thekkumkara, T J; Pons, G

    1988-01-01

    Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency. Images PMID:3140238

  17. Cloning and mRNA Expression of NADH Dehydrogenase during Ochlerotatus taeniorhynchus Development and Pesticide Response

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NADH dehydrogenase, the largest of the respiratory complexes, is the first enzyme of the mitochondrial electron transport chain. We have cloned and sequenced cDNA of NADH dehydrogenase gene from Ochlerotatus (Ochlerotatus) taeniorhynchus (Wiedemann) adult (GeneBank Accession number: FJ458415). The ...

  18. The influence of oxygen on radiation-induced structural and functional changes in glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Rodacka, Aleksandra; Serafin, Eligiusz; Bubinski, Michal; Krokosz, Anita; Puchala, Mieczyslaw

    2012-07-01

    Proteins are major targets for oxidative damage due to their abundance in cells and high reactivity with free radicals. In the present study we examined the influence of oxygen on radiation-induced inactivation and structural changes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH). We chose these two enzymes because they occur at high concentrations and participate in the most important processes in organisms; furthermore, they show considerable similarity in their structure. Protein solutions were irradiated with X-rays in doses ranging from 0.1 to 0.7 kGy, in air and N2O. The much higher radiation inactivation of GAPDH as compared to LDH is correlated with substantially greater structural changes in this protein, mainly involving the loss of free thiol groups (-SH). Of lesser importance in the differentiation of the radiosensitivity of the studied enzymes are tryptophan residues. Molecular oxygen, present during irradiation, increased to a significantly greater extent the inactivation and structural changes of GAPDH than that of LDH. The results suggest that the greater effect of oxygen on GAPDH is due to the higher efficiency of the superoxide radical, the higher amount of hydroperoxides generated, and the higher degree of unfolding of this protein.

  19. Electrochemical conversion of carbon dioxide to methanol with the assistance of formate dehydrogenase and methanol dehydrogenase as biocatalysts

    SciTech Connect

    Kuwabata, Susumu; Tsuda, Ryo; Yoneyama, Hiroshi )

    1994-06-15

    Electrolysis at potentials between -0.7 and -0.9 V vs SCE of carbon dioxide-saturated phosphate buffer solutions (pH7) containing formate dehydrogenase (FDH) and either methyl viologen (MV[sup 2+]) or pyrroloquinolinequinone (PQQ) as an electron mediator yielded formate with current efficiencies as high as 90%. The enzyme was durable as long as the electrolysis was carried out in the dark. Electrolysis of phosphate buffer solutions containing sodium formate in the presence of methanol dehydrogenase (MDH) and MV[sup 2+] at -0.7 V vs SCE yielded formaldehyde if the concentration of the enzyme used was low, whereas both formaldehyde and methanol were produced for relatively high concentrations of the enzyme where the methanol production began to occur when the formaldehyde produced accumulated. The use of PQQ in place of MV[sup 2+] as the electron mediator exclusively produced methanol alone after some induction period in the electrolysis. On the basis of these results, successful attempts have been made to reduce carbon dioxide to methanol with cooperative assistance of FDH and MDH in the presence of PQQ as the electron mediator. The role of enzyme and mediator in these reduction processes is discussed in detail. 34 refs., 10 figs., 2 tabs.

  20. Evaluation of alcohol dehydrogenase and aldehyde dehydrogenase enzymes as bi-enzymatic anodes in a membraneless ethanol microfluidic fuel cell

    NASA Astrophysics Data System (ADS)

    Galindo-de-la-Rosa, J.; Arjona, N.; Arriaga, L. G.; Ledesma-García, J.; Guerra-Balcázar, M.

    2015-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldH) enzymes were immobilized by covalent binding and used as the anode in a bi-enzymatic membraneless ethanol hybrid microfluidic fuel cell. The purpose of using both enzymes was to optimize the ethanol electro-oxidation reaction (EOR) by using ADH toward its direct oxidation and AldH for the oxidation of aldehydes as by-products of the EOR. For this reason, three enzymatic bioanode configurations were evaluated according with the location of enzymes: combined, vertical and horizontally separated. In the combined configuration, a current density of 16.3 mA cm-2, a voltage of 1.14 V and a power density of 7.02 mW cm-2 were obtained. When enzymes were separately placed in a horizontal and vertical position the ocp drops to 0.94 V and to 0.68 V, respectively. The current density also falls to values of 13.63 and 5.05 mA cm-2. The decrease of cell performance of bioanodes with separated enzymes compared with the combined bioanode was of 31.7% and 86.87% for the horizontal and the vertical array.

  1. Structural and functional properties of a yeast xylitol dehydrogenase, a Zn2+-containing metalloenzyme similar to medium-chain sorbitol dehydrogenases.

    PubMed Central

    Lunzer, R; Mamnun, Y; Haltrich, D; Kulbe, K D; Nidetzky, B

    1998-01-01

    The NAD+-dependent xylitol dehydrogenase from the xylose-assimilating yeast Galactocandida mastotermitis has been purified in high yield (80%) and characterized. Xylitol dehydrogenase is a heteronuclear multimetal protein that forms homotetramers and contains 1 mol of Zn2+ ions and 6 mol of Mg2+ ions per mol of 37.4 kDa protomer. Treatment with chelating agents such as EDTA results in the removal of the Zn2+ ions with a concomitant loss of enzyme activity. The Mg2+ ions are not essential for activity and are removed by chelation or extensive dialysis without affecting the stability of the enzyme. Results of initial velocity studies at steady state for d-sorbitol oxidation and d-fructose reduction together with the characteristic patterns of product inhibition point to a compulsorily ordered Theorell-Chance mechanism of xylitol dehydrogenase in which coenzyme binds first and leaves last. At pH 7.5, the binding of NADH (Ki approximately 10 microM) is approx. 80-fold tighter than that of NAD+. Polyhydroxyalcohols require at least five carbon atoms to be good substrates of xylitol dehydrogenase, and the C-2 (S), C-3 (R) and C-4 (R) configuration is preferred. Therefore xylitol dehydrogenase shares structural and functional properties with medium-chain sorbitol dehydrogenases. PMID:9806889

  2. Amphibian alcohol dehydrogenase, the major frog liver enzyme. Relationships to other forms and assessment of an early gene duplication separating vertebrate class I and class III alcohol dehydrogenases

    SciTech Connect

    Cederlund, E.; Joernvall, H. ); Peralba, J.M.; Pares, X. )

    1991-03-19

    Submammalian alcohol dehydrogenase structures can be used to evaluate the origins and functions of different types of the mammalian enzyme. Two avian forms were recently reported, and the authors now define the major amphibian alcohol dehydrogenase. The enzyme from the liver of the Green frog Rana perezi was purified, carboxymethylated, and submitted to amino acid sequence determination by peptide analysis of six different digest. The protein has a 375-residue subunit and is a class I alcohol dehydrogenase, bridging the gap toward the original separation of the classes that are observable in the human alcohol dehydrogenase system. In relation to the human class I enzyme, the amphibian protein has residue identities exactly halfway (68%) between those for the corresponding avian enzyme (74%) and the human class III enzyme (62%), suggesting an origin of the alcohol dehnydrogenase classes very early in or close to the evolution of the vertebrate line. This conclusion suggests that these enzyme classes are more universal among animals than previously realized and constitutes the first real assessment of the origin of the duplications leading to the alcohol dehydrogenase classes. In conclusion, the amphibian enzyme allows a rough positioning of the divergence of the alcohol dehydrogenase classes, shows that the class I type is widesprread in vertebrates, and functionally conforms with greater variations at the substrate-binding than the coenzyme-binding site.

  3. Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation.

    PubMed Central

    Engeland, K; Höög, J O; Holmquist, B; Estonius, M; Jörnvall, H; Vallee, B L

    1993-01-01

    The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. The activities of the recombinant native and mutant enzymes toward ethanol are essentially identical, but mutagenesis greatly decreases the kcat/Km values for glutathione-dependent formaldehyde oxidation. The catalytic efficiency for the Asp variant is < 0.1% that of the unmutated enzyme, due to both a higher Km and a lower kcat value. As with the native enzyme, neither mutant can oxidize methanol, be saturated by ethanol, or be inhibited by 4-methylpyrazole; i.e., they retain these class III characteristics. In contrast, however, their activation by fatty acids, another characteristic unique to class III alcohol dehydrogenase, is markedly attenuated. The Ala mutant is activated only slightly, but the Asp mutant is not activated at all. The results strongly indicate that Arg-115 in class III alcohol dehydrogenase is a component of the binding site for activating fatty acids and is critical for the binding of S-hydroxymethylglutathione in glutathione-dependent formaldehyde dehydrogenase activity. PMID:8460164

  4. Changing kinetic properties of glucose-6-phosphate dehydrogenase from pea chloroplasts during photosynthetic induction

    SciTech Connect

    Yuan, X.; Anderson, L.E.

    1987-04-01

    The first enzyme of the oxidative pentose phosphate pathway, glucose-6-P dehydrogenase (EC 1.1.1.49), is inactivated when pea chloroplasts are irradiated. They have examined the kinetics of light inactivation of glucose-6-P dehydrogenase in intact chloroplasts during photosynthetic induction and the kinetic parameters of the active (dark) and less active (light) form of the dehydrogenase. Light inactivation of the dehydrogenase is rapid and occurs before photosynthetic O/sub 2/ evolution is measureable in intact chloroplasts. Likewise dark activation is quite rapid. The major change in the kinetic parameters of glucose-6-phosphate dehydrogenase is in maximal velocity. This light inactivation probably prevents operation of a futile cycle involving glucose-6-P, NADPH and oxidative and reductive pentose phosphate pathway enzymes.

  5. [Characterization of aldehyde dehydrogenase gene fragment from mung bean Vigna radiata using the polymerase chain reaction].

    PubMed

    Ponomarev, A G; Bubiakina, V V; Tatarinova, T D; Zelenin, S M

    1998-01-01

    Two degenerate oligonucleotide sequence primers and polymerase chain reactions on total DNA have been utilized to clone on 651--bp gene fragment coding the central part of amino acid sequence of an earlier unknown aldehyde dehydrogenase (ALDH) from mung bean. The deduced partial amino acid sequence for this aldehyde dehydrogenase shows about 65% sequence identity to ALDHs of Vibrio cholerae Rhodococcus sp., Alcaligenes eutrophus and about 45% sequence identity to mammalian ALDHs 1 and 2, ALDHs of Aspergillus niger and A, nidulans, the betain aldehyde dehydrogenase from spinach. Alignment of the mung bean aldehyde dehydrogenase partial amino acid sequence with the sequence of 16 NAD(P)(+)-dependent aldehyde dehydrogenases has demonstrated that all strictly conserved amino acid residues and all three conservative regions are identical. PMID:9778740

  6. Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7.

    PubMed Central

    Suzuki, T; Inoki, Y; Yamagishi, A; Iwasaki, T; Wakagi, T; Oshima, T

    1997-01-01

    The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family. PMID:9023199

  7. Increased IMP dehydrogenase gene expression in solid tumor tissues and tumor cell lines

    SciTech Connect

    Collart, F.R.; Chubb, C.B.; Mirkin, B.L.; Huberman, E.

    1992-07-10

    IMP dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this possibility, we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results are consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.

  8. Analysis of rat cytosolic 9-cis-retinol dehydrogenase activity and enzymatic characterization of rat ADHII.

    PubMed

    Popescu, G; Napoli, J L

    2000-01-01

    We report the characterization of two enzymes that catalyze NAD(+)-dependent 9-cis-retinol dehydrogenase activity in rat liver cystol. Alcohol dehydrogenase class I (ADHI) contributes > 80% of the NA D+-dependent 9-cis-retinol dehydrogenase activity recovered, whereas alcohol dehydrogenase class II (ADHII), not identified previously at the protein level, nor characterized enzymatically in rat, accounts for approximately 2% of the activity. Rat ADHII exhibits properties different from those described for human ADHII. Moreover, rat ADHII-catalyzed rates of ethanol dehydrogenation are markedly lower than octanol or retinoid dehydrogenation rates. Neither ethanol nor 4-methylpyrazole inhibits the 9-cis-retinol dehydrogenase activity of rat ADHII. We propose that ADHII represents the previously observed additional retinoid oxidation activity of rat liver cytosol which occurred in the presence of either ethanol or 4-methylpyrazole. We also show that human and rat ADHII differ considerably in enzymatic properties. PMID:10606766

  9. Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis.

    PubMed Central

    Winberg, J O; Hovik, R; McKinley-McKee, J S; Juan, E; Gonzalez-Duarte, R

    1986-01-01

    Purified Drosophila lebanonensis alcohol dehydrogenase (Adh) revealed one enzymically active zone in starch gel electrophoresis at pH 8.5. This zone was located on the cathode side of the origin. Incubation of D. lebanonensis Adh with NAD+ and acetone altered the electrophoretic pattern to more anodal migrating zones. D. lebanonensis Adh has an Mr of 56,000, a subunit of Mr of 28 000 and is a dimer with two active sites per enzyme molecule. This agrees with a polypeptide chain of 247 residues. Metal analysis by plasma emission spectroscopy indicated that this insect alcohol dehydrogenase is not a metalloenzyme. In studies of the substrate specificity and stereospecificity, D. lebanonensis Adh was more active with secondary than with primary alcohols. Both alkyl groups in the secondary alcohols interacted hydrophobically with the alcohol binding region of the active site. The catalytic centre activity for propan-2-ol was 7.4 s-1 and the maximum velocity of most secondary alcohols was approximately the same and indicative of rate-limiting enzyme-coenzyme dissociation. For primary alcohols the maximum velocity varied and was much lower than for secondary alcohols. The catalytic centre activity for ethanol was 2.4 s-1. With [2H6]ethanol a primary kinetic 2H isotope effect of 2.8 indicated that the interconversion of the ternary complexes was rate-limiting. Pyrazole was an ethanol-competitive inhibitor of the enzyme. The difference spectra of the enzyme-NAD+-pyrazole complex gave an absorption peak at 305 nm with epsilon 305 14.5 X 10(3) M-1 X cm-1. Concentrations and amounts of active enzyme can thus be determined. A kinetic rate assay to determine the concentration of enzyme active sites is also presented. This has been developed from active site concentrations established by titration at 305 nm of the enzyme and pyrazole with NAD+. In contrast with the amino acid composition, which indicated that D. lebanonensis Adh and the D. melanogaster alleloenzymes were not

  10. Nomenclature of glucose-6-phosphate dehydrogenase in man*

    PubMed Central

    1967-01-01

    The World Health Organization convened in Geneva from 5 to 10 December 1966 a Scientific Group on Standardization of Procedures for the Study of Glucose-6-Phosphate Dehydrogenase1 (EC 1.1.1.49; D-glucose-6-phosphate: NAPD oxidoreductase; G6PD). Variants of this enzyme have attracted international attention both as causes of various haemolytic disorders and as useful genetic markers in man. In the course of the meeting the variants of this enzyme thus far described were extensively reviewed. There was unanimous agreement that a consistent system of nomenclature would be desirable, and that as G6PD variants were only one example of similar polymorphisms in man, a nomenclature should be devised which might conceivably be applied to other enzymes. The Group included the following recommendations on nomenclature in its report, which will be published in full in World Health Organization: Technical Report Series, 1967, 366. PMID:5299754

  11. Microbial metabolic activity in soil as measured by dehydrogenase determinations

    NASA Technical Reports Server (NTRS)

    Casida, L. E., Jr.

    1977-01-01

    The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 C incubation with either glucose or yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.

  12. Human Lactate Dehydrogenase A Inhibitors: A Molecular Dynamics Investigation

    PubMed Central

    Shi, Yun; Pinto, B. Mario

    2014-01-01

    Lactate dehydrogenase A (LDHA) is an important enzyme in fermentative glycolysis, generating most energy for cancer cells that rely on anaerobic respiration even under normal oxygen concentrations. This renders LDHA a promising molecular target for the treatment of various cancers. Several efforts have been made recently to develop LDHA inhibitors with nanomolar inhibition and cellular activity, some of which have been studied in complex with the enzyme by X-ray crystallography. In this work, we present a molecular dynamics (MD) study of the binding interactions of selected ligands with human LDHA. Conventional MD simulations demonstrate different binding dynamics of inhibitors with similar binding affinities, whereas steered MD simulations yield discrimination of selected LDHA inhibitors with qualitative correlation between the in silico unbinding difficulty and the experimental binding strength. Further, our results have been used to clarify ambiguities in the binding modes of two well-known LDHA inhibitors. PMID:24466056

  13. IMP Dehydrogenase: Structural Schizophrenia and an Unusual Base

    SciTech Connect

    Hedstrom,L.; Gan, L.

    2006-01-01

    Textbooks describe enzymes as relatively rigid templates for the transition state of a chemical reaction, and indeed an enzyme such as chymotrypsin, which catalyzes a relatively simple hydrolysis reaction, is reasonably well described by this model. Inosine monophosphate dehydrogenase (IMPDH) undergoes a remarkable array of conformational transitions in the course of a complicated catalytic cycle, offering a dramatic counterexample to this view. IMPDH displays several other unusual mechanistic features, including an Arg residue that may act as a general base catalyst and a dynamic monovalent cation site. Further, IMPDH appears to be involved in 'moon-lighting' functions that may require additional conformational states. How the balance between conformational states is maintained and how the various conformational states interconvert is only beginning to be understood.

  14. 17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

    SciTech Connect

    Mendoza-Hernandez, G.; Lopez-Solache, I.; Rendon, J.L.; Diaz-Sanchez, V.; Diaz-Zagoya, J.C.

    1988-04-15

    The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively.

  15. Aldehyde dehydrogenase (ALDH) in Alzheimer's and Parkinson's disease.

    PubMed

    Grünblatt, Edna; Riederer, Peter

    2016-02-01

    Evidence suggests that aldehyde dehydrogenase (ALDH; E.C. 1.2.1.3) gene, protein expression and activity are substantially decreased in the substantia nigra of patients with Parkinson's disease (PD). This holds especially true for cytosolic ALDH1A1, while mitochondrial ALDH2 is increased in the putamen of PD. Similarly, in Alzheimer's disease (AD) several studies in genetic, transcriptomic, protein and animal models suggest ALDH involvement in the neurodegeneration processes. Such data are in line with findings of increased toxic aldehydes, like for example malondialdehyde, nonenal, 3,4-dihydroxyphenylacetaldehyde and others. Genetic, transcriptomic and protein alterations may contribute to such data. Also in vitro and in vivo experimental work points to an important role of ALDH in the pathology of neurodegenerative disorders. Aims at investigating dysfunctions of aldehyde detoxification are suitable to define genetic/molecular targets for new therapeutic strategies balancing amine metabolism in devastating disorders like PD and probably also AD. PMID:25298080

  16. Method To Identify Specific Inhibiutors Of Imp Dehydrogenase

    DOEpatents

    Collart, Frank R.; Huberman, Eliezer

    2000-11-28

    This invention relates to methods to identify specific inhibitors of the purine nucleotide synthesis enzyme, IMP dehydrogenase (IMPDH). IMPDH is an essential enzyme found in all free-living organisms from humans to bacteria and is an important therapeutic target. The invention allows the identification of specific inhibitors of any IMPDH enzyme which can be expressed in a functional form in a recombinant host cell. A variety of eukaryotic or prokaryotic host systems commonly used for the expression of recombinant proteins are suitable for the practice of the invention. The methods are amenable to high throughput systems for the screening of inhibitors generated by combinatorial chemistry or other methods such as antisense molecule production. Utilization of exogenous guanosine as a control component of the methods allows for the identification of inhibitors specific for IMPDH rather than other causes of decreased cell proliferation.

  17. Idiopathic intracranial hypertension, hormones, and 11β-hydroxysteroid dehydrogenases

    PubMed Central

    Markey, Keira A; Uldall, Maria; Botfield, Hannah; Cato, Liam D; Miah, Mohammed A L; Hassan-Smith, Ghaniah; Jensen, Rigmor H; Gonzalez, Ana M; Sinclair, Alexandra J

    2016-01-01

    Idiopathic intracranial hypertension (IIH) results in raised intracranial pressure (ICP) leading to papilledema, visual dysfunction, and headaches. Obese females of reproductive age are predominantly affected, but the underlying pathological mechanisms behind IIH remain unknown. This review provides an overview of pathogenic factors that could result in IIH with particular focus on hormones and the impact of obesity, including its role in neuroendocrine signaling and driving inflammation. Despite occurring almost exclusively in obese women, there have been a few studies evaluating the mechanisms by which hormones and adipokines exert their effects on ICP regulation in IIH. Research involving 11β-hydroxysteroid dehydrogenase type 1, a modulator of glucocorticoids, suggests a potential role in IIH. Improved understanding of the complex interplay between adipose signaling factors such as adipokines, steroid hormones, and ICP regulation may be key to the understanding and future management of IIH. PMID:27186074

  18. Engineered PQQ-Glucose Dehydrogenase as a Universal Biosensor Platform.

    PubMed

    Guo, Zhong; Murphy, Lindy; Stein, Viktor; Johnston, Wayne A; Alcala-Perez, Siro; Alexandrov, Kirill

    2016-08-17

    Biosensors with direct electron output hold promise for nearly seamless integration with portable electronic devices. However, so far, they have been based on naturally occurring enzymes that significantly limit the spectrum of detectable analytes. Here, we present a novel biosensor architecture based on analyte-driven intermolecular recombination and activity reconstitution of a re-engineered component of glucometers: PQQ-glucose dehydrogenase. We demonstrate that this sensor architecture can be rapidly adopted for the detection of immunosuppressant drugs, α-amylase protein, or protease activity of thrombin and Factor Xa. The biosensors could be stored in dried form without appreciable loss of activity. We further show that ligand-induced activity of the developed biosensors could be directly monitored by chronoamperometry, enabling construction of disposable sensory electrodes. We expect that this architecture could be expanded to the detection of other biochemical activities, post-translational modifications, nucleic acids, and inorganic molecules. PMID:27463000

  19. Glucose dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus.

    PubMed Central

    Giardina, P; de Biasi, M G; de Rosa, M; Gambacorta, A; Buonocore, V

    1986-01-01

    Glucose dehydrogenase has been purified to homogeneity from cell extracts of the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme utilizes both NAD+ and NADP+ as coenzyme and catalyses the oxidation of several monosaccharides to the corresponding glyconic acid. Substrate specificity and oxidation rate depend on the coenzyme present; when NAD+ is used, the enzyme binds and oxidizes specifically sugars presenting equatorial orientation of hydroxy groups at C-2, C-3 and C-4. The Mr of the native enzyme is 124,000 and decreases to about 60,000 in the presence of 6 M-guanidinium chloride and to about 30,000 in the presence of 5% (w/v) SDS. The enzyme shows maximal activity at pH 9, 77 degrees C and 20 mM-Mg2+, -Mn2+ or -Ca2+ and is fairly stable in the presence of chaotropic agents and water-miscible organic solvents such as methanol or acetone. PMID:3827812

  20. Separation of turkey lactate dehydrogenase isoenzymes using isoelectric focusing technique.

    PubMed

    Heinová, Dagmar; Kostecká, Zuzana; Csank, Tomáš

    2016-01-01

    Native polyacrylamide gel electrophoresis at pH 8.8 did not allow to separate lactate dehydrogenase (LDH) isoenzymes of turkey origin. Five electrophoretically distinguishable forms of the enzyme were detected in serum and tissues of turkey using IEF technique in a pH range of 3-9. Generally, three different groups were seen: (i) those having an anodic domination (heart, kidney, pancreas, and erythrocytes) with mainly LDH-1 fraction, (ii) those having a cathodic domination (breast muscle and serum) with prevalence of LDH-5, and (iii) those with a more uniform distribution (liver, spleen, lung, and brain). The specific enzyme activity was the highest in the breast muscle, followed by heart muscle, and brain. Low activities were detected in serum, kidney, and liver. PMID:26471476

  1. Mapping of the glucose dehydrogenase gene in Bacillus subtilis.

    PubMed Central

    Chaudhry, G R; Halpern, Y S; Saunders, C; Vasantha, N; Schmidt, B J; Freese, E

    1984-01-01

    A 4.0-kilobase DNA fragment containing the developmentally regulated gene for glucose dehydrogenase (gdh) from Bacillus subtilis was incorporated into the plasmid pGX345, which contains a marker conferring chloramphenicol resistance (cat). The resistance marker of the resulting integration vector was used to map the gdh gene on the B. subtilis chromosome. Using PBS1 transduction, the gene order was determined to be aroI cat (gdh) mtlB dal. The cat (gdh) marker was also cotransformable with mtlB. The genetic location of the gdh gene established by this indirect method was confirmed by the fact that the original phage lambda EF2, containing a 10-kilobase B. subtilis DNA fragment from which the 4-kilobase gdh region had been subcloned, also contained the mtlB gene. Images PMID:6438057

  2. Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

    2000-01-01

    Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For

  3. Electrocatalytic hydrocarbon hydroxylation by ethylbenzene dehydrogenase from Aromatoleum aromaticum.

    PubMed

    Kalimuthu, Palraj; Heider, Johann; Knack, Daniel; Bernhardt, Paul V

    2015-02-26

    We report the electrocatalytic activity of ethylbenzene dehydrogenase (EBDH) from the β-proteobacterium Aromatoleum aromaticum. EBDH is a complex 155 kDa heterotrimeric molybdenum/iron-sulfur/heme protein which catalyzes the enantioselective hydroxylation of nonactivated ethylbenzene to (S)-1-phenylethanol without molecular oxygen as cosubstrate. Furthermore, it oxidizes a wide range of other alkyl-substituted aromatic and heterocyclic compounds to their secondary alcohols. Hydroxymethylferrocenium (FM) is used as an artificial electron acceptor for EBDH in an electrochemically driven catalytic system. Electrocatalytic activity of EBDH is demonstrated with both its native substrate ethylbenzene and the related substrate p-ethylphenol. The catalytic system has been modeled by electrochemical simulation across a range of sweep rates and concentrations of each substrate, which provides new insights into the kinetics of the EBDH catalytic mechanism. PMID:25635950

  4. Fabricating polystyrene fiber-dehydrogenase assemble as a functional biocatalyst.

    PubMed

    An, Hongjie; Jin, Bo; Dai, Sheng

    2015-01-01

    Immobilization of the enzymes on nano-structured materials is a promising approach to enhance enzyme stabilization, activation and reusability. This study aimed to develop polystyrene fiber-enzyme assembles to catalyze model formaldehyde to methanol dehydrogenation reaction, which is an essential step for bioconversion of CO2 to a renewable bioenergy. We fabricated and modified electrospun polystyrene fibers, which showed high capability to immobilize dehydrogenase for the fiber-enzyme assembles. Results from evaluation of biochemical activities of the fiber-enzyme assemble showed that nitriation with the nitric/sulfuric acid ratio (v/v, 10:1) and silanization treatment delivered desirable enzyme activity and long-term storage stability, showing great promising toward future large-scale applications. PMID:25435501

  5. [Effect Of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase].

    PubMed

    Dubrovsky, A V; Musina, E V; Kim, A L; Tikhonenko, S A

    2016-01-01

    Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 M and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect. PMID:27266256

  6. Circadian rhythm of lactate dehydrogenase in rat testis.

    PubMed

    Vermouth, N T; Ponce, R H; Carriazo, C S; Blanco, A

    1984-01-01

    Activity of total lactate dehydrogenase (LDH) and of the isozyme X (LDH X or C4) have been determined at 2 hr intervals during 24 hr cycles in testis of adult rats maintained since birth in a photoperiod of 14 hr light: 10 hr dark. LDH X activity of epididymal sections (caput, corpus and cauda) from the same animals was also determined. Total LDH and LDH X activities in testis exhibited circadian rhythms with different timing. LDH X in the three portions of epididymis showed diurnal variations similar to those in testis. Rats subjected to constant light or constant dark presented marked modifications of LDH X profiles, indicating that the photoperiod plays a synchronizer role. While total soluble proteins did not show variations in testis of rats exposed to the photoperiod, a circadian rhythm was demonstrated in animals maintained in constant light or dark. PMID:6467917

  7. A Case of Hyperammonemia Associated with High Dihydropyrimidine Dehydrogenase Activity.

    PubMed

    Nagaharu, Keiki; Ikemura, Kenji; Yamashita, Yoshiki; Oda, Hiroyasu; Ishihara, Mikiya; Sugawara, Yumiko; Tamaru, Satoshi; Mizuno, Toshiro; Katayama, Naoyuki

    2016-01-01

    Over the past decades, 5-Fluorouracil (5-FU) has been widely used to treat several types of carcinoma, including esophageal squamous cell carcinoma. In addition to its common side effects, including diarrhea, mucositis, neutropenia, and anemia, 5-FU treatment has also been reported to cause hyperammonemia. However, the exact mechanism responsible for 5-FU-induced hyperammonemia remains unknown. We encountered an esophageal carcinoma patient who developed hyperammonemia when receiving 5-FU-containing chemotherapy but did not exhibit any of the other common adverse effects of 5-FU treatment. At the onset of hyperammonemia, laboratory tests revealed high dihydropyrimidine dehydrogenase (DPD) activity and rapid 5-FU clearance. Our findings suggested that 5-FU hypermetabolism may be one of the key mechanisms responsible for hyperammonemia during 5-FU treatment. PMID:27195162

  8. Protein-mediated assembly of succinate dehydrogenase and its cofactors.

    PubMed

    Van Vranken, Jonathan G; Na, Un; Winge, Dennis R; Rutter, Jared

    2015-01-01

    Succinate dehydrogenase (or complex II; SDH) is a heterotetrameric protein complex that links the tribarboxylic acid cycle with the electron transport chain. SDH is composed of four nuclear-encoded subunits that must translocate independently to the mitochondria and assemble into a mature protein complex embedded in the inner mitochondrial membrane. Recently, it has become clear that failure to assemble functional SDH complexes can result in cancer and neurodegenerative syndromes. The effort to thoroughly elucidate the SDH assembly pathway has resulted in the discovery of four subunit-specific assembly factors that aid in the maturation of individual subunits and support the assembly of the intact complex. This review will focus on these assembly factors and assess the contribution of each factor to the assembly of SDH. Finally, we propose a model of the SDH assembly pathway that incorporates all extant data. PMID:25488574

  9. SDHAF4 promotes mitochondrial succinate dehydrogenase activity and prevents neurodegeneration

    PubMed Central

    Van Vranken, Jonathan G.; Bricker, Daniel K.; Dephoure, Noah; Gygi, Steven P.; Cox, James E.; Thummel, Carl S.; Rutter, Jared

    2014-01-01

    SUMMARY Succinate dehydrogenase (SDH) occupies a central place in cellular energy production, linking the tricarboxylic cycle with the electron transport chain. As a result, a subset of cancers and neuromuscular disorders result from mutations affecting any of the four SDH structural subunits or either of two known SDH assembly factors. Herein we characterize a novel evolutionarily conserved SDH assembly factor designated Sdh8/SDHAF4, using yeast, Drosophila, and mammalian cells. Sdh8 interacts specifically with the catalytic Sdh1 subunit in the mitochondrial matrix, facilitating its association with Sdh2 and the subsequent assembly of the SDH holocomplex. These roles for Sdh8 are critical for preventing motility defects and neurodegeneration in Drosophila as well as the excess ROS generated by free Sdh1. These studies provide insights into the mechanisms by which SDH is assembled and raise the possibility that some forms of neuromuscular disease may be associated with mutations that affect this SDH assembly factor. PMID:24954416

  10. Protein-mediated assembly of succinate dehydrogenase and its cofactors

    PubMed Central

    Van Vranken, Jonathan G.; Na, Un; Winge, Dennis R.; Rutter, Jared

    2015-01-01

    Succinate dehydrogenase (or Complex II; SDH) is a heterotetrameric protein complex that links the tribarboxylic acid cycle with the electron transport chain. SDH is composed of four nuclear-encoded subunits that must translocate independently to the mitochondria and assemble into a mature protein complex embedded in the inner mitochondrial membrane. Recently, it has become clear that failure to assemble functional SDH complexes can result in cancer and neurodegenerative syndromes. The effort to thoroughly elucidate the SDH assembly pathway has resulted in the discovery of four subunit-specific assembly factors that aid in the maturation of individual subunits and support the assembly of the intact complex. This review will focus on these assembly factors and assess the contribution of each factor to the assembly of SDH. Finally, we propose a model of the SDH assembly pathway that incorporates all extant data. PMID:25488574

  11. Light and Acetate Regulate a Mitochondrial Malate Dehydrogenase 1

    PubMed Central

    Struck, Friedhelm; Grölz-Krug, Sabine; Boschek, Bruce; Zetsche, Klaus

    1987-01-01

    A malate dehydrogenase was purified from the unicellular green alga Chlorogonium elongatum Dangeard. The enzyme was localized in the mitochondria by immunogold electron microscopy and was found to be present on the cristae. The concentration of the enzyme is regulated by acetate and light. In cells cultured heterotrophically with acetate as carbon source the activity and the concentration of the enzyme is 5- to 6-fold higher than in autotrophic cells. In mixotrophically cultured cells (light and acetate) the enzyme level attains only half of the value of that in heterotrophic cells. Acetate induces an increase of the enzyme concentration while light has an inhibitory effect on this process. Images Fig. 2 Fig. 3 PMID:16665643

  12. Alcohol dehydrogenase polymorphism in barrel cactus populations of Drosophila mojavensis.

    PubMed

    Cleland, S; Hocutt, G D; Breitmeyer, C M; Markow, T A; Pfeiler, E

    1996-07-01

    Starch gel electrophoresis revealed that the alcohol dehydrogenase (ADH-2) locus was polymorphic in two populations (from Agua Caliente, California and the Grand Canyon, Arizona) of cactophilic Drosophila mojavensis that utilize barrel cactus (Ferocactus acanthodes) as a host plant. Electromorphs representing products of a slow (S) and a fast (F) allele were found in adult flies. The frequency of the slow allele was 0.448 in flies from Agua Caliente and 0.659 in flies from the Grand Canyon. These frequencies were intermediate to those of the low (Baja California peninsula, Mexico) and high (Sonora, Mexico and southern Arizona) frequency Adh-2S populations of D. mojavensis that utilize different species of host cacti. PMID:8765684

  13. Encapsulation of Alcohol Dehydrogenase in Mannitol by Spray Drying

    PubMed Central

    Shiga, Hirokazu; Joreau, Hiromi; Neoh, Tze Loon; Furuta, Takeshi; Yoshii, Hidefumi

    2014-01-01

    The retention of the enzyme activity of alcohol dehydrogenase (ADH) has been studied in various drying processes such as spray drying. The aim of this study is to encapsulate ADH in mannitol, either with or without additive in order to limit the thermal denaturation of the enzyme during the drying process. The retention of ADH activity was investigated at different drying temperatures. When mannitol was used, the encapsulated ADH was found inactive in all the dried powders. This is presumably due to the quick crystallization of mannitol during spray drying that resulted in the impairment of enzyme protection ability in comparison to its amorphous form. Maltodextin (dextrose equivalent = 11) was used to reduce the crystallization of mannitol. The addition of maltodextrin increased ADH activity and drastically changed the powder X-ray diffractogram of the spray-dried powders. PMID:24662364

  14. A Case of Hyperammonemia Associated with High Dihydropyrimidine Dehydrogenase Activity

    PubMed Central

    Nagaharu, Keiki; Ikemura, Kenji; Yamashita, Yoshiki; Oda, Hiroyasu; Ishihara, Mikiya; Sugawara, Yumiko; Tamaru, Satoshi; Mizuno, Toshiro; Katayama, Naoyuki

    2016-01-01

    Over the past decades, 5-Fluorouracil (5-FU) has been widely used to treat several types of carcinoma, including esophageal squamous cell carcinoma. In addition to its common side effects, including diarrhea, mucositis, neutropenia, and anemia, 5-FU treatment has also been reported to cause hyperammonemia. However, the exact mechanism responsible for 5-FU-induced hyperammonemia remains unknown. We encountered an esophageal carcinoma patient who developed hyperammonemia when receiving 5-FU-containing chemotherapy but did not exhibit any of the other common adverse effects of 5-FU treatment. At the onset of hyperammonemia, laboratory tests revealed high dihydropyrimidine dehydrogenase (DPD) activity and rapid 5-FU clearance. Our findings suggested that 5-FU hypermetabolism may be one of the key mechanisms responsible for hyperammonemia during 5-FU treatment. PMID:27195162

  15. Frostbite: A Novel Presentation of Glucose-6-Phosphate Dehydrogenase Deficiency?

    PubMed

    Bowles, Justin M; Joas, Chris; Head, Steven

    2015-01-01

    Acute hemolytic anemia (AHA) due to glucose 6-phosphate dehydrogenase (G6PD) deficiency has rarely been recognized as a contributor to the development of frostbite. We discuss a case of frostbite in a 32-year-old male Marine with G6PD deficiency during military training on Mount McKinley in Alaska, which eventually led to a permanent disability. In this report, the pathophysiology of G6PD deficiency, the effects of hemolytic anemia, and factors that contribute to frostbite will be discussed, as well as the clinical findings, treatment course, and the outcome of this case. The patient was evacuated and admitted to Alaska Regional Hospital. He was treated for fourth-degree frostbite, ultimately resulting in the complete or partial amputation of all toes. Although it cannot be proved that AHA occurred in this patient, this case potentially adds frostbite to the list of rare but possible clinical presentations of G6PD deficiency. PMID:26360347

  16. Kawasaki disease with Glucose-6-Phosphate Dehydrogenase deficiency, case report.

    PubMed

    Obeidat, Hesham Radi; Al-Dossary, Sahar; Asseri, Abdulsalam

    2015-09-01

    Kawasaki disease (KD) is an acute, self-limited vasculitis of unknown etiology that occurs predominantly in infants and children younger than 5 years of age. Coronary artery abnormalities are the most serious complication. Based on the literatures infusion of Intravenous Immunoglobulin of 2 g/kg and a high dose of oral aspirin up to 100 mg/kg/day are the standard treatment for Kawasaki disease in the acute stage, and should be followed by antiplatelet dose of aspirin for thrombocytosis. Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is an inherited X-linked hereditary disorder, and aspirin can induce hemolysis in patients with G6PD deficiency. We report a case of a 5 year and 8 month old male with KD and G6PD deficiency. PMID:27134550

  17. Cardiac-specific succinate dehydrogenase deficiency in Barth syndrome.

    PubMed

    Dudek, Jan; Cheng, I-Fen; Chowdhury, Arpita; Wozny, Katharina; Balleininger, Martina; Reinhold, Robert; Grunau, Silke; Callegari, Sylvie; Toischer, Karl; Wanders, Ronald Ja; Hasenfuß, Gerd; Brügger, Britta; Guan, Kaomei; Rehling, Peter

    2015-01-01

    Barth syndrome (BTHS) is a cardiomyopathy caused by the loss of tafazzin, a mitochondrial acyltransferase involved in the maturation of the glycerophospholipid cardiolipin. It has remained enigmatic as to why a systemic loss of cardiolipin leads to cardiomyopathy. Using a genetic ablation of tafazzin function in the BTHS mouse model, we identified severe structural changes in respiratory chain supercomplexes at a pre-onset stage of the disease. This reorganization of supercomplexes was specific to cardiac tissue and could be recapitulated in cardiomyocytes derived from BTHS patients. Moreover, our analyses demonstrate a cardiac-specific loss of succinate dehydrogenase (SDH), an enzyme linking the respiratory chain with the tricarboxylic acid cycle. As a similar defect of SDH is apparent in patient cell-derived cardiomyocytes, we conclude that these defects represent a molecular basis for the cardiac pathology in Barth syndrome. PMID:26697888

  18. Transformation linked decrease of pyruvate dehydrogenase complex in human epidermis.

    PubMed

    Eboli, M L; Pasquini, A

    1994-10-14

    Epidermis exhibits glycolytic features peculiar to cancer cells. The activity of pyruvate dehydrogenase complex, both active (PDHa) and total (PDHt) forms, has been investigated and compared in epidermis and epidermal carcinomas from human source. Low or undetectable PDHa is found in either normal and neoplastic tissue. PDHt is unchanged in human epidermis between the second and seventh decades of life but is dramatically decreased following neoplastic transformation (0.107 and 0.026 units/g fresh tissue for epidermis and epidermal carcinoma, respectively). As PDH plays a key role in mitochondrial carbohydrate metabolism, the decrease of total enzymic capacity found in tumors suggest that different mechanisms regulate PDH expression and, in turn, glycolytic mechanisms of epidermis and cancer cells. PMID:7954343

  19. Animal models for glutaryl-CoA dehydrogenase deficiency.

    PubMed

    Koeller, D M; Sauer, S; Wajner, M; de Mello, C F; Goodman, S I; Woontner, M; Mühlhausen, C; Okun, J G; Kölker, S

    2004-01-01

    In vitro studies suggest that excitotoxic cell damage is an underlying mechanism for the acute striatal damage in glutaryl-CoA dehydrogenase (GCDH) deficiency. It is believed to result from an imbalance of glutamatergic and GABAergic neurotransmission induced by the accumulating organic acids 3-hydroxyglutaric acid (3-OH-GA) and to a lesser extent glutaric acid (GA). Stereotaxic administration of 3-OH-GA and GA into the rat striatum have confirmed these results, but may not truly represent the effect of chronic exposure to these compounds. In an attempt to better understand the pathophysiology of GCDH deficiency in vivo , two animal models have been utilized. A mouse that lacks GCDH activity in all tissues was generated by gene targeting in embryonic stem cells. These animals develop the characteristic biochemical phenotype of the human disease. Pathologically, these mice have a diffuse spongiform myelinopathy similar to that in human patients; however, there is no evidence for acute striatal damage or sensitivity to acute encephalopathy induced by catabolism or inflammatory cytokines. A naturally occurring animal model, the fruit-eating bat Rousettus aegypticus, lacks hepatic and renal GCDH activity, but retains cerebral enzyme activity. Like the mouse, these bats develop the characteristic biochemical phenotype of glutaryl-CoA dehydrogenase deficiency, but lack overt neurological symptoms such as dystonia. It is not known whether they also develop the spongiform myelinopathy seen in the Gcdh-deficient mice. Otherwise, these constellations would suggest that cerebral GCDH deficiency is responsible for the development of neuronal damage. The lack of striatal damage in these two rodent models may also be related to species differences. However, they also highlight our lack of a comprehensive understanding of additional factors that might modulate the susceptibiliy of neurons to accumulating 3-OH-GA and GA in GCDH deficiency. Unravelling these mechanisms may be the

  20. Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family.

    PubMed

    Duester, G; Farrés, J; Felder, M R; Holmes, R S; Höög, J O; Parés, X; Plapp, B V; Yin, S J; Jörnvall, H

    1999-08-01

    The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies. PMID:10424757

  1. STRUCTURE AND KINETICS OF MONOFUNCTIONAL PROLINE DEHYDROGENASE FROM THERMUS THERMOPHILUS

    PubMed Central

    White, Tommi A.; Krishnan, Navasona; Becker, Donald F.; Tanner, John J.

    2009-01-01

    Proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria, but are fused into bifunctional enzymes known as Proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0 Å resolution structure of Thermus thermophilus PRODH reveals a distorted (βα)8 barrel catalytic core domain and a hydrophobic α-helical domain located above the carboxyl terminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent exposed compared to PutA due to a 4-Å shift of helix 8. Structure-based sequence analysis of the PutA/PRODH family led us to identify 9 conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is −75 mV and the kinetic parameters for proline are Km=27 mM and kcat=13 s−1. 3,4-dehydro-L-proline was found to be an efficient substrate and L-tetrahydro-2-furoic acid is a competitive inhibitor (KI=1.0 mM). Finally, we demonstrate that T. thermophilus PRODH reacts with O2 producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs. PMID:17344208

  2. Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum

    PubMed Central

    Vitale, Annalisa; Thorne, Natasha; Lovell, Scott; Battaile, Kevin P.; Hu, Xin; Shen, Min; D'Auria, Sabato; Auld, Douglas S.

    2013-01-01

    In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn2+) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn2+ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn2+ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes. PMID:23755111

  3. Catalytic properties of Sepharose-bound L-alanine dehydrogenase from Bacillus cereus.

    PubMed

    Mureşan, L; Vancea, D; Presecan, E; Porumb, H; Lascu, I; Oargă, M; Matinca, D; Abrudan, I; Bârzu, O

    1983-02-15

    (1) L-Alanine dehydrogenase from Bacillus cereus was purified by a two-step chromatographic procedure involving Cibacron-Blue 3G-A Sepharose 4B-CL, and Sepharose 6B-CL, and immobilized on CNBr-activated Sepharose 4B. (2) Following immobilization via two of the six subunits, L-alanine dehydrogenase retained 66% of the specific activity of the soluble enzyme. The affinity of the immobilized enzyme for NH4+, pyruvate and L-alanine, was not different to that of the soluble form. The Km of the Sepharose-bound L-alanine dehydrogenase for pyridine coenzymes was 6-8-times higher than in the soluble case. (3) The stability of L-alanine dehydrogenase towards urea or thermal denaturation was increased by immobilization. (4) The incubation at 37 degrees C for 24 h of the immobilized L-alanine dehydrogenase with 3 M NH4Cl/NH4OH buffer (pH 9) released 70% of the enzyme. The specific activity and the affinity of the 'solubilized' L-alanine dehydrogenase for the pyridine coenzymes was the same as that obtained with the original, soluble L-alanine dehydrogenase. PMID:6404304

  4. Glyceraldehyde-3-phosphate dehydrogenase-catalyzed chain oxidation of reduced nicotinamide adenine dinucleotide by perhydroxyl radicals

    SciTech Connect

    Chan, P.C.

    1980-02-10

    The chain oxidation of glyceraldehyde-3-phosphate dehydrogenase NADH by perhydroxyl radicals and propagated by molecular oxygen was studied by the xanthine-xanthine oxidase system, /sup 60/Co ..gamma..-ray, and pulse radiolysis. The chain length, amount of NADH oxidized per HO/sub 2/ generated, increases with increasing acidity of the medium and reaches a value of 73 at pH 5.0. The rate constant for the oxidation of the glyceraldehyde-3-phosphate dehydrogenase NADH complex by HO/sub 2/ was estimated to be 2 x 10/sup 7/ m/sup -1/s/sup -1/ at ambient temperatures (23-24/sup 0/C). Rate studies as a function of pH indicate that O/sub 2//sup -/ is unreactive toward the glyceraldehyde-3-phosphate dehydrogenase NADH complex. Other dehydrogenases (malate dehydrogenase, glutamate dehydrogenase, and isocitric dehydrogenase) studied showed no catalytic activity in the oxidation of NADH by HO/sub 2//O/sub 2//sup -/.

  5. Improved Production of Propionic Acid in Propionibacterium jensenii via Combinational Overexpression of Glycerol Dehydrogenase and Malate Dehydrogenase from Klebsiella pneumoniae

    PubMed Central

    Liu, Long; Zhuge, Xin; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua

    2015-01-01

    Microbial production of propionic acid (PA), an important chemical building block used as a preservative and chemical intermediate, has gained increasing attention for its environmental friendliness over traditional petrochemical processes. In previous studies, we constructed a shuttle vector as a useful tool for engineering Propionibacterium jensenii, a potential candidate for efficient PA synthesis. In this study, we identified the key metabolites for PA synthesis in P. jensenii by examining the influence of metabolic intermediate addition on PA synthesis with glycerol as a carbon source under anaerobic conditions. We also further improved PA production via the overexpression of the identified corresponding enzymes, namely, glycerol dehydrogenase (GDH), malate dehydrogenase (MDH), and fumarate hydratase (FUM). Compared to those in wild-type P. jensenii, the activities of these enzymes in the engineered strains were 2.91- ± 0.17- to 8.12- ± 0.37-fold higher. The transcription levels of the corresponding enzymes in the engineered strains were 2.85- ± 0.19- to 8.07- ± 0.63-fold higher than those in the wild type. The coexpression of GDH and MDH increased the PA titer from 26.95 ± 1.21 g/liter in wild-type P. jensenii to 39.43 ± 1.90 g/liter in the engineered strains. This study identified the key metabolic nodes limiting PA overproduction in P. jensenii and further improved PA titers via the coexpression of GDH and MDH, making the engineered P. jensenii strain a potential industrial producer of PA. PMID:25595755

  6. Stereoselective carveol dehydrogenase from Rhodococcus erythropolis DCL14. A novel nicotinoprotein belonging to the short chain dehydrogenase/reductase superfamily.

    PubMed

    van der Werf, M J; van der Ven, C; Barbirato, F; Eppink, M H; de Bont, J A; van Berkel, W J

    1999-09-10

    A novel nicotinoprotein, catalyzing the dichlorophenolindophenol-dependent oxidation of carveol to carvone, was purified to homogeneity from Rhodococcus erythropolis DCL14. The enzyme is specifically induced after growth on limonene and carveol. Dichlorophenolindophenol-dependent carveol dehydrogenase (CDH) is a homotetramer of 120 kDa with each subunit containing a tightly bound NAD(H) molecule. The enzyme is optimally active at pH 5.5 and 50 degrees C and displays a broad substrate specificity with a preference for substituted cyclohexanols. When incubated with a diastereomeric mixture of (4R)- or (4S)-carveol, CDH stereoselectively catalyzes the conversion of the (6S)-carveol stereoisomers only. Kinetic studies with pure stereoisomers showed that this is due to large differences in V(max)/K(m) values and simultaneous product inhibition by (R)- or (S)-carvone. The R. erythropolis CDH gene (limC) was identified in an operon encoding the enzymes involved in limonene degradation. The CDH nucleotide sequence revealed an open reading frame of 831 base pairs encoding a 277-amino acid protein with a deduced mass of 29,531 Da. The CDH primary structure shares 10-30% sequence identity with members of the short chain dehydrogenase/reductase superfamily. Structure homology modeling with trihydroxynaphthalene reductase from Magnaporthe grisea suggests that CDH from R. erythropolis DCL14 is an alpha/beta one-domain protein with an extra loop insertion involved in NAD binding and a flexible C-terminal part involved in monoterpene binding. PMID:10473585

  7. Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide

    PubMed Central

    Barry, Standish; O'Carra, Pádraig

    1973-01-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD+ through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD+ (probably through the 8 position of the adenine residue) to a number of different spacer-arm–agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD+ derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD+. Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD+-binding site of this enzyme. Problems

  8. Human gastric alcohol dehydrogenase activity: effect of age, sex, and alcoholism.

    PubMed Central

    Seitz, H K; Egerer, G; Simanowski, U A; Waldherr, R; Eckey, R; Agarwal, D P; Goedde, H W; von Wartburg, J P

    1993-01-01

    As various isoenzymes of gastric alcohol dehydrogenase exist and as the effect of sex and age on these enzymes is unknown, this study measured the activity of gastric alcohol dehydrogenase at high and low ethanol concentrations in endoscopic biopsy specimens from a total of 290 patients of various ages and from 10 patients with chronic alcoholism. Gastric alcohol dehydrogenase was also detected by immunohistological tests in biopsy specimens from 40 patients by the use of a polyclonal rabbit antibody against class I alcohol dehydrogenase. A significant correlation was found between the immunohistological reaction assessed by the intensity of the colour reaction in the biopsy specimen and the activity of alcohol dehydrogenase measured at 580 mM ethanol. While alcohol dehydrogenase activity measured at 16 mM ethanol was not significantly affected by age and sex, both factors influenced alcohol dehydrogenase activity measured at 580 mM ethanol. Young women below 50 years of age had significantly lower alcohol dehydrogenase activities in the gastric corpus and antrum when compared with age matched controls (SEM) (6.4 (0.7) v 8.8 (0.6) nmol/min/mg protein; p < 0.001 and 6.0 (1.3) v 9.5 (1.3) nmol/min/mg protein; p < 0.001). Over 50 years of age this sex difference was no longer detectable, as high Km gastric alcohol dehydrogenase activity decreases with age only in men and not in women. In addition, extremely low alcohol dehydrogenase activities have been found in gastric biopsy specimens from young male alcoholics (2.2 (0.5) nmol/min/mg protein), which returned to normal after two to three weeks of abstinence. The activity of alcohol dehydrogenase in the human stomach measured at 580 mM ethanol is decreased in young women, in elderly men, and in the subject with alcoholism. This decrease in alcohol dehydrogenase activity may contribute to the reduced first pass metabolism of ethanol associated with raised ethanol blood concentrations seen in these people. Images Figure

  9. AB104. Glucose-6 phospate dehydrogenase deficiency among mongolian neonates

    PubMed Central

    Batjargal, Khishigjargal; Nansal, Gerelmaa; Zagd, Gerelmaa; Ganbaatar, Erdenetuya

    2015-01-01

    Background and objective Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency in humans, affecting 400 million people worldwide and a high prevalence in persons of African, Middle Asian countries. The most common clinical manifestations are neonatal jaundice and acute hemolytic anemia, which is caused by the impairment of erythrocyte’s ability to remove harmful oxidative stress triggered by exogenous agents such as drugs, infection, or fava bean ingestion. Neonatal hyperbilirubinemia caused by G6PD is strongly associated with mortality and long-term neurodevelopmental impairment. The study aims to determine a level of G6PD in healthy neonates. Methods We obtained blood spot samples from 268 infants around 24-72 hours in their age who has unsuspected intranatal and neonatal disorders. Glucose 6 phosphate dehydrogenase “Perkin Elmer, Finland” level is determined by Victor 2D Fluorometer assay, developing of neonatal jaundice is examined by recall. Results The76.5% of all participants (n=205) was assessed 4.36±1.15 Ug/Hb in normal reference range of G6PD, other 23.5% (n=63) was 0.96±0.51 Ug/Hb with G6PD deficiency. In the both sex, 51.5% of male 0.88±0.46 Ug/Hb (n=33) and 47.6% of female (n=30) 0.97±0.55 Ug/Hb was assessed with G6PD deficiency. Developing Jaundice period in number of 63 neonates with G6PD deficiency, 86% of neonates (n=54) was in 1-4 days, 4% of neonates (n=3) was in 5-7 days and there is no sign of jaundice in 9% (n=6). Therefore neonates with G6PD deficiency, 53.9% (n=34) continued jaundice more than two weeks. Conclusions G6PD deficiency was determined in male neonates (51.5%) more than female (47.6%). The 76.5% of all participants (n=205) was assessed 4.36±1.15 Ug/Hb in normal reference range of G6PDH other 23.5% (n=63) of all participants was 0.96±0.51 Ug/Hb with G6PD deficiency. It shows that G6PD might be one potential risk of neonatal jaundice and hyperbilirubinemia in neonates in Mongolia.

  10. Improvement of the soy formate dehydrogenase properties by rational design.

    PubMed

    Kargov, I S; Kleimenov, S Y; Savin, S S; Tishkov, V I; Alekseeva, A A

    2015-06-01

    Previous experiments on substitution of the residue Phe290 to Asp, Asn and Ser in NAD(+)-dependent formate dehydrogenase from soya Glycine max (SoyFDH) showed important role of the residue in enzyme thermal stability and catalytic properties (Alekseeva et al. Prot. Eng. Des. Sel., 2012a; 25: :781-88). In this work, we continued site-directed mutagenesis experiments of the Phe290 and the residue was changed to Ala, Thr, Tyr, Glu and Gln. All amino acid changes resulted in increase of catalytic constant from 2.9 to 3.5-4.7 s(-1). The substitution Phe290Ala led to KM (NAD+) decrease from 13.3 to 8.6 μM, and substitutions Phe290Tyr and Phe290Glu resulted in decrease and increase of KM (HCOO-) from 1.5 to 0.9 and -2.9 mM, respectively. The highest improvement of catalytic properties was observed for SoyFDH Phe290Ala which showed 2-fold higher catalytic efficiency with both substrates. Stability of mutants was examined by study of thermal inactivation kinetics and differential scanning calorimetry (DSC). All five amino acids provided increase of thermal stability of mutant SoyFDH in comparison with wild-type enzyme. Mutant SoyFDH Phe290Glu showed the highest improvement-the stabilization effect was 43 at 56°C. The DSC data agree with results of thermal inactivation kinetics. Substitutions Phe290Tyr, Phe290Thr, Phe290Gln and Phe290Glu provided Tm value increase 0.6°-6.6°. SoyFDH Phe290Glu and previously prepared SoyFDH Phe290Asp show similar thermal stability as enzymes from Candida boidinii and Mycobacterium vaccae N10 and have higher catalytic efficiency with NAD(+) compared with all described FDHs. Therefore, these mutants are very perspective enzymes for coenzyme regeneration in processes of chiral synthesis with dehydrogenases. PMID:25744036

  11. A simple method for the rapid determination of the stereospecificity of NAD-dependent dehydrogenases applied to mammalian IMP dehydrogenase and bacterial NADH peroxidase.

    PubMed

    Cooney, D; Hamel, E; Cohen, M; Kang, G J; Dalal, M; Marquez, V

    1987-11-01

    The stereospecificity of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205) from two different sources was determined. The enzyme preparations were obtained from murine lymphoblasts and from Escherichia coli. Both enzymes transferred the 2-3H of IMP to the pro-S position of carbon atom C-4 of the nicotinamide ring in NAD. Thus, B-sided stereospecificity is common to the enzyme from two very different species. In addition, the studies described here demonstrate that alcohol dehydrogenase and NADH peroxidase, used as auxiliary enzymes, in combination with a microdistillation procedure, should permit rapid determination of the stereospecificity of any NAD-dependent dehydrogenase for which the appropriate tritiated substrate is available. PMID:2889473

  12. Redesigning alcohol dehydrogenases/reductases for more efficient biosynthesis of enantiopure isomers.

    PubMed

    Zhang, Rongzhen; Xu, Yan; Xiao, Rong

    2015-12-01

    Alcohol dehydrogenases/reductases predominantly catalyze the asymmetric biosynthesis of optically pure stereoisomers because of their unique chiral constitutions. The enantioselectivities of alcohol dehydrogenases/reductases are substrate- and cofactor-dependent, and therefore they usually catalyze specific reactions with high enantioselectivity under physiological conditions; this may not be suitable for asymmetric biosynthesis with non-natural substrates or non-natural cofactors, and under nonphysiological conditions. It is therefore necessary to modify alcohol dehydrogenases/reductases using various redesigning tools such as directed evolution and rational design, and their combinations, as well as engineering enzyme modules for more efficient production of "non-natural" products. In this article, progress in these aspects of alcohol dehydrogenase/reductase design is reviewed, and future challenges are discussed. PMID:26320091

  13. Glucose-6-phosphate dehydrogenase deficiency presented with convulsion: a rare case.

    PubMed

    Merdin, Alparslan; Avci, Fatma; Guzelay, Nihal

    2014-01-29

    Red blood cells carry oxygen in the body and Glucose-6-Phosphate Dehydrogenase protects these cells from oxidative chemicals. If there is a lack of Glucose-6-Phosphate Dehydrogenase, red blood cells can go acute hemolysis. Convulsion is a rare presentation for acute hemolysis due to Glucose-6-Phosphate Dehydrogenase deficiency. Herein, we report a case report of a Glucose-6-Phosphate Dehydrogenase deficiency diagnosed patient after presentation with convulsion. A 70 year-old woman patient had been hospitalized because of convulsion and fatigue. She has not had similar symptoms before. She had ingested fava beans in the last two days. Her hypophyseal and brain magnetic resonance imaging were normal. Blood transfusion was performed and the patient recovered. PMID:24711919

  14. DEVELOPMENTAL EXPRESSION OF ALDEHYDE DEHYDROGENASE IN RAT: A COMPARISON OF LIVER AND LUNG DEVELOPMENT

    EPA Science Inventory

    Metabolism is one of the major determinants for age-related susceptibility changes to chemicals. Aldehydes are highly reactive molecules present in the environment and can be produced during biotransformation of xenobiotics. Aldehyde dehydrogenases (ALDH) are important in aldehyd...

  15. Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

    NASA Technical Reports Server (NTRS)

    2003-01-01

    Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

  16. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis

    DOEpatents

    Ingram, Lonnie O.; Conway, Tyrrell

    1992-01-01

    The alcohol dehydrogenase II gene from Zymomonas mobilis has been cloned and sequenced. This gene can be expressed at high levels in other organisms to produce acetaldehyde or to convert acetaldehyde to ethanol.

  17. Triazaspirodimethoxybenzoyls as Selective Inhibitors of Mycobacterial Lipoamide Dehydrogenase

    SciTech Connect

    Bryk, Ruslana; Arango, Nancy; Venugopal, Aditya; Warren, J. David; Park, Yun-Hee; Patel, Mulchand S.; Lima, Christopher D.; Nathan, Carl

    2010-06-25

    Mycobacterium tuberculosis (Mtb) remains the leading single cause of death from bacterial infection. Here we explored the possibility of species-selective inhibition of lipoamide dehydrogenase (Lpd), an enzyme central to Mtb's intermediary metabolism and antioxidant defense. High-throughput screening of combinatorial chemical libraries identified triazaspirodimethoxybenzoyls as high-nanomolar inhibitors of Mtb's Lpd that were noncompetitive versus NADH, NAD{sup +}, and lipoamide and >100-fold selective compared to human Lpd. Efficacy required the dimethoxy and dichlorophenyl groups. The structure of an Lpd-inhibitor complex was resolved to 2.42 {angstrom} by X-ray crystallography, revealing that the inhibitor occupied a pocket adjacent to the Lpd NADH/NAD{sup +} binding site. The inhibitor did not overlap with the adenosine moiety of NADH/NAD{sup +} but did overlap with positions predicted to bind the nicotinamide rings in NADH and NAD{sup +} complexes. The dimethoxy ring occupied a deep pocket adjacent to the FAD flavin ring where it would block coordination of the NADH nicotinamide ring, while the dichlorophenyl group occupied a more exposed pocket predicted to coordinate the NAD{sup +} nicotinamide. Several residues that are not conserved between the bacterial enzyme and its human homologue were predicted to contribute both to inhibitor binding and to species selectivity, as confirmed for three residues by analysis of the corresponding mutant Mtb Lpd proteins. Thus, nonconservation of residues lining the electron-transfer tunnel in Mtb Lpd can be exploited for development of species-selective Lpd inhibitors.

  18. Yeast cell-based analysis of human lactate dehydrogenase isoforms.

    PubMed

    Mohamed, Lulu Ahmed; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

    2015-12-01

    Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc/LDH strains are expected to be of use for versatile analyses of human LDH. PMID:26126931

  19. Serum hydroxybutyrate dehydrogenase (HBD) assays in the clinical laboratory

    PubMed Central

    Montazemi, K.; Lines, J. G.

    1972-01-01

    Measurement of serum hydroxybutyrate dehydrogenase (HBD) activity for suspected myocardial infarction will nowadays usually be carried out with a commercially available test kit. Four such kits have been compared and evaluated: British Drug Houses HBDH set, Boehringer Corporation LDH-1-Iso-enzyme `α-HBDH' kit, Calbiochem α-HBDH Statpack, and Eskalab α-HBDH reagent tablets. Of the 100 sera examined, 60 were from patients believed to have suffered a recent myocardial infarction, and 40 samples were from patients aged between 45 and 65 years who had no known hepatic, renal, or cardiac pathology, these being used to determine the normal range for each kit. Under standardized conditions the activity found differed from kit to kit; the clinical value of the various assay systems in terms of their ability to discriminate between normal and pathological sera is assessed; and finally the cost and the convenience of use of each kit procedure are discussed. Theoretical and experimental evidence is provided in support of an assay temperature of 30°C, and a plea is made for international agreement on enzyme assay conditions. PMID:5017443

  20. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling

    PubMed Central

    Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  1. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    SciTech Connect

    Huo, Lu; Davis, Ian; Liu, Fange; Andi, Babak; Esaki, Shingo; Iwaki, Hiroaki; Hasegawa, Yoshie; Orville, Allen M.; Liu, Aimin

    2015-01-07

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation.

  2. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    DOE PAGESBeta

    Huo, Lu; Davis, Ian; Liu, Fange; Andi, Babak; Esaki, Shingo; Iwaki, Hiroaki; Hasegawa, Yoshie; Orville, Allen M.; Liu, Aimin

    2015-01-07

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacylmore » intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation.« less

  3. Properties of microtubule bundles induced by Glyceraldehyde-3-phosphate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Somers, Marijke; Engelborghs, Yves

    1991-05-01

    The binding of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) to microtubules causes the microtubules to assemble into large bundles. This bundling can be considered as a further step in the assembly of supramolecular structures. The rate of bundle formation, after addition of GAPDH to preformed microtubules, is not dependent on the GAPDH concentration and reflects bundling kinetics. Bundle disassembly can be studied by the addition of 1 mM adenosine 5'-(β, -imidotri-phosphate) (AMPPNP) to bundled microtubules, and is extremely fast. Bundling reduces the rate of association of tubulin dimers to microtubules, as well as the dissocation from the microtubles. Both rates are reduced to the same extent. This is in agreement with the fact that the critical concentration of tubulin is practically not influenced by the binding of the enzyme. Adding microtubule associated proteins (at I=0.1 M) does not appreciably influence the affinity for GAPDH, but reduces bundle formation possibly for sterical reasons.

  4. The reaction of ozone with glyceraldehyde-3-phosphate dehydrogenase

    SciTech Connect

    Knight, K.L.; Mudd, J.B.

    1984-02-15

    Inactivation of glyceraldehyde-3-phosphate dehydrogenase (GPDH) by ozone can be correlated with oxidation of the active-site -SH residue. Oxidation of peripheral -SH groups, and tryptophan, methionine, and histidine residues occurs concomitantly, but loss of activity depends solely on active-site oxidation. Inactivation is only slightly reversible by dithiothreitol. Kinetic studies show that inhibition of GPDH by ozone mimics noncompetitive inhibition and is characterized as irreversible enzyme inactivation. Analysis of products resulting from ozone oxidation of glutathione suggests that cysteic acid is the product of protein-SH oxidation. Despite oxidation of the active-site -SH, no significant decrease in the Racker band absorbance occurs. This is explained by the appearance of a new chromophore in this region of the absorbance spectrum. Increased absorbance at 322 nm following ozone treatment indicates that tryptophan is converted quantitatively to N-formylkynurenine. When the active-site -SH is reversibly blocked by tetrathionate, enzyme activity is completely recoverable following reaction of the derivatized enzyme with a 1.3X excess of ozone over enzyme monomer. Activity is fully recovered despite the oxidation of peripheral -SH, tryptophan, and histidine residues. Circular dichroism spectra of ozone-treated enzyme show that reaction of GPDH with up to a threefold excess of ozone over enzyme monomer results in no significant disruption of protein secondary structure. Spectra in the near-uv show distinct changes that reflect tryptophan oxidation.

  5. Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii.

    PubMed

    Guo, Qi; Gakhar, Lokesh; Wickersham, Kyle; Francis, Kevin; Vardi-Kilshtain, Alexandra; Major, Dan T; Cheatum, Christopher M; Kohen, Amnon

    2016-05-17

    The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented. PMID:27100912

  6. Aldehyde dehydrogenase 1A1 in stem cells and cancer

    PubMed Central

    Tomita, Hiroyuki; Tanaka, Kaori; Tanaka, Takuji; Hara, Akira

    2016-01-01

    The human genome contains 19 putatively functional aldehyde dehydrogenase (ALDH) genes, which encode enzymes critical for detoxification of endogenous and exogenous aldehyde substrates through NAD(P)+-dependent oxidation. ALDH1 has three main isotypes, ALDH1A1, ALDH1A2, and ALDH1A3, and is a marker of normal tissue stem cells (SC) and cancer stem cells (CSC), where it is involved in self-renewal, differentiation and self-protection. Experiments with murine and human cells indicate that ALDH1 activity, predominantly attributed to isotype ALDH1A1, is tissue- and cancer-specific. High ALDH1 activity and ALDH1A1 overexpression are associated with poor cancer prognosis, though high ALDH1 and ALDH1A1 levels do not always correlate with highly malignant phenotypes and poor clinical outcome. In cancer therapy, ALDH1A1 provides a useful therapeutic CSC target in tissue types that normally do not express high levels of ALDH1A1, including breast, lung, esophagus, colon and stomach. Here we review the functions and mechanisms of ALDH1A1, the key ALDH isozyme linked to SC populations and an important contributor to CSC function in cancers, and we outline its potential in future anticancer strategies. PMID:26783961

  7. An animal model of human aldehyde dehydrogenase deficiency

    SciTech Connect

    Chang, C.; Mann, J.; Yoshida, A.

    1994-09-01

    The genetic deficiency of ALDH2, a major mitochondrial aldehyde dehydrogenase, is intimately related to alcohol sensitivity and the degree of predisposition to alcoholic diseases in humans. The ultimate biological role of ALDH2 can be exposed by knocking out the ALDH2 gene in an animal model. As the first step for this line of studies, we cloned and characterized the ALDH2 gene from mouse C57/6J strain which is associated with a high alcohol preference. The gene spans 26 kbp and is composed of 13 exons. Embryonic stem cells were transfected with a replacement vector which contains a partially deleted exon3, a positive selection cassette (pPgk Neo), exon 4 with an artificial stop codon, exons 5, 6, 7, and a negative selection cassette (pMCI-Tk). Genomic DNAs prepared from drug resistant clones were analyzed by polymerase chain reaction and by Southern blot analysis to distinguish random integration from homologous recombination. Out of 132 clones examined, 8 had undergone homologous recombination at one of the ALDH2 alleles. The cloned transformed embryonic stem cells with a disrupted ALDH2 allele were injected into blastocysts. Transplantation of the blastocysts into surrogate mother mice yielded chimeric mice. The role of ALDH2 in alcohol preference, alcohol sensitivity and other biological and behavioral characteristics can be elucidated by examining the heterozygous and homozygous mutant strains produced by breeding of chimeric mice.

  8. Lactate Dehydrogenase in Hepatocellular Carcinoma: Something Old, Something New

    PubMed Central

    Faloppi, Luca; Bianconi, Maristella; Memeo, Riccardo; Casadei Gardini, Andrea; Giampieri, Riccardo; Bittoni, Alessandro; Andrikou, Kalliopi; Del Prete, Michela; Cascinu, Stefano; Scartozzi, Mario

    2016-01-01

    Hepatocellular carcinoma (HCC) is the most common primary liver tumour (80–90%) and represents more than 5.7% of all cancers. Although in recent years the therapeutic options for these patients have increased, clinical results are yet unsatisfactory and the prognosis remains dismal. Clinical or molecular criteria allowing a more accurate selection of patients are in fact largely lacking. Lactic dehydrogenase (LDH) is a glycolytic key enzyme in the conversion of pyruvate to lactate under anaerobic conditions. In preclinical models, upregulation of LDH has been suggested to ensure both an efficient anaerobic/glycolytic metabolism and a reduced dependence on oxygen under hypoxic conditions in tumour cells. Data from several analyses on different tumour types seem to suggest that LDH levels may be a significant prognostic factor. The role of LDH in HCC has been investigated by different authors in heterogeneous populations of patients. It has been tested as a potential biomarker in retrospective, small, and nonfocused studies in patients undergoing surgery, transarterial chemoembolization (TACE), and systemic therapy. In the major part of these studies, high LDH serum levels seem to predict a poorer outcome. We have reviewed literature in this setting trying to resume basis for future studies validating the role of LDH in this disease. PMID:27314036

  9. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    PubMed Central

    Bemer, Meagan J.; Risler, Linda J.; Phillips, Brian R.; Wang, Joanne; Storer, Barry E.; Sandmaier, Brenda M.; Duan, Haichuan; Raccor, Brianne S.; Boeckh, Michael J.; McCune, Jeannine S.

    2014-01-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5’- monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNC) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T-cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation, but not with chronic GVHD, relapse, non-relapse mortality, or overall mortality. We conclude that quantitation of the recipient’s pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient’s sensitivity to MMF, but confirmatory studies are needed. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  10. Pyruvate dehydrogenase kinase regulates hepatitis C virus replication.

    PubMed

    Jung, Gwon-Soo; Jeon, Jae-Han; Choi, Yeon-Kyung; Jang, Se Young; Park, Soo Young; Kim, Sung-Woo; Byun, Jun-Kyu; Kim, Mi-Kyung; Lee, Sungwoo; Shin, Eui-Cheol; Lee, In-Kyu; Kang, Yu Na; Park, Keun-Gyu

    2016-01-01

    During replication, hepatitis C virus (HCV) utilizes macromolecules produced by its host cell. This process requires host cellular metabolic reprogramming to favor elevated levels of aerobic glycolysis. Therefore, we evaluated whether pyruvate dehydrogenase kinase (PDK), a mitochondrial enzyme that promotes aerobic glycolysis, can regulate HCV replication. Levels of c-Myc, hypoxia-inducible factor-1α (HIF-1α), PDK1, PDK3, glucokinase, and serine biosynthetic enzymes were compared between HCV-infected and uninfected human liver and Huh-7.5 cells infected with or without HCV. Protein and mRNA expression of c-Myc, HIF-1α, and glycolytic enzymes were significantly higher in HCV-infected human liver and hepatocytes than in uninfected controls. This increase was accompanied by upregulation of serine biosynthetic enzymes, suggesting cellular metabolism was altered toward facilitated nucleotide synthesis essential for HCV replication. JQ1, a c-Myc inhibitor, and dichloroacetate (DCA), a PDK inhibitor, decreased the expression of glycolytic and serine synthetic enzymes in HCV-infected hepatocytes, resulting in suppressed viral replication. Furthermore, when co-administered with IFN-α or ribavirin, DCA further inhibited viral replication. In summary, HCV reprograms host cell metabolism to favor glycolysis and serine biosynthesis; this is mediated, at least in part, by increased PDK activity, which provides a surplus of nucleotide precursors. Therefore, blocking PDK activity might have therapeutic benefits against HCV replication. PMID:27471054

  11. Clinical research on neuroblastoma based on serum lactate dehydrogenase.

    PubMed

    Pang, Q M; Li, K; Ma, L J; Sun, R P

    2015-01-01

    In recent years, more and more scholars tend to study neuroblastoma (NB) since it possesses increasing morbidity, but lack of effective treatment. This paper aims to investigate variation and clinical significance of the neuron-specific enolase (NSE) and lactic dehydrogenase (LDH) level in serum of children with NB before and after Auto Peripheral Blood Stem Cell Transplantation (APBSCT). A total of 90 children with NB from various hospitals were included in this research, and we analyzed the relationship between levels of NSE and LDH and the change of disease by comparing the two levels before and after APBSCT treatment. The results indicated that the positive rate of NSE in serum was high before treatment, and the levels of NSE and LDH were remarkably higher than those when the treatment was valid; after comprehensive treatment of chemotherapy, excision and radiotherapy, there was a significant difference of NSE and LDH levels in serum between children with complete remission (CR) and those with partial remission (PR); however, no significant differences of NSE and LDH levels were found among children in progressive stage compared to before treatment. It is believed that NSE and LDH levels are associated to the recurrence and treatment effect of NB, proving that both can reflect tumor load, therefore they can be taken as the auxiliary indicators for monitoring curative effects of NB treatment. PMID:25864749

  12. Undetected Toxicity Risk in Pharmacogenetic Testing for Dihydropyrimidine Dehydrogenase

    PubMed Central

    Falvella, Felicia Stefania; Caporale, Marta; Cheli, Stefania; Martinetti, Antonia; Berenato, Rosa; Maggi, Claudia; Niger, Monica; Ricchini, Francesca; Bossi, Ilaria; Di Bartolomeo, Maria; Sottotetti, Elisa; Bernardi, Francesca Futura; de Braud, Filippo; Clementi, Emilio; Pietrantonio, Filippo

    2015-01-01

    Fluoropyrimidines, the mainstay agents for the treatment of colorectal cancer, alone or as a part of combination therapies, cause severe adverse reactions in about 10%–30% of patients. Dihydropyrimidine dehydrogenase (DPD), a key enzyme in the catabolism of 5-fluorouracil, has been intensively investigated in relation to fluoropyrimidine toxicity, and several DPD gene (DPYD) polymorphisms are associated with decreased enzyme activity and increased risk of fluoropyrimidine-related toxicity. In patients carrying non-functional DPYD variants (c.1905+1G>A, c.1679T>G, c.2846A>T), fluoropyrimidines should be avoided or reduced according to the patients’ homozygous or heterozygous status, respectively. For other common DPYD variants (c.496A>G, c.1129-5923C>G, c.1896T>C), conflicting data are reported and their use in clinical practice still needs to be validated. The high frequency of DPYD polymorphism and the lack of large prospective trials may explain differences in studies’ results. The epigenetic regulation of DPD expression has been recently investigated to explain the variable activity of the enzyme. DPYD promoter methylation and its regulation by microRNAs may affect the toxicity risk of fluoropyrimidines. The studies we reviewed indicate that pharmacogenetic testing is promising to direct personalised dosing of fluoropyrimidines, although further investigations are needed to establish the role of DPD in severe toxicity in patients treated for colorectal cancer. PMID:25906475

  13. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    PubMed Central

    Huo, Lu; Davis, Ian; Liu, Fange; Andi, Babak; Esaki, Shingo; Iwaki, Hiroaki; Hasegawa, Yoshie; Orville, Allen M.; Liu, Aimin

    2015-01-01

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation. PMID:25565451

  14. Pyruvate dehydrogenase kinase regulates hepatitis C virus replication

    PubMed Central

    Jung, Gwon-Soo; Jeon, Jae-Han; Choi, Yeon-Kyung; Jang, Se Young; Park, Soo Young; Kim, Sung-Woo; Byun, Jun-Kyu; Kim, Mi-Kyung; Lee, Sungwoo; Shin, Eui-Cheol; Lee, In-Kyu; Kang, Yu Na; Park, Keun-Gyu

    2016-01-01

    During replication, hepatitis C virus (HCV) utilizes macromolecules produced by its host cell. This process requires host cellular metabolic reprogramming to favor elevated levels of aerobic glycolysis. Therefore, we evaluated whether pyruvate dehydrogenase kinase (PDK), a mitochondrial enzyme that promotes aerobic glycolysis, can regulate HCV replication. Levels of c-Myc, hypoxia-inducible factor-1α (HIF-1α), PDK1, PDK3, glucokinase, and serine biosynthetic enzymes were compared between HCV-infected and uninfected human liver and Huh-7.5 cells infected with or without HCV. Protein and mRNA expression of c-Myc, HIF-1α, and glycolytic enzymes were significantly higher in HCV-infected human liver and hepatocytes than in uninfected controls. This increase was accompanied by upregulation of serine biosynthetic enzymes, suggesting cellular metabolism was altered toward facilitated nucleotide synthesis essential for HCV replication. JQ1, a c-Myc inhibitor, and dichloroacetate (DCA), a PDK inhibitor, decreased the expression of glycolytic and serine synthetic enzymes in HCV-infected hepatocytes, resulting in suppressed viral replication. Furthermore, when co-administered with IFN-α or ribavirin, DCA further inhibited viral replication. In summary, HCV reprograms host cell metabolism to favor glycolysis and serine biosynthesis; this is mediated, at least in part, by increased PDK activity, which provides a surplus of nucleotide precursors. Therefore, blocking PDK activity might have therapeutic benefits against HCV replication. PMID:27471054

  15. Nitrated carbon nanoblisters for high-performance glucose dehydrogenase bioanodes.

    PubMed

    de Souza, João C P; Iost, Rodrigo M; Crespilho, Frank N

    2016-03-15

    Recently, many strategies are being explored for efficiently wiring glucose dehydrogenase (GDh) enzymes capable of glucose (fuel) oxidation. For instance, the use of GDh NAD(+)-dependent for glucose oxidation is of great interest in biofuel cell technology because the enzyme are unaffected by the presence of molecular oxygen commonly present in electrolyte. Here we present the fabrication of flexible carbon fibers modified with nitrated carbon nanoblisters and their application as high-performance GDh bioanodes. These bioelectrodes could electro-oxidize glucose at -360 mV (vs. Ag/AgClsat) in the presence of a molecular oxygen saturated electrolyte with current densities higher than 1.0 mAcm(-2) at 0.0 V. It is corroborated by open circuit potential, where a potential stabilization occurs at -150 mV in a long term stability current-transient experiment. This value is in agreement with the quasi-steady current obtained at very low scan rate (0.1 mVs(-1)), where the onset potential for glucose oxidation is -180 mV. X-ray photoelectron spectroscopy and scanning electron microscopy revealed that the nitrated blisters and edge-like carbon structures, enabling highly efficient enzyme immobilization and low overpotential for electron transfer, allowing for glucose oxidation with potential values close to the thermodynamic cofactor. PMID:26516686

  16. Crystallographic Investigation and Selective Inhibition of Mutant Isocitrate Dehydrogenase

    PubMed Central

    2013-01-01

    Mutations in isocitrate dehydrogenase (IDH), a key enzyme in the tricarboxylic acid cycle, have recently been found in ∼75% glioma and ∼20% acute myeloid leukemia. Different from the wild-type enzyme, mutant IDH1 catalyzes the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid. Strong evidence has shown mutant IDH1 represents a novel target for this type of cancer. We found two 1-hydroxypyridin-2-one compounds that are potent inhibitors of R132H and R132C IDH1 mutants with Ki values as low as 120 nM. These compounds exhibit >60-fold selectivity against wild-type IDH1 and can inhibit the production of d-2-hydroxyglutaric acid in IDH1 mutated cells, representing novel chemical probes for cancer biology studies. We also report the first inhibitor-bound crystal structures of IDH1(R132H), showing these inhibitors have H-bond, electrostatic, and hydrophobic interactions with the mutant enzyme. Comparison with the substrate-bound IDH1 structures revealed the structural basis for the high enzyme selectivity of these compounds. PMID:23795241

  17. Crystallographic Investigation and Selective Inhibition of Mutant Isocitrate Dehydrogenase.

    PubMed

    Zheng, Baisong; Yao, Yuan; Liu, Zhen; Deng, Lisheng; Anglin, Justin L; Jiang, Hong; Prasad, B V Venkataram; Song, Yongcheng

    2013-06-13

    Mutations in isocitrate dehydrogenase (IDH), a key enzyme in the tricarboxylic acid cycle, have recently been found in ~75% glioma and ~20% acute myeloid leukemia. Different from the wild-type enzyme, mutant IDH1 catalyzes the reduction of α-ketoglutaric acid to D-2-hydroxyglutaric acid. Strong evidence has shown mutant IDH1 represents a novel target for this type of cancer. We found two 1-hydroxypyridin-2-one compounds that are potent inhibitors of R132H and R132C IDH1 mutants with Ki values as low as 120 nM. These compounds exhibit >60-fold selectivity against wild-type IDH1 and can inhibit the production of D-2-hydroxyglutaric acid in IDH1 mutated cells, representing novel chemical probes for cancer biology studies. We also report the first inhibitor-bound crystal structures of IDH1(R132H), showing these inhibitors have H-bond, electrostatic and hydrophobic interactions with the mutant enzyme. Comparison with the substrate-bound IDH1 structures revealed the structural basis for the high enzyme selectivity of these compounds. PMID:23795241

  18. 11β-Hydroxysteroid Dehydrogenase 2 in Preeclampsia

    PubMed Central

    Główka, Franciszek K.

    2016-01-01

    Preeclampsia is a serious medical problem affecting the mother and her child and influences their health not only during the pregnancy, but also many years after. Although preeclampsia is a subject of many research projects, the etiology of the condition remains unclear. One of the hypotheses related to the etiology of preeclampsia is the deficiency in placental 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), the enzyme which in normal pregnancy protects the fetus from the excess of maternal cortisol. The reduced activity of the enzyme was observed in placentas from pregnancies complicated with preeclampsia. That suggests the overexposure of the developing child to maternal cortisol, which in high levels exerts proapoptotic effects and reduces fetal growth. The fetal growth restriction due to the diminished placental 11β-HSD2 function may be supported by the fact that preeclampsia is often accompanied with fetal hypotrophy. The causes of the reduced function of 11β-HSD2 in placental tissue are still discussed. This paper summarizes the phenomena that may affect the activity of the enzyme at various steps on the way from the gene to the protein. PMID:27200090

  19. Membrane-Associated Quinoprotein Formaldehyde Dehydrogenase from Methylococcus capsulatus Bath

    PubMed Central

    Zahn, James A.; Bergmann, David J.; Boyd, Jeffery M.; Kunz, Ryan C.; DiSpirito, Alan A.

    2001-01-01

    A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 μmol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)+-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159–167, 1999; Vorholt et al., J. Bacteriol. 180:5351–5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b559/569 complex as the physiological electron acceptor. PMID:11698372

  20. Membrane-associated quinoprotein formaldehyde dehydrogenase from Methylococcus capsulatus Bath.

    PubMed

    Zahn, J A; Bergmann, D J; Boyd, J M; Kunz, R C; DiSpirito, A A

    2001-12-01

    A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159-167, 1999; Vorholt et al., J. Bacteriol. 180:5351-5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b(559/569) complex as the physiological electron acceptor. PMID:11698372

  1. Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum

    PubMed Central

    Ding, Wei; Si, Meiru; Zhang, Weipeng; Zhang, Yaoling; Chen, Can; Zhang, Lei; Lu, Zhiqiang; Chen, Shaolin; Shen, Xihui

    2015-01-01

    Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30C, and interestingly, it could utilize NAD+ and NADP+ as coenzymes with similar efficiency and showed no obvious difference toward NAD+ and NADP+. In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum. PMID:25622822

  2. Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli.

    PubMed

    Bzik, D J; Fox, B A; Gonyer, K

    1993-05-01

    A Plasmodium falciparum gene is described which encodes lactate dehydrogenase activity (P. falciparum LDH). The P. falciparum LDH gene contains no introns and is present in a single copy on chromosome 13. P. falciparum LDH was expressed in all asexual blood stages as a 1.6-kb mRNA. The predicted 316 amino acid protein coding region of P. falciparum LDH was inserted into the prokaryotic expression vector pKK223-3 and a 33-kDa protein having LDH activity was synthesized in Escherichia coli. P. falciparum LDH primary structure displays high amino acid similarity (50-57%) to vertebrate and bacterial LDH, but lacks the amino terminal extension observed in all vertebrate LDH. The majority of amino acid residues implicated in substrate and coenzyme binding and catalysis of other LDH are well conserved in P. falciparum LDH. However, several notable differences in amino acid composition were observed. P. falciparum LDH contained several distinctive single amino acid insertions and deletions compared to other LDH enzymes, and most remarkably, it contained a novel insertion of 5 amino acids within the conserved mobile loop region near arginine residue 109, a residue which is known to make contact with pyruvate in the ternary complex of other LDH. These results suggest that novel features of P. falciparum LDH primary structure may be correlated with previously characterized and distinctive kinetic, biochemical, immunochemical, and electrophoretic properties of P. falciparum LDH. PMID:8515777

  3. Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase*

    PubMed Central

    Takahashi-Íñiguez, Tóshiko; Aburto-Rodríguez, Nelly; Vilchis-González, Ana Laura; Flores, María Elena

    2016-01-01

    Malate dehydrogenase (MDH) is an enzyme widely distributed among living organisms and is a key protein in the central oxidative pathway. It catalyzes the interconversion between malate and oxaloacetate using NAD+ or NADP+ as a cofactor. Surprisingly, this enzyme has been extensively studied in eukaryotes but there are few reports about this enzyme in prokaryotes. It is necessary to review the relevant information to gain a better understanding of the function of this enzyme. Our review of the data generated from studies in bacteria shows much diversity in their molecular properties, including weight, oligomeric states, cofactor and substrate binding affinities, as well as differences in the direction of the enzymatic reaction. Furthermore, due to the importance of its function, the transcription and activity of this enzyme are rigorously regulated. Crystal structures of MDH from different bacterial sources led to the identification of the regions involved in substrate and cofactor binding and the residues important for the dimer-dimer interface. This structural information allows one to make direct modifications to improve the enzyme catalysis by increasing its activity, cofactor binding capacity, substrate specificity, and thermostability. A comparative analysis of the phylogenetic reconstruction of MDH reveals interesting facts about its evolutionary history, dividing this superfamily of proteins into two principle clades and establishing relationships between MDHs from different cellular compartments from archaea, bacteria, and eukaryotes.

  4. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    SciTech Connect

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D.

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  5. Green tea polyphenols modulate insulin secretion by inhibiting glutamate dehydrogenase.

    PubMed

    Li, Changhong; Allen, Aron; Kwagh, Jae; Doliba, Nicolai M; Qin, Wei; Najafi, Habiba; Collins, Heather W; Matschinsky, Franz M; Stanley, Charles A; Smith, Thomas J

    2006-04-14

    Insulin secretion by pancreatic beta-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED(50) values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that EGCG inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the beta-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH. PMID:16476731

  6. Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis

    PubMed Central

    Locasale, Jason W.; Grassian, Alexandra R.; Melman, Tamar; Lyssiotis, Costas A.; Mattaini, Katherine R.; Bass, Adam J.; Heffron, Gregory; Metallo, Christian M.; Muranen, Taru; Sharfi, Hadar; Sasaki, Atsuo T.; Anastasiou, Dimitrios; Mullarky, Edouard; Vokes, Natalie I.; Sasaki, Mika; Beroukhim, Rameen; Stephanopoulos, Gregory; Ligon, Azra H.; Meyerson, Matthew; Richardson, Andrea L; Chin, Lynda; Wagner, Gerhard; Asara, John M; Brugge, Joan S.; Cantley, Lewis C.; Vander Heiden, Matthew G.

    2013-01-01

    Most tumors display increased glucose metabolism compared to that of normal tissues. The preferential conversion of glucose to lactate in cancer cells (the Warburg Effect) has been emphasized1; however, the extent to which metabolic fluxes originating from glucose are utilized for alternative processes is poorly understood2,3. Here we used a combination of mass spectrometry and NMR with stable isotope labeling to investigate the alternate pathways derived from glucose metabolism in cancer cells. We found that in some cancer cells, a relatively large amount of glycolytic carbon is diverted into serine and glycine biosynthesis through phosphoglycerate dehydrogenase (PHGDH). A bioinformatics analysis of 3131 human cancers revealed that the gene PHGDH at 1p12 is recurrently amplified in a genomic region of focal copy number gain most commonly found in melanoma in which amplification was associated with increased protein expression. Decreased PHGDH expression by RNA interference impaired growth and flux into serine metabolism in PHGDH-amplified cell lines. Increased expression was also associated with breast cancer subtypes and ectopic expression of PHGDH in mammary epithelial cells (MCF-10a) disrupted acinar morphogenesis, induced loss of polarity, and preserved the viability of the extracellular matrix-deprived cells, each being phenotypic alterations that may predispose cells to transformation. Our findings demonstrate that altered metabolic flux from glucose into a specific alternate pathway can be selected during tumor development and may contribute to the pathogenesis of human cancer. PMID:21804546

  7. Detailed kinetics and regulation of mammalian 2-oxoglutarate dehydrogenase

    PubMed Central

    2011-01-01

    Background Mitochondrial 2-oxoglutarate (α-ketoglutarate) dehydrogenase complex (OGDHC), a key regulatory point of tricarboxylic acid (TCA) cycle, plays vital roles in multiple pathways of energy metabolism and biosynthesis. The catalytic mechanism and allosteric regulation of this large enzyme complex are not fully understood. Here computer simulation is used to test possible catalytic mechanisms and mechanisms of allosteric regulation of the enzyme by nucleotides (ATP, ADP), pH, and metal ion cofactors (Ca2+ and Mg2+). Results A model was developed based on an ordered ter-ter enzyme kinetic mechanism combined with con-formational changes that involve rotation of one lipoic acid between three catalytic sites inside the enzyme complex. The model was parameterized using a large number of kinetic data sets on the activity of OGDHC, and validated by comparison of model predictions to independent data. Conclusions The developed model suggests a hybrid rapid-equilibrium ping-pong random mechanism for the kinetics of OGDHC, consistent with previously reported mechanisms, and accurately describes the experimentally observed regulatory effects of cofactors on the OGDHC activity. This analysis provides a single consistent theoretical explanation for a number of apparently contradictory results on the roles of phosphorylation potential, NAD (H) oxidation-reduction state ratio, as well as the regulatory effects of metal ions on ODGHC function. PMID:21943256

  8. Regulation of human class I alcohol dehydrogenases by bile acids

    PubMed Central

    Langhi, Cédric; Pedraz-Cuesta, Elena; Haro, Diego; Marrero, Pedro F.; Rodríguez, Joan C.

    2013-01-01

    Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol and vitamin A (retinol) metabolism in the liver. Because previous studies have shown that human ADH1 enzymes may participate in bile acid metabolism, we investigated whether the bile acid-activated nuclear receptor farnesoid X receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064 increased ADH1 mRNA, protein, and activity. Moreover, overexpression of a constitutively active form of FXR induced ADH1A and ADH1B expression, whereas silencing of FXR abolished the effects of FXR agonists on ADH1 expression and activity. Transient transfection studies and electrophoretic mobility shift assays revealed functional FXR response elements in the ADH1A and ADH1B proximal promoters, thus indicating that both genes are direct targets of FXR. These findings provide the first evidence for direct connection of bile acid signaling and alcohol metabolism. PMID:23772048

  9. Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens.

    PubMed Central

    Vancurová, I; Vancura, A; Volc, J; Neuzil, J; Flieger, M; Basarová, G; Bĕhal, V

    1988-01-01

    Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl. Images PMID:3182727

  10. Structural analysis of oncogenic mutation of isocitrate dehydrogenase 1.

    PubMed

    Rajendran, Vidya

    2016-06-21

    Arginine to histidine mutation at position 132 (R132H) in isocitrate dehydrogenase 1 (IDH1) led to reduced affinity of the respective enzymes for isocitrate and increased affinity for α-ketoglutarate (AKG) and NADPH. This phenomenon retarded oxidative decarboxylation of isocitrate to AKG and conferred a novel enzymatic activity that facilitated the reduction of AKG to d-2-hydroxyglutarate (d-2HG). The loss of isocitrate utilization and gain of 2HG production from IDH1 R132H had been taken up as a fundamental problem and to solve this, structural biology approaches were adopted. Interaction analysis was carried out to investigate the IDH1 substrate binding environment. The altered behaviour of mutant and native IDH1 in interaction analysis was explored by performing long-term molecular dynamics simulations (∼300 ns). This study reports a comprehensive atomic behaviour of the gain-of-function mutation (R132H) in the IDH1 enzyme which in turn provides a direction towards new therapeutics. PMID:27194485

  11. Emerging concepts in the flavinylation of succinate dehydrogenase.

    PubMed

    Kim, Hyung J; Winge, Dennis R

    2013-05-01

    The Succinate Dehydrogenase (SDH) heterotetrameric complex catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle and in the aerobic respiratory chains of eukaryotes and bacteria. Essential in this catalysis is the covalently-linked cofactor flavin adenine dinucleotide (FAD) in subunit1 (Sdh1) of the SDH enzyme complex. The mechanism of FAD insertion and covalent attachment to Sdh1 is unknown. Our working concept of this flavinylation process has relied mostly on foundational works from the 1990s and by applying the principles learned from other enzymes containing a similarly linked FAD. The discovery of the flavinylation factor Sdh5, however, has provided new insight into the possible mechanism associated with Sdh1 flavinylation. This review focuses on encapsulating prior and recent advances towards understanding the mechanism associated with flavinylation of Sdh1 and how this flavinylation process affects the overall assembly of SDH. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease. PMID:23380393

  12. Isocitrate dehydrogenase mutations: new opportunities for translational research

    PubMed Central

    Keum, Young-Sam; Choi, Bu Young

    2015-01-01

    Over the last decade, comprehensive genome-wide sequencing studies have enabled us to find out unexpected genetic alterations of metabolism in cancer. An example is the identification of arginine missense mutations of isocitrate dehydrogenases-1 and -2 (IDH1/2) in glioma, acute myeloid leukemia (AML), chondrosarcomas, and cholangiocarcinoma. These alterations are closely associated with the production of a new stereospecific metabolite, (R)-2-hydroxyglutarate (R-2HG). A large number of follow-up studies have been performed to address the molecular mechanisms of IDH1/2 mutations underlying how these events contribute to malignant transformation. In the meanwhile, the development of selective mutant IDH1/2 chemical inhibitors is being actively pursued in the scientific community and pharmaceutical industry. The present review article briefly discusses the important findings that highlight the molecular mechanisms of IDH1/2 mutations in cancer and provides a current status for development of selective mutant IDH1/2 chemical inhibitors. [BMB Reports 2015; 48(5): 266-270] PMID:25787993

  13. [Succinate dehydrogenase (SDH)-deficient renal cell carcinoma].

    PubMed

    Agaimy, A

    2016-03-01

    Succinate dehydrogenase (SDH) represents a type II mitochondrial complex related to the respiratory chain and Krebs cycle. The complex is composed of four major subunits, SDHA, SDHB, SDHC and SDHD. The oncogenic role of this enzyme complex has only recently been recognized and the complex is currently considered an important oncogenic signaling pathway with tumor suppressor properties. In addition to the familial paraganglioma syndromes (types 1-5) as prototypical SDH-related diseases, many other tumors have been defined as SDH-deficient, in particular a subset of gastrointestinal stromal tumors (GIST), rare hypophyseal adenomas, a subset of pancreatic neuroendocrine neoplasms (recently added) and a variety of other tumor entities, the latter mainly described as rare case reports. As a central core subunit responsible for the integrity of the SDH complex, the expression of SDHB is lost in all SDH-deficient neoplasms irrespective of the specific SDH subunit affected by a genetic mutation in addition to concurrent loss of the subunit specifically affected by genetic alteration. Accordingly, all SDH-deficient neoplasms are by definition SDHB-deficient. The SDH-deficient renal cell carcinoma (RCC) has only recently been well-characterized and it is included as a specific subtype of RCC in the new World Health Organization (WHO) classification published in 2016. In this review, the major clinicopathological, immunohistochemical and genetic features of this rare disease entity are presented and discussed in the context of the broad differential diagnosis. PMID:26979428

  14. Succinic semialdehyde dehydrogenase deficiency: Lessons from mice and men

    PubMed Central

    Pearl, P. L.; Gibson, K. M.; Cortez, M. A.; Wu, Y.; Snead, O. Carter; Knerr, I.; Forester, K.; Pettiford, J. M.; Jakobs, C.; Theodore, W. H.

    2009-01-01

    Summary Succinic semialdehyde dehydrogenase (SSADH) deficiency, a disorder of GABA degradation with subsequent elevations in brain GABA and GHB, is a neurometabolic disorder with intellectual disability, epilepsy, hypotonia, ataxia, sleep disorders, and psychiatric disturbances. Neuroimaging reveals increased T2-weighted MRI signal usually affecting the globus pallidus, cerebellar dentate nucleus, and subthalamic nucleus, and often cerebral and cerebellar atrophy. EEG abnormalities are usually generalized spike-wave, consistent with a predilection for generalized epilepsy. The murine phenotype is characterized by failure-to-thrive, progressive ataxia, and a transition from generalized absence to tonic-clonic to ultimately fatal convulsive status epilepticus. Binding and electrophysiological studies demonstrate use-dependent downregulation of GABA(A) and (B) receptors in the mutant mouse. Translational human studies similarly reveal downregulation of GABAergic activity in patients, utilizing flumazenil-PET and transcranial magnetic stimulation for GABA(A) and (B) activity, respectively. Sleep studies reveal decreased stage REM with prolonged REM latencies and diminished percentage of stage REM. An ad libitum ketogenic diet was reported as effective in the mouse model, with unclear applicability to the human condition. Acute application of SGS–742, a GABA(B) antagonist, leads to improvement in epileptiform activity on electrocorticography. Promising mouse data using compounds available for clinical use, including taurine and SGS–742, form the framework for human trials. PMID:19172412

  15. SERUM VALUES OF ALKALINE PHOSPHATASE AND LACTATE DEHYDROGENASE IN OSTEOSARCOMA

    PubMed Central

    ZUMÁRRAGA, JUAN PABLO; BAPTISTA, ANDRÉ MATHIAS; ROSA, LUIS PABLO DE LA; CAIERO, MARCELO TADEU; CAMARGO, OLAVO PIRES DE

    2016-01-01

    ABSTRACT Objective: To study the relationship between the pre and post chemotherapy (CT) serum levels of alkaline phosphatase (AP) and lactate dehydrogenase (LDH), and the percentage of tumor necrosis (TN) found in specimens after the pre surgical CT in patients with osteosarcoma. Methods: Series of cases with retrospective evaluation of patients diagnosed with osteosarcoma. Participants were divided into two groups according to serum values of both enzymes. The values of AP and LDH were obtained before and after preoperative CT. The percentage of tumor necrosis (TN) of surgical specimens of each patient was also included. Results: One hundred and thirty seven medical records were included from 1990 to 2013. Both the AP as LDH decreased in the patients studied, being the higher in pre CT than post CT. The average LHD decrease was 795.12U/L and AP decrease was 437.40 U/L. The average TN was 34.10 %. There was no statistically significant correlation between the serums values and the percentage of tumoral necrosis. Conclusion: The serum levels values of AP and LDH are not good predictors for the chemotherapy-induced necrosis in patients with osteosarcoma. Level of Evidence IV, Case Series. PMID:27217815

  16. Succinate Dehydrogenase Deficiency in Pediatric and Adult Gastrointestinal Stromal Tumors

    PubMed Central

    Belinsky, Martin G.; Rink, Lori; von Mehren, Margaret

    2013-01-01

    Gastrointestinal stromal tumors (GISTs) in adults are generally driven by somatic gain-of-function mutations in KIT or PDGFRA, and biological therapies targeted to these receptor tyrosine kinases comprise part of the treatment regimen for metastatic and inoperable GISTs. A minority (10–15%) of GISTs in adults, along with ∼85% of pediatric GISTs, lacks oncogenic mutations in KIT and PDGFRA. Not surprisingly these wild type (WT) GISTs respond poorly to kinase inhibitor therapy. A subset of WT GISTs shares a set of distinguishing clinical and pathological features, and a flurry of recent reports has convincingly demonstrated shared molecular characteristics. These GISTs have a distinct transcriptional profile including over-expression of the insulin-like growth factor-1 receptor, and exhibit deficiency in the succinate dehydrogenase (SDH) enzyme complex. The latter is often but not always linked to bi-allelic inactivation of SDH subunit genes, particularly SDHA. This review will summarize the molecular, pathological, and clinical connections that link this group of SDH-deficient neoplasms, and offer a view toward understanding the underlying biology of the disease and the therapeutic challenges implicit to this biology. PMID:23730622

  17. Hypohidrotic ectodermal dysplasia associated with glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    Ermertcan, Aylin Türel; Yaşar, Ali; Kayhan, Tuba Çelebı; Gülen, Hüseyin; Ertan, Pelin

    2011-09-01

    Hypohidrotic ectodermal dysplasia (HED) is a syndrome characterized by hypodontia, hypotrichosis, and partial or total ecrine sweat gland deficiency. The most prevalent form of HED is inherited as an X linked pattern. Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is an X-linked recessive disease, which leads to hemolytic anemia and jaundice. It is expressed in males, while heterozygous females are usually clinically normal. A 12-year-old boy with the complaints of hair and eyebrow disturbances, teeth disfigurement, decreased sweating, and xerosis presented to the outpatient clinic. Dermatological examination revealed sparse hair and eyebrows, conical-shaped teeth, xerosis, syndactylia, transverse grooves, and discoloration of nails. Laboratory findings indicated anemia. His 3-year-old sister also had sparse hair and eyebrows, xerosis, and syndactylia. We learned that the patient had a previous history of neonatal jaundice and a diagnosis of G-6-PD deficiency. Although it has been shown that loci of ectodermal dysplasia and G-6-PD deficiency genes are near to one another, we did not find any case study reporting on occurrence of these two genetic diseases together. With the aspect of this rare and interesting case, the relationship between HED and G-6-PD deficiency was defined. PMID:22028581

  18. Hypohidrotic Ectodermal Dysplasia Associated with Glucose-6-Phosphate Dehydrogenase Deficiency

    PubMed Central

    Yaşar, Ali; Kayhan, Tuba Çelebİ; Gülen, Hüseyin; Ertan, Pelin

    2011-01-01

    Hypohidrotic ectodermal dysplasia (HED) is a syndrome characterized by hypodontia, hypotrichosis, and partial or total ecrine sweat gland deficiency. The most prevalent form of HED is inherited as an X linked pattern. Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is an X-linked recessive disease, which leads to hemolytic anemia and jaundice. It is expressed in males, while heterozygous females are usually clinically normal. A 12-year-old boy with the complaints of hair and eyebrow disturbances, teeth disfigurement, decreased sweating, and xerosis presented to the outpatient clinic. Dermatological examination revealed sparse hair and eyebrows, conical-shaped teeth, xerosis, syndactylia, transverse grooves, and discoloration of nails. Laboratory findings indicated anemia. His 3-year-old sister also had sparse hair and eyebrows, xerosis, and syndactylia. We learned that the patient had a previous history of neonatal jaundice and a diagnosis of G-6-PD deficiency. Although it has been shown that loci of ectodermal dysplasia and G-6-PD deficiency genes are near to one another, we did not find any case study reporting on occurrence of these two genetic diseases together. With the aspect of this rare and interesting case, the relationship between HED and G-6-PD deficiency was defined. PMID:22028581

  19. Intracellular localization of aldehyde dehydrogenase in rat liver

    PubMed Central

    Marjanen, Leo

    1972-01-01

    1. Distribution of aldehyde dehydrogenase activity in rat liver was studied by measuring the rate of disappearance of acetaldehyde in the presence of each of the subcellular fractions. These were obtained by rough separation of particulate fractions from the soluble portion of the cell, by differential centrifugation, and by isopycnic gradient centrifugation. 2. The maximal rate of acetaldehyde oxidation was 3.7 μmol/min per g, with an apparent Km value below 10−5m. The highest rate of activity was observed in phosphate buffers of high Pi concentration (above 60mm). 3. The activity measured was completely dependent on NAD+. 4. The microsomal fraction and the nuclei were inactive in the assay. Of the total activity 80% was found in the mitochondrial fraction and the remaining 20% in the cytoplasm. 5. The distribution pattern is important from the point of view of acetaldehyde oxidation during ethanol metabolism. The apparent discrepancy of the results obtained by different workers and the localization of acetaldehyde oxidation in vivo is discussed. PMID:4346744

  20. Transcriptional regulation of the Bacillus subtilis glucitol dehydrogenase gene.

    PubMed Central

    Ye, R; Wong, S L

    1994-01-01

    The regulatory region of the Bacillus subtilis glucitol dehydrogenase (gutB) gene was divided into three subregions: a promoter, an upstream positive regulatory region, and a downstream negative regulatory region. Data from primer extension, deletion, and site-directed mutagenesis analyses were consistent with two possible models for the gutB promoter. It is either a sigma A-type promoter with an unusually short spacer region (15 bp) or a special sigma A promoter which requires only the hexameric -10 sequence for its function. Sequence carrying just the promoter region (from -48 to +6) failed to direct transcription in vivo. An upstream regulatory sequence was essential for glucitol induction. When this sequence was inserted in a high-copy-number plasmid, an effect characteristic of titration of a transcriptional activator was seen. Downstream from the promoter, there is an imperfect, AT-rich inverted repeat sequence. Deletion of this element did not lead to constitutive expression of gutB. However, the induced gutB expression level was enhanced three- to fourfold. Images PMID:8195086

  1. Acetate Utilization in Lactococcus lactis Deficient in Lactate Dehydrogenase: a Rescue Pathway for Maintaining Redox Balance

    PubMed Central

    Hols, Pascal; Ramos, Ana; Hugenholtz, Jeroen; Delcour, Jean; de Vos, Willem M.; Santos, Helena; Kleerebezem, Michiel

    1999-01-01

    Acetate was shown to improve glucose fermentation in Lactococcus lactis deficient in lactate dehydrogenase. 13C and 1H nuclear magnetic resonance studies using [2-13C]glucose and [2-13C]acetate as substrates demonstrated that acetate was exclusively converted to ethanol. This novel pathway provides an alternative route for NAD+ regeneration in the absence of lactate dehydrogenase. PMID:10464231

  2. Modulation of the interaction between aldolase and glycerol-phosphate dehydrogenase by fructose phosphates.

    PubMed

    Vértessy, B G; Orosz, F; Ovádi, J

    1991-06-24

    Kinetics of fructose-1,6-disphosphate aldolase (EC 4.1.2.13) catalyzed conversion of fructose phosphates was analyzed by coupling the aldolase reactions to the metabolically sequential enzyme, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), which interacts with aldolase. At low enzyme concentration poly(ethylene glycol) was added to promote complex formation of aldolase and glycerol-phosphate dehydrogenase resulting in a 3-fold increase in KM of fructose-1,6-bisphosphate and no change in Vmax. Kinetic parameters for fructose-1-phosphate conversion changed inversely upon complex formation: Vmax increased while KM remained unchanged. Gel penetration and ion-exchange chromatographic experiments showed positive modulation of the interaction of aldolase and dehydrogenase by fructose-1,6-bisphosphate. The dissociation constant of the heterologous enzyme complex decreased 10-fold in the presence of this substrate. Fructose-1-phosphate or dihydroxyacetone phosphate had no effect on the dissociation constant of the aldolase-dehydrogenase complex. In addition, titration of fluorescein-labelled glycerol-phosphate dehydrogenase with aldolase indicated that both fructose-1,6-bisphosphate and fructose-2,6-biphosphate enhanced the affinity of aldolase to glycerol-phosphate dehydrogenase. The results of the kinetic and binding experiments suggest that binding of the C-6 phosphate group of fructose-1,6-bisphosphate to aldolase complexed with dehydrogenase is sterically impeded while saturation of the C-6 phosphate group site increases the affinity of aldolase for dehydrogenase. The possible molecular mechanism of the fructose-1,6-bisphosphate modulated interaction is discussed. PMID:2065091

  3. Crystal structure of Pseudomonas fluorescens mannitol 2-dehydrogenase binary and ternary complexes. Specificity and catalytic mechanism.

    PubMed

    Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

    2002-11-01

    Long-chain mannitol dehydrogenases are secondary alcohol dehydrogenases that are of wide interest because of their involvement in metabolism and potential applications in agriculture, medicine, and industry. They differ from other alcohol and polyol dehydrogenases because they do not contain a conserved tyrosine and are not dependent on Zn(2+) or other metal cofactors. The structures of the long-chain mannitol 2-dehydrogenase (54 kDa) from Pseudomonas fluorescens in a binary complex with NAD(+) and ternary complex with NAD(+) and d-mannitol have been determined to resolutions of 1.7 and 1.8 A and R-factors of 0.171 and 0.176, respectively. These results show an N-terminal domain that includes a typical Rossmann fold. The C-terminal domain is primarily alpha-helical and mediates mannitol binding. The electron lone pair of Lys-295 is steered by hydrogen-bonding interactions with the amide oxygen of Asn-300 and the main-chain carbonyl oxygen of Val-229 to act as the general base. Asn-191 and Asn-300 are involved in a web of hydrogen bonding, which precisely orients the mannitol O2 proton for abstraction. These residues also aid in stabilizing a negative charge in the intermediate state and in preventing the formation of nonproductive complexes with the substrate. The catalytic lysine may be returned to its unprotonated state using a rectifying proton tunnel driven by Glu-292 oscillating among different environments. Despite low sequence homology, the closest structural neighbors are glycerol-3-phosphate dehydrogenase, N-(1-d-carboxylethyl)-l-norvaline dehydrogenase, UDP-glucose dehydrogenase, and 6-phosphogluconate dehydrogenase, indicating a possible evolutionary relationship among these enzymes. PMID:12196534

  4. The Crystal Structure of Aquifex aeolicus Prephenate Dehydrogenase Reveals the Mode of Tyrosine Inhibition

    SciTech Connect

    Sun, Warren; Shahinas, Dea; Bonvin, Julie; Hou, Wenjuan; Kimber, Matthew S.; Turnbull, Joanne; Christendat, Dinesh

    2009-08-14

    TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD(+) plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering.

  5. Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Korotchkina, Lioubov; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand

    2003-01-01

    The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

  6. Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane.

    PubMed Central

    Serrano, A.; Cordoba, F.; Gonzalez-Reyes, J. A.; Navas, P.; Villalba, J. M.

    1994-01-01

    Highly purified plasma membrane fractions were obtained from onion (Allium cepa L.) roots and used as a source for purification of redox proteins. Plasma membranes solubilized with Triton X-100 contained two distinct polypeptides showing NAD(P)H-dependent dehydrogenase activities. Dehydrogenase I was purified by gel filtration in Sephacryl S-300 HR, ion-exchange chromatography in DEAE-Sepharose CL-6B, and dye-ligand affinity chromatography in Blue-Sepharose CL-6B after biospecific elution with NADH. Dehydrogenase I consisted of a single polypeptide of about 27 kD and an isoelectric point of about 6. Dehydrogenase II was purified from the DEAE-unbound fraction by chromatography in Blue-Sepharose CL-6B and affinity elution with NADH. Dehydrogenase II consisted of a single polypeptide of about 31 kD and an isoelectric point of about 8. Purified dehydrogenase I oxidized both NADPH and NADH, although higher rates of electron transfer were obtained with NADPH. Maximal activity was achieved with NADPH as donor and juglone or coenzyme Q as acceptor. Dehydrogenase II was specific for NADH and exhibited maximal activity with ferricyanide. Optimal pH for both dehydrogenases was about 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltrifluoroacetone, and the thiol reagent N-ethyl-maleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifluoroacetone, and the thiol reagent p-hydroxymercuribenzoate. PMID:12232306

  7. Monoterpene metabolism. Cloning, expression, and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermint and spearmint.

    PubMed

    Ringer, Kerry L; Davis, Edward M; Croteau, Rodney

    2005-03-01

    The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (-)-trans-isopiperitenol to (-)-isopiperitenone in peppermint and (-)-trans-carveol to (-)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (-)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (-)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (-)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5'-truncated cDNA encoding the spearmint homolog, (-)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5'-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (-)-trans-isopiperitenol and (-)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene

  8. Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

    PubMed

    Winberg, J O; McKinley-McKee, J S

    1998-02-01

    Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain

  9. Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

    PubMed Central

    Winberg, J O; McKinley-McKee, J S

    1998-01-01

    Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain

  10. Targeting Aldehyde Dehydrogenase Cancer Stem Cells in Ovarian Cancer

    PubMed Central

    Landen, Charles N.; Goodman, Blake; Katre, Ashwini A.; Steg, Adam D.; Nick, Alpa M.; Stone, Rebecca L.; Miller, Lance D.; Mejia, Pablo Vivas; Jennings, Nicolas B.; Gershenson, David M.; Bast, Robert C.; Coleman, Robert L.; Lopez-Berestein, Gabriel; Sood, Anil K.

    2010-01-01

    Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines, we found that ALDH1A1 expression and activity was significantly higher in taxane and platinum-resistant cell lines. In patient samples, 72.9% of ovarian cancers had ALDH1A1 expression, in whom the percent of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 v 13.81 months, p<0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor initiating studies, where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly, tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations, but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer, ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy, significantly reducing tumor growth in mice compared to chemotherapy alone (a 74–90% reduction, p<0.015). These data demonstrate that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients, and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced, but not absolute, tumorigenicity, but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer. PMID:20889728

  11. Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis.

    PubMed

    Barretto, O C de O; Oshiro, M; Oliveira, R A G; Fedullo, J D L; Nonoyama, K

    2006-05-01

    In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 +/- 38 IU g-1 Hb-1 min-1 at 37 degrees C, compared to the human erythrocyte activity of 12 +/- 2 IU g-1 Hb-1 min-1 at 37 degrees C. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 microM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 microM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate. PMID:16648898

  12. Aldehyde dehydrogenases in cellular responses to oxidative/electrophilic stress.

    PubMed

    Singh, Surendra; Brocker, Chad; Koppaka, Vindhya; Chen, Ying; Jackson, Brian C; Matsumoto, Akiko; Thompson, David C; Vasiliou, Vasilis

    2013-03-01

    Reactive oxygen species (ROS) are continuously generated within living systems and the inability to manage ROS load leads to elevated oxidative stress and cell damage. Oxidative stress is coupled to the oxidative degradation of lipid membranes, also known as lipid peroxidation. This process generates over 200 types of aldehydes, many of which are highly reactive and toxic. Aldehyde dehydrogenases (ALDHs) metabolize endogenous and exogenous aldehydes and thereby mitigate oxidative/electrophilic stress in prokaryotic and eukaryotic organisms. ALDHs are found throughout the evolutionary gamut, from single-celled organisms to complex multicellular species. Not surprisingly, many ALDHs in evolutionarily distant, and seemingly unrelated, species perform similar functions, including protection against a variety of environmental stressors such as dehydration and ultraviolet radiation. The ability to act as an "aldehyde scavenger" during lipid peroxidation is another ostensibly universal ALDH function found across species. Upregulation of ALDHs is a stress response in bacteria (environmental and chemical stress), plants (dehydration, salinity, and oxidative stress), yeast (ethanol exposure and oxidative stress), Caenorhabditis elegans (lipid peroxidation), and mammals (oxidative stress and lipid peroxidation). Recent studies have also identified ALDH activity as an important feature of cancer stem cells. In these cells, ALDH expression helps abrogate oxidative stress and imparts resistance against chemotherapeutic agents such as oxazaphosphorine, taxane, and platinum drugs. The ALDH superfamily represents a fundamentally important class of enzymes that contributes significantly to the management of electrophilic/oxidative stress within living systems. Mutations in various ALDHs are associated with a variety of pathological conditions in humans, highlighting the fundamental importance of these enzymes in physiological and pathological processes. PMID:23195683

  13. Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase.

    SciTech Connect

    Zhang, R.; Evans, G.; Rotella, F. J.; Westbrook, E. M.; Beno, D.; Huberman, E.; Joachimiak, A.; Collart, F. R.

    1999-01-01

    IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the K{sub m} for NAD (1180 {mu}M) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 {angstrom} with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne's Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione {beta}-synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors.

  14. Mechanism of Thermal Adaptation in the Lactate Dehydrogenases.

    PubMed

    Peng, Huo-Lei; Egawa, Tsuyoshi; Chang, Eric; Deng, Hua; Callender, Robert

    2015-12-10

    The mechanism of thermal adaptation of enzyme function at the molecular level is poorly understood but is thought to lie within the structure of the protein or its dynamics. Our previous work on pig heart lactate dehydrogenase (phLDH) has determined very high resolution structures of the active site, via isotope edited IR studies, and has characterized its dynamical nature, via laser-induced temperature jump (T-jump) relaxation spectroscopy on the Michaelis complex. These particular probes are quite powerful at getting at the interplay between structure and dynamics in adaptation. Hence, we extend these studies to the psychrophilic protein cgLDH (Champsocephalus gunnari; 0 °C) and the extreme thermophile tmLDH (Thermotoga maritima LDH; 80 °C) for comparison to the mesophile phLDH (38-39 °C). Instead of the native substrate pyruvate, we utilize oxamate as a nonreactive substrate mimic for experimental reasons. Using isotope edited IR spectroscopy, we find small differences in the substate composition that arise from the detailed bonding patterns of oxamate within the active site of the three proteins; however, we find these differences insufficient to explain the mechanism of thermal adaptation. On the other hand, T-jump studies of reduced β-nicotinamide adenine dinucleotide (NADH) emission reveal that the most important parameter affecting thermal adaptation appears to be enzyme control of the specific kinetics and dynamics of protein motions that lie along the catalytic pathway. The relaxation rate of the motions scale as cgLDH > phLDH > tmLDH in a way that faithfully matches kcat of the three isozymes. PMID:26556099

  15. Structural and functional consequences of succinate dehydrogenase subunit B mutations.

    PubMed

    Kim, E; Rath, E M; Tsang, V H M; Duff, A P; Robinson, B G; Church, W B; Benn, D E; Dwight, T; Clifton-Bligh, R J

    2015-06-01

    Mitochondrial dysfunction, due to mutations of the gene encoding succinate dehydrogenase (SDH), has been implicated in the development of adrenal phaeochromocytomas, sympathetic and parasympathetic paragangliomas, renal cell carcinomas, gastrointestinal stromal tumours and more recently pituitary tumours. Underlying mechanisms behind germline SDH subunit B (SDHB) mutations and their associated risk of disease are not clear. To investigate genotype-phenotype correlation of SDH subunit B (SDHB) variants, a homology model for human SDH was developed from a crystallographic structure. SDHB mutations were mapped, and biochemical effects of these mutations were predicted in silico. Results of structural modelling indicated that many mutations within SDHB are predicted to cause either failure of functional SDHB expression (p.Arg27*, p.Arg90*, c.88delC and c.311delAinsGG), or disruption of the electron path (p.Cys101Tyr, p.Pro197Arg and p.Arg242His). GFP-tagged WT SDHB and mutant SDHB constructs were transfected (HEK293) to determine biological outcomes of these mutants in vitro. According to in silico predictions, specific SDHB mutations resulted in impaired mitochondrial localisation and/or SDH enzymatic activity. These results indicated strong genotype-functional correlation for SDHB variants. This study reveals new insights into the effects of SDHB mutations and the power of structural modelling in predicting biological consequences. We predict that our functional assessment of SDHB mutations will serve to better define specific consequences for SDH activity as well as to provide a much needed assay to distinguish pathogenic mutations from benign variants. PMID:25972245

  16. Maize cytokinin dehydrogenase isozymes are localized predominantly to the vacuoles.

    PubMed

    Zalabák, David; Johnová, Patricie; Plíhal, Ondřej; Šenková, Karolina; Šamajová, Olga; Jiskrová, Eva; Novák, Ondřej; Jackson, David; Mohanty, Amitabh; Galuszka, Petr

    2016-07-01

    The maize genome encompasses 13 genes encoding for cytokinin dehydrogenase isozymes (CKXs). These enzymes are responsible for irreversible degradation of cytokinin plant hormones and thus, contribute regulating their levels. Here, we focus on the unique aspect of CKXs: their diverse subcellular distribution, important in regulating cytokinin homeostasis. Maize CKXs were tagged with green fluorescent protein (GFP) and transiently expressed in maize protoplasts. Most of the isoforms, namely ZmCKX1, ZmCKX2, ZmCKX4a, ZmCKX5, ZmCKX6, ZmCKX8, ZmCKX9, and ZmCKX12, were associated with endoplasmic reticulum (ER) several hours after transformation. GFP-fused CKXs were observed to accumulate in putative prevacuolar compartments. To gain more information about the spatiotemporal localization of the above isoforms, we prepared stable expression lines of all ZmCKX-GFP fusions in Arabidopsis thaliana Ler suspension culture. All the ER-associated isoforms except ZmCKX1 and ZmCKX9 were found to be targeted primarily to vacuoles, suggesting that ER-localization is a transition point in the intracellular secretory pathway and vacuoles serve as these isoforms' final destination. ZmCKX9 showed an ER-like localization pattern similar to those observed in the transient maize assay. Apoplastic localization of ZmCKX1 was further confirmed and ZmCKX10 showed cytosolic/nuclear localization due to the absence of the signal peptide sequence as previously reported. Additionally, we prepared GFP-fused N-terminal signal deletion mutants of ZmCKX2 and ZmCKX9 and clearly demonstrated that the localization pattern of these mutant forms was cytosolic/nuclear. This study provides the first complex model for spatiotemporal localization of the key enzymes of the cytokinin degradation/catabolism in monocotyledonous plants. PMID:27031423

  17. Dysfunctional TCA-Cycle Metabolism in Glutamate Dehydrogenase Deficient Astrocytes.

    PubMed

    Nissen, Jakob D; Pajęcka, Kamilla; Stridh, Malin H; Skytt, Dorte M; Waagepetersen, Helle S

    2015-12-01

    Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and α-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from α-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids. PMID:26221781

  18. Phylogenetic analysis of vertebrate lactate dehydrogenase (LDH) multigene families.

    PubMed

    Li, Yi-Ju; Tsoi, Stephen C-M; Mannen, Hideyuka; Shoei-lung Li, Steven

    2002-05-01

    In this paper we analyzed 49 lactate dehydrogenase (LDH) sequences, mostly from vertebrates. The amino acid sequence differences were found to be larger for a human-killifish pair than a human-lamprey pair. This indicates that some protein sequence convergence may occur and reduce the sequence differences in distantly related species. We also examined transitions and transversions separately for several species pairs and found that the transitions tend to be saturated in the distantly related species pair, while transversions are increasing. We conclude that transversions maintain a conservative rate through the evolutionary time. Kimura's two-parameter model for multiple-hit correction on transversions only was used to derive a distance measure and then construct a neighbor-joining (NJ) tree. Three findings were revealed from the NJ tree: (i) the branching order of the tree is consistent with the common branch pattern of major vertebrates; (ii) Ldh-A and Ldh-B genes were duplicated near the origin of vertebrates; and (iii) Ldh-C and Ldh-A in mammals were produced by an independent gene duplication in early mammalian history. Furthermore, a relative rate test showed that mammalian Ldh-C evolved more rapidly than mammalian Ldh-A. Under a two-rate model, this duplication event was calibrated to be approximately 247 million years ago (mya), dating back to the Triassic period. Other gene duplication events were also discovered in Xenopus, the first duplication occurring approximately 60-70 mya in both Ldh-A and Ldh-B, followed by another recent gene duplication event, approximately 20 mya, in Ldh-B. PMID:11965434

  19. THE HEME BINDING PROPERTIES OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

    PubMed Central

    Hannibal, Luciana; Collins, Daniel; Brassard, Julie; Chakravarti, Ritu; Vempati, Rajesh; Dorlet, Pierre; Santolini, Jérôme; Dawson, John H.; Stuehr, Dennis J.

    2012-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for cellular heme insertion into inducible nitric oxide synthase (Chakravarti et al, PNAS 2010, 107(42):18004-9), we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (1 heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418 and 537 nm, and when reduced to ferrous gave maxima at 424, 527 and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were kon =17,800 M−1s−1 and koff1 = 7.0 × 10−3 s−1; koff2 = 3.3 × 10−4 s−1 respectively, giving approximate affinities of 19–390 nM. Ferrous heme bound more poorly to GAPDH and dissociated with a koff = 4.2 × 10−3 s−1. Magnetic circular dichroism (MCD), resonance Raman (rR) and EPR spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in ferric complex was not displaced by CN− or N3− but in ferrous complex was displaceable by CO at a rate of 1.75 s−1 (for [CO]>0.2 mM). Studies with heme analogs revealed selectivity toward the coordinating metal and porphyrin ring structure. GAPDH-heme was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-amino levulinic acid. Our finding of heme binding to GAPDH expands the protein’s potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH is consistent with it performing heme sensing or heme chaperone-like functions in cells. PMID:22957700

  20. Short-chain dehydrogenases/reductases in cyanobacteria.

    PubMed

    Kramm, Anneke; Kisiela, Michael; Schulz, Rüdiger; Maser, Edmund

    2012-03-01

    The short-chain dehydrogenases/reductases (SDRs) represent a large superfamily of enzymes, most of which are NAD(H)-dependent or NADP(H)-dependent oxidoreductases. They display a wide substrate spectrum, including steroids, alcohols, sugars, aromatic compounds, and xenobiotics. On the basis of characteristic sequence motifs, the SDRs are subdivided into two main (classical and extended) and three smaller (divergent, intermediate, and complex) families. Despite low residue identities in pairwise comparisons, the three-dimensional structure among the SDRs is conserved and shows a typical Rossmann fold. Here, we used a bioinformatics approach to determine whether and which SDRs are present in cyanobacteria, microorganisms that played an important role in our ecosystem as the first oxygen producers. Cyanobacterial SDRs could indeed be identified, and were clustered according to the SDR classification system. Furthermore, because of the early availability of its genome sequence and the easy application of transformation methods, Synechocystis sp. PCC 6803, one of the most important cyanobacterial strains, was chosen as the model organism for this phylum. Synechocystis sp. SDRs were further analysed with bioinformatics tools, such as hidden Markov models (HMMs). It became evident that several cyanobacterial SDRs show remarkable sequence identities with SDRs in other organisms. These so-called 'homologous' proteins exist in plants, model organisms such as Drosophila melanogaster and Caenorhabditis  elegans, and even in humans. As sequence identities of up to 60% were found between Synechocystis and humans, it was concluded that SDRs seemed to have been well conserved during evolution, even after dramatic terrestrial changes such as the conversion of the early reducing atmosphere to an oxidizing one by cyanobacteria. PMID:22251568

  1. Thiosulfate Dehydrogenase (TsdA) from Allochromatium vinosum

    PubMed Central

    Brito, José A.; Denkmann, Kevin; Pereira, Inês A. C.; Archer, Margarida; Dahl, Christiane

    2015-01-01

    Although the oxidative condensation of two thiosulfate anions to tetrathionate constitutes a well documented and significant part of the natural sulfur cycle, little is known about the enzymes catalyzing this reaction. In the purple sulfur bacterium Allochromatium vinosum, the reaction is catalyzed by the periplasmic diheme c-type cytochrome thiosulfate dehydrogenase (TsdA). Here, we report the crystal structure of the “as isolated” form of A. vinosum TsdA to 1.98 Å resolution and those of several redox states of the enzyme to different resolutions. The protein contains two typical class I c-type cytochrome domains wrapped around two hemes axially coordinated by His53/Cys96 and His164/Lys208. These domains are very similar, suggesting a gene duplication event during evolution. A ligand switch from Lys208 to Met209 is observed upon reduction of the enzyme. Cys96 is an essential residue for catalysis, with the specific activity of the enzyme being completely abolished in several TsdA-Cys96 variants. TsdA-K208N, K208G, and M209G variants were catalytically active in thiosulfate oxidation as well as in tetrathionate reduction, pointing to heme 2 as the electron exit point. In this study, we provide spectroscopic and structural evidence that the TsdA reaction cycle involves the transient presence of heme 1 in the high-spin state caused by movement of the Sγ atom of Cys96 out of the iron coordination sphere. Based on the presented data, we draw important conclusions about the enzyme and propose a possible reaction mechanism for TsdA. PMID:25673691

  2. Targeting Aldehyde Dehydrogenase: a Potential Approach for Cell labeling

    PubMed Central

    Vaidyanathan, Ganesan; Song, Haijing; Affleck, Donna; McDougald, Darryl L.; Storms, Robert W.; Zalutksy, Michael R.; Chin, Bennett B.

    2009-01-01

    Introduction To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative, and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. Methods We developed schemes for the synthesis of two 3radioiodinated aldehdyes—N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)—at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. Results The average radiochemical yields for the synthesis [125I]FMIC and [125I]DEIBA were 70 ± 5% and 47 ± 14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. Conclusion To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells. PMID:19875048

  3. Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules.

    PubMed

    Andrade, Josefa; Pearce, Sandy Timm; Zhao, Hu; Barroso, Margarida

    2004-12-01

    Previously, we have shown that p22, an EF-hand Ca2+-binding protein, interacts indirectly with microtubules in an N-myristoylation-dependent and Ca2+-independent manner. In the present study, we report that N-myristoylated p22 interacts with several microtubule-associated proteins within the 30-100 kDa range using overlay blots of microtubule pellets containing cytosolic proteins. One of those p22-binding partners, a 35-40 kDa microtubule-binding protein, has been identified by MS as GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Several lines of evidence suggest a functional relationship between GAPDH and p22. First, endogenous p22 interacts with GAPDH by immunoprecipitation. Secondly, p22 and GAPDH align along microtubule tracks in analogous punctate structures in BHK cells. Thirdly, GAPDH facilitates the p22-dependent interactions between microtubules and microsomal membranes, by increasing the ability of p22 to bind microtubules but not membranes. We have also shown a direct interaction between N-myristoylated p22 and GAPDH in vitro with a K(D) of approximately 0.5 microM. The removal of either the N-myristoyl group or the last six C-terminal amino acids abolishes the binding of p22 to GAPDH and reduces the ability of p22 to associate with microtubules. In summary, we report that GAPDH is involved in the ability of p22 to facilitate microtubule-membrane interactions by affecting the p22-microtubule, but not the p22-membrane, association. PMID:15312048

  4. Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases*

    PubMed Central

    Beretta, Matteo; Gruber, Karl; Kollau, Alexander; Russwurm, Michael; Koesling, Doris; Goessler, Walter; Keung, Wing Ming; Schmidt, Kurt; Mayer, Bernd

    2008-01-01

    Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle. PMID:18450747

  5. Origin and evolution of medium chain alcohol dehydrogenases.

    PubMed

    Jörnvall, Hans; Hedlund, Joel; Bergman, Tomas; Kallberg, Yvonne; Cederlund, Ella; Persson, Bengt

    2013-02-25

    Different lines of alcohol dehydrogenases (ADHs) have separate superfamily origins, already recognized but now extended and re-evaluated by re-screening of the latest databank update. The short-chain form (SDR) is still the superfamily with most abundant occurrence, most multiple divergence, most prokaryotic emphasis, and most non-complicated architecture. This pattern is compatible with an early appearance at the time of the emergence of prokaryotic cellular life. The medium-chain form (MDR) is also old but second in terms of all the parameters above, and therefore compatible with a second emergence. However, this step appears seemingly earlier than previously considered, and may indicate sub-stages of early emergences at the increased resolution available from the now greater number of data entries. The Zn-MDR origin constitutes a third stage, possibly compatible with the transition to oxidative conditions on earth. Within all these three lines, repeated enzymogeneses gave the present divergence. MDR-ADH origin(s), at a fourth stage, may also be further resolved in multiple or extended modes, but the classical liver MDR-ADH of the liver type can still be traced to a gene duplication ~550 MYA (million years ago), at the early vertebrate radiation, compatible with the post-eon-shift, "Cambrian explosion". Classes and isozymes correspond to subsequent and recent duplicatory events, respectively. They illustrate a peculiar pattern with functional and emerging evolutionary distinctions between parent and emerging lines, suggesting a parallelism between duplicatory and mutational events, now also visible at separate sub-stages. Combined, all forms show distinctive patterns at different levels and illustrate correlations with global events. They further show that simple molecular observations on patterns, multiplicities and occurrence give much information, suggesting common divergence rules not much disturbed by horizontal gene transfers after the initial origins. PMID

  6. Bifunctional aldehyde/alcohol dehydrogenase (ADHE) in chlorophyte algal mitochondria.

    PubMed

    Atteia, Ariane; van Lis, Robert; Mendoza-Hernández, Guillermo; Henze, Katrin; Martin, William; Riveros-Rosas, Hector; González-Halphen, Diego

    2003-09-01

    Protein profiles of mitochondria isolated from the heterotrophic chlorophyte Polytomella sp. grown on ethanol at pH 6.0 and pH 3.7 were analyzed by Blue Native and denaturing polyacrylamide gel electrophoresis. Steady-state levels of oxidative phosphorylation complexes were influenced by external pH. Levels of an abundant, soluble, mitochondrial protein of 85 kDa and its corresponding mRNA increased at pH 6.0 relative to pH 3.7. N-terminal and internal sequencing of the 85 kDa mitochondrial protein together with the corresponding cDNA identified it as a bifunctional aldehyde/alcohol dehydrogenase (ADHE) with strong similarity to homologues from eubacteria and amitochondriate protists. A mitochondrial targeting sequence of 27 amino acids precedes the N-terminus of the mature mitochondrial protein. A gene encoding an ADHE homologue was also identified in the genome of Chlamydomonas reinhardtii, a photosynthetic relative of Polytomella. ADHE reveals a complex picture of sequence similarity among homologues. The lack of ADHE from archaebacteria indicates a eubacterial origin for the eukaryotic enzyme. Among eukaryotes, ADHE has hitherto been characteristic of anaerobes since it is essential to cytosolic energy metabolism of amitochondriate protists such as Giardia intestinalis and Entamoeba histolytica. Its abundance and expression pattern suggest an important role for ADHE in mitochondrial metabolism of Polytomella under the conditions studied. The current data are compatible with the view that Polytomella ADHE could be involved either in ethanol production or assimilation, or both, depending upon environmental conditions. Presence of ADHE in an oxygen-respiring algal mitochondrion and co-expression at ambient oxygen levels with respiratory chain components is unexpected with respect to the view that eukaryotes acquired ADHE genes specifically as an adaptation to an anaerobic lifestyle. PMID:14756315

  7. Furosemide and 11beta-hydroxysteroid dehydrogenase activity, in man.

    PubMed

    Palermo, M; Armanini, D; Shackleton, C H L; Sorba, G; Cossu, M; Roitman, E; Scaroni, C; Delitala, G

    2002-09-01

    Mineralocorticoid receptors possess the same affinity for aldosterone and for cortisol and preferential binding of aldosterone is modulated by the 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) enzyme, which converts cortisol to its inactive metabolite cortisone. Several endogenous or exogenous compounds able to inhibit the enzyme have been described and, as a consequence, produce the syndrome of apparent mineralocorticoid excess (AME) characterized by hypertension, hypokalemia, volume repletion and suppression of the renin-angiotensin-aldosterone system. High doses of furosemide, a diuretic that works in the luminal surface of the thick ascending limb of Henle's loop, have been reported to inhibit 11 beta-OHSD activity to the same extent as licorice in vivo and in vitro, in rat. The aim of our study was to verify the effect of the drug on 11 beta-OHSD activity in man at the doses currently used in clinical practice. We tested the activity of 11 beta-OHSD following both acute and protracted administration of furosemide. In the acute study, the drug was administered at low (40 mg i.v. in bolo) and high doses (infusion of 10 mg/kg bw i.v for six hours); the protracted furosemide administration consisted in 50 mg/day for 20 days, by mouth. The ratios between the cortisol metabolites tetrahydrocortisol plus allo-tetrahydrocortisol to tetra-hydrocortisone and urinary free cortisol to urinary free cortisone were used to measure the activity of 11 beta-OHSD. Urinary cortisol, cortisone and their metabolites were tested by a gas-chromatographic/mass spectrometric method. Neither acute nor prolonged administration of furosemide did affect the activity of 11 beta-OHSD although the drug was able to modify plasma aldosterone and PRA secretion and to determine hypokalemia. Our results suggest that furosemide does not play a significant role in 11 beta-OHSD modulation in humans, at least at the dosage used in clinical practice. PMID:12373630

  8. Induced fit and the catalytic mechanism of isocitrate dehydrogenase.

    PubMed

    Gonçalves, Susana; Miller, Stephen P; Carrondo, Maria A; Dean, Anthony M; Matias, Pedro M

    2012-09-11

    NADP(+) dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) belongs to a large family of α-hydroxyacid oxidative β-decarboxylases that catalyze similar three-step reactions, with dehydrogenation to an oxaloacid intermediate preceding β-decarboxylation to an enol intermediate followed by tautomerization to the final α-ketone product. A comprehensive view of the induced fit needed for catalysis is revealed on comparing the first "fully closed" crystal structures of a pseudo-Michaelis complex of wild-type Escherichia coli IDH (EcoIDH) and the "fully closed" reaction product complex of the K100M mutant with previously obtained "quasi-closed" and "open" conformations. Conserved catalytic residues, binding the nicotinamide ring of NADP(+) and the metal-bound substrate, move as rigid bodies during domain closure by a hinge motion that spans the central β-sheet in each monomer. Interactions established between Thr105 and Ser113, which flank the "phosphorylation loop", and the nicotinamide mononucleotide moiety of NADP(+) establish productive coenzyme binding. Electrostatic interactions of a Lys100-Leu103-Asn115-Glu336 tetrad play a pivotal role in assembling a catalytically competent active site. As predicted, Lys230* is positioned to deprotonate/reprotonate the α-hydroxyl in both reaction steps and Tyr160 moves into position to protonate C3 following β-decarboxylation. A proton relay from the catalytic triad Tyr160-Asp307-Lys230* connects the α-hydroxyl of isocitrate to the bulk solvent to complete the picture of the catalytic mechanism. PMID:22891681

  9. Structural Basis of Cooperativity in Human UDP-Glucose Dehydrogenase

    PubMed Central

    Rajakannan, Venkatachalam; Lee, Hui-Sun; Chong, Seon-Ha; Ryu, Han-Bong; Bae, Ji-Young; Whang, Eun-Young; Huh, Jae-Wan; Cho, Sung-Woo; Kang, Lin-Woo; Choe, Han; Robinson, Robert C.

    2011-01-01

    Background UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics. Methodology Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle. Conclusion In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme. PMID:21984906

  10. Purification and characterization of guinea pig liver morphine 6-dehydrogenase.

    PubMed

    Yamano, S; Kageura, E; Ishida, T; Toki, S

    1985-05-10

    Morphine 6-dehydrogenase, which catalyzes the dehydrogenation of morphine to morphinone, has been purified about 440-fold from the soluble fraction of guinea pig liver with a yield of 38%. The purified enzyme was a homogeneous protein on polyacrylamide gel disc electrophoresis and isoelectric focusing. The molecular weight and isoelectric point of the enzyme were 29,000 and 7.6, respectively. The enzyme utilizes both NAD and NADP as a cofactor, and the Km values were 0.12 mM for NAD and 0.42 mM for NADP. The Vmax values for morphine were 588 milliunits/mg of protein (with NAD) and 1600 milliunits/mg of protein (with NADP). The Km values for morphine were 0.12 mM (with NAD) and 0.49 mM (with NADP). The enzyme also exhibited activity for morphine-related compounds: nalorphine, normorphine, codeine, and ethylmorphine; however, 7,8-saturated congeners such as dihydromorphine and dihydrocodeine were poor substrates. The enzyme was inactivated by removal of 2-mercaptoethanol from the enzyme solution. The inactivated enzyme was rapidly recovered by the addition of 2-mercaptoethanol. Phenylarsine oxide and CdCl2 (dithiol modifiers) inhibited competitively toward cofactor binding and noncompetitively toward morphine binding. These results suggest that the enzyme possesses the essential thiol groups, probably vicinal dithiol, at or near the cofactor-binding site. Using the partially purified enzyme, 8-(2-hydroxyethylthio)dihydromorphinone was isolated as the product and identified by UV, mass, and NMR spectra. It was confirmed that morphinone proposed as the dehydrogenation product was nonenzymatically and covalently bound to 2-mercaptoethanol. Accordingly, the isolated morphinone-2-mercaptoethanol conjugate must be formed by two steps: enzymatic production of morphinone from morphine and then nonenzymatic binding of 2-mercaptoethanol to morphinone. PMID:2580834

  11. Vascular Bioactivation of Nitroglycerin by Aldehyde Dehydrogenase-2

    PubMed Central

    Lang, Barbara S.; Gorren, Antonius C. F.; Oberdorfer, Gustav; Wenzl, M. Verena; Furdui, Cristina M.; Poole, Leslie B.; Mayer, Bernd; Gruber, Karl

    2012-01-01

    Aldehyde dehydrogenase-2 (ALDH2) catalyzes the bioactivation of nitroglycerin (glyceryl trinitrate, GTN) in blood vessels, resulting in vasodilation by nitric oxide (NO) or a related species. Because the mechanism of this reaction is still unclear we determined the three-dimensional structures of wild-type (WT) ALDH2 and of a triple mutant of the protein that exhibits low denitration activity (E268Q/C301S/C303S) in complex with GTN. The structure of the triple mutant showed that GTN binds to the active site via polar contacts to the oxyanion hole and to residues 268 and 301 as well as by van der Waals interactions to hydrophobic residues of the catalytic pocket. The structure of the GTN-soaked wild-type protein revealed a thionitrate adduct to Cys-302 as the first reaction intermediate, which was also found by mass spectrometry (MS) experiments. In addition, the MS data identified sulfinic acid as the irreversibly inactivated enzyme species. Assuming that the structures of the triple mutant and wild-type ALDH2 reflect binding of GTN to the catalytic site and the first reaction step, respectively, superposition of the two structures indicates that denitration of GTN is initiated by nucleophilic attack of Cys-302 at one of the terminal nitrate groups, resulting in formation of the observed thionitrate intermediate and release of 1,2-glyceryl dinitrate. Our results shed light on the molecular mechanism of the GTN denitration reaction and provide useful information on the structural requirements for high affinity binding of organic nitrates to the catalytic site of ALDH2. PMID:22988236

  12. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

    PubMed Central

    Jean, Elise; Laoudj-Chenivesse, Dalila; Notarnicola, Cécile; Rouger, Karl; Serratrice, Nicolas; Bonnieu, Anne; Gay, Stéphanie; Bacou, Francis; Duret, Cédric; Carnac, Gilles

    2011-01-01

    Abstract Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56+ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non-human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H2O2)-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. PMID:19840193

  13. MAPPING OF SUCCINATE DEHYDROGENASE LOSSES IN 2258 EPITHELIAL NEOPLASMS

    PubMed Central

    Miettinen, Markku; Sarlomo-Rikala, Maarit; Cue, Peter Mc.; Czapiewski, Piotr; Langfor, Renata; Waloszczyk, Piotr; Wazny, Krzysztof; Biernat, Wojciech; Lasota, Jerzy; Wang, Zengfeng

    2013-01-01

    Losses in the succinate dehydrogenase (SDH) complex characterize 20–30% of extra-adrenal paragangliomas and 7–8% of gastric GISTs, and rare renal cell carcinomas. This loss is reflected as lack of the normally ubiquitous immunohistochemical expression of the SDH subunit B (SDHB). In paragangliomas, SDHB loss correlates with homozygous loss of any of the SDH subunits, typically by loss-of-function mutations. The occurrence of SDHB losses in other epithelial malignancies is unknown. In this study, we immunohistochemically examined 2258 epithelial, mostly malignant neoplasms including common carcinomas of all sites. Among renal cell carcinomas, SDHB loss was observed in 4 of 711 cases (0.6%) including a patient with an SDHB-deficient GIST. Histologically the SDHB-negative renal carcinomas varied. There was one clear cell carcinoma with a high nuclear grade, one papillary carcinoma type 2, one unclassified carcinoma with a glandular pattern, and one oncocytoid low-grade carcinoma as previously described for SDHB-negative renal carcinoma. None of these patients was known to have paragangliomas or had loss of SDHA expression in the tumor. Three of these patients had metastases at presentation (2 in the adrenal, one in the retroperitoneal lymph nodes). There were no cases with SDHB-loss among 64 renal oncocytomas. SDHB-losses were not seen in other carcinomas, except in one prostatic adenocarcinoma (1/57), one lymphoepithelial carcinoma of the stomach, and one (1/40) seminoma. Based on this study, SDHB-losses occur in 0.6% of renal cell carcinomas and extremely rarely in other carcinomas. Some of these renal carcinomas may be clinically aggressive. The clinical significance and molecular genetics of these SDHB-negative tumors requires further study. PMID:23531856

  14. 11 beta-Hydroxysteroid dehydrogenase activity in hypothalamic obesity.

    PubMed

    Tiosano, Dov; Eisentein, Israel; Militianu, Daniela; Chrousos, George P; Hochberg, Ze'ev

    2003-01-01

    After extensive suprasellar operations for hypothalamic tumor removal, some patients develop Cushing-like morbid obesity while they receive replacement doses of glucocorticoids. In this study, we examined the hypothesis that target tissue conversion of inactive 11-ketosteroids to active 11 beta-OH glucocorticoids might explain the obesity of some patients with hypothalamic lesions. Toward this aim, we studied 10 patients with hypothalamic obesity and secondary adrenal insufficiency and 6 control Addisonian patients while they were on glucocorticoid replacement therapy. Pituitary hormone deficiencies were replaced when medically indicated. Twenty-four-hour urine was collected after a single oral dose of 12 mg/m(2) hydrocortisone acetate. The ratios of free and conjugated cortisol (F) to cortisone (E) and their metabolites, [tetrahydrocortisol (THF)+5 alpha THF]/tetrahyrdocortisone (THE), dihydrocortisols/dihydrocortisones, cortols/cortolones, and (F+E)/(THF+THE+5 alpha THF), were considered to represent 11 beta-hydroxysteroid dehydrogenase (HSD) activity. The 11-OH/11-oxo ratios were significantly higher in the urine of patients with hypothalamic obesity. The 11-OH/11-oxo ratios, however, did not correlate with the degree of obesity, yet a significant correlation was found between conjugated F/E and the ratio of visceral fat to sc fat measured by computerized tomography at the umbilical level. The consequence of increased 11 beta-HSD1 activity and the shift of the interconversion toward cortisol may contribute to the effects of the latter in adipose tissue. We propose that deficiency of hypothalamic messengers after surgical injury induces a paracrine/autocrine effect of enhanced glucocorticoid activity due to up-regulated 11 beta-HSD1 activity. PMID:12519880

  15. Action of shear on enzymes: studies with alcohol dehydrogenase.

    PubMed

    Thomas, C R; Nienow, A W; Dunnill, P

    1979-12-01

    Yeast alcohol dehydrogenase (ADH) solutions (approximately 1 mg/ml, pH 7) were sheared in a coaxial cylindrical viscometer. This was fitted with a lid sealing the contents from the atmosphere and preventing evaporation. At 30 degrees C after a total of 5 hr intermittent shearing at 683 sec-1 no losses of activity were observed. No losses were found after 5 hr continuous shearing and in a no-shear control. At 40 degrees C and 683 sec-1 there were only small activity losses in 5 hr. Shearing at 3440 sec-1 no measurable losses of activity were found with a 1.03 mg/ml solution in 5 hr at 30 degrees C, a 1.03 mg/ml solution in 8 hr at 5 degrees C, and with a 3.89 mg/ml solution in 3 hr at 5 degrees C. In all these cases, however, a white precipitate formed that was not observed in zero shear control experiments. The sheared 3.89 mg/ml solution was clarified by centrifugation. It was shown that there were no ADH aggregates in the supernatant and that the precipitate was less than 2% of the original protein. At 30 degrees C under adverse pH conditions (pH 8.8) there was no significant difference in activity losses of an approximately 1 mg/ml solution sheared at 65 and 744 sec-1. An approximately 0.5 mg/ml ADH solution, pH 7, was agitated in a small reactor with no free air-liquid interface. Peak shear rates near the impeller were estimated to be about 9000 sec-1. Only a small decrease in specific activity was observed until over 15 hr total running at 5 degrees C. PMID:42450

  16. 15-Hydroxyprostaglandin Dehydrogenase (15-PGDH) and Lung Cancer

    PubMed Central

    Tai, Hsin-Hsiung; Tong, Min; Ding, Yunfei

    2007-01-01

    15-Hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes NAD+-linked oxidation of 15 (S)-hydroxyl group of prostaglandins and lipoxins and is the key enzyme responsible for the biological inactivation of these eicosanoids. The enzyme was found to be under-expressed as opposed to cyclooxygenase-2 (COX-2) being over-expressed in lung and other tumors. A549 human lung adenocarcinoma cells were used as a model system to study the role of 15-PGDH in lung tumorigenesis. Up-regulation of COX-2 expression by pro-inflammatory cytokines in A549 cells was accompanied by a down-regulation of 15-PGDH expression. Over-expression of COX-2 but not COX-1 by adenoviral-mediated approach also attenuated 15-PGDH expression. Similarly, over-expression of 15-PGDH by the same strategy inhibited IL-1β-induced COX-2 expression. It appears that the expression of COX-2 and 15-PGDH is regulated reciprocally. Adenoviral-mediated transient over-expression of 15-PGDH in A549 cells resulted in apoptosis. Xenograft studies in nude mice also showed tumor suppression with cells transiently over-expressing 15-PGDH. However, cells stably over-expressing 15-PGDH generated tumors faster than those control cells. Examination of different clones of A549 cells stably expressing different levels of 15-PGDH indicated that the levels of 15-PGDH expression correlated positively with those of mesenchymal markers, and negatively with those of epithelial markers. It appears that the stable expression of 15-PGDH induces epithelial-mesenchymal transition (EMT) which may account for the tumor promotion in xenograft studies. A number of anti-cancer agents, such as transforming growth factor-β1 (TGF-β1), glucocorticoids and some histone deacetylase inhibitors were found to induce 15-PGDH expression. These results suggest that tumor suppressive action of these agents may, in part, be related to their ability to induce 15-PGDH expression. PMID:17481556

  17. Urinary Bladder Paragangliomas: Analysis of Succinate Dehydrogenase and Outcome.

    PubMed

    Gupta, Sounak; Zhang, Jun; Rivera, Michael; Erickson, Lori A

    2016-09-01

    Paragangliomas of the urinary bladder can arise sporadically or as a part of hereditary syndromes including those with underlying mutations in the succinate dehydrogenase (SDH) genes, which serve as tumor suppressors. SDH deficiency can be screened for by absence of immunohistochemical detection of SDHB. In this study of 11 cases, clinical follow-up was available for 9/11 cases. The cases were reviewed and graded based on the grading system for adrenal pheochromocytomas and paragangliomas (GAPP) criteria. Immunohistochemistry was performed for Ki67 and SDHB. Proliferative index was calculated by quantification of Ki67-positive cells at hot spots. The medical record was accessed for documentation of germline SDH mutations. Urinary bladder paragangliomas had a female predilection (8/11 cases), and 5/11 cases exhibited metastatic behavior. Patients with metastatic disease tended to be younger (mean age 43 vs 49 years), have larger lesions (5.8 vs 1.5 cm), and presented with catecholamine excess (4/4 vs 2/6 patients with non-metastatic lesions). Patients with metastatic disease had a higher mean Ki67 proliferation rate (4.9 vs 1.3 %) and GAPP score (mean of 5.8 vs 3.8) (p = 0.01). IHC for SDHB expression revealed loss of expression in 2/6 cases of non-metastatic paragangliomas compared to 4/5 patients with metastatic paragangliomas. Interestingly, of these four patients, two had a documented mutation of SDHB, one patient had a SDHC mutation, and another patient had a history of familial disease without mutation analysis being performed. Our study, suggests that SDH loss was suggestive of metastatic behavior in addition to younger age at diagnosis, larger tumor size, and higher Ki67 proliferation rate and catecholamine type. PMID:27262318

  18. Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus.

    PubMed

    Porumb, H; Vancea, D; Mureşan, L; Presecan, E; Lascu, I; Petrescu, I; Porumb, T; Pop, R; Bârzu, O

    1987-04-01

    Alanine dehydrogenase from Bacillus cereus, a non-allosteric enzyme composed of six identical subunits, was purified to homogeneity by chromatography on blue-Sepharose and Sepharose 6B-CL. Like other pyridine-linked dehydrogenases, alanine dehydrogenase is inhibited by Cibacron blue, competitively with respect to NADH and noncompetitively with respect to pyruvate. The enzyme was inactivated by 0.1 M glycine/HCl (pH 2) and reactivated by 0.1 M phosphate (pH 8) supplemented with NAD+ or NADH. The reactivation was characterized by sigmoidal kinetics indicating a complex mechanism involving rate-limiting folding and association steps. Cibacron blue interfered with renaturation, presumably by competition with NADH. Chromatography on Sepharose 6B-CL of the partially renatured alanine dehydrogenase led to the separation of several intermediates, but only the hexamer was characterized by enzymatic activity. By immobilization on Sepharose 4B, alanine dehydrogenase from B. cereus retained 66% of the specific activity of the soluble enzyme. After denaturation of immobilized alanine dehydrogenase with 7 M urea, 37% of the initial protein was still bound to Sepharose, indicating that on the average the hexamer was attached to the matrix via, at most, two subunits. The ability of the denatured, immobilized subunits to pick up subunits from solution shows their capacity to fold back to the native conformation after urea treatment. The formation of "hybrids" between subunits of enzyme from B. cereus and Bacillus subtilis demonstrates the close resemblance of the tertiary and quaternary structures of alanine dehydrogenases from these species. PMID:3104322

  19. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    PubMed

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. PMID:25280628

  20. Orchestration of Enzymatic Processing by Thiazole/Oxazole-Modified Microcin Dehydrogenases

    PubMed Central

    Melby, Joel O.; Li, Xiangpo; Mitchell, Douglas A.

    2014-01-01

    Thiazole/oxazole-modified microcins (TOMMs) comprise a structurally diverse family of natural products with varied bioactivities linked by the presence of posttranslationally installed thiazol(in)e and oxazol(in)e heterocycles. The detailed investigation of the TOMM biosynthetic enzymes from Bacillus sp. Al Hakam (Balh) has provided significant insight into heterocycle biosynthesis. Thiazoles and oxazoles are installed by the successive action of an ATP-dependent cyclodehydratase (C- and D-protein) and a FMN-dependent dehydrogenase (B-protein), which are responsible for azoline formation and azoline oxidation, respectively. Although several studies have focused on the mechanism of azoline formation, many details regarding the role of the dehydrogenase (B-protein) in overall substrate processing remain unknown. In this work, we evaluated the involvement of the dehydrogenase in determining the order of ring formation, as well as the promiscuity of the Balh and microcin B17 cyclodehydratases to accept a panel of noncognate dehydrogenases. In support of the observed promiscuity, a fluorescence polarization assay was utilized to measure binding of the dehydrogenase to the cyclodehydratase using the intrinsic fluorescence of the FMN cofactor. Ultimately, the noncognate dehydrogenases were shown to possess cyclodehydratase-independent activity. A previous study identified a conserved Lys-Tyr motif to be important for dehydrogenase activity. Using the tools developed in this study, the Lys-Tyr motif was shown to not alter complex formation with the cyclodehydratase nor the reduction potential. Taken with the known crystal structure of a homolog, our data suggest that the Lys-Tyr motif is of catalytic importance. Overall, this study provides a greater level of insight into the complex orchestration of enzymatic activity during TOMM biosynthesis. PMID:24364559

  1. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    PubMed

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  2. Differential effects of polyamine on the cytosolic and mitochondrial NADP-isocitrate dehydrogenases.

    PubMed

    Murakami, Keiko; Haneda, Miyako; Iwata, Shouko; Yoshino, Masataka

    2012-01-01

    Two isozymes of NADP-dependent isocitrate dehydrogenases (EC 1.1.1.42) exist in mammalian tissues: mitochondrial (ICD1) and cytosolic (ICD2). Effects of polyamines such as spermine, spermidine, and putrescine on the cytosolic and mitochondrial NADP-isocitrate dehydrogenases were analyzed kinetically. Spermine activated ICD2, the cytosolic NADP-isocitrate dehydrogenase from rat liver with the increase in the maximal velocity and the decrease in the affinity for the substrates isocitrate and NADP. The activating action of spermine can be explained by "the uncompetitive effect," and the dissociation constant of spermine for the enzyme-substrate complex was determined to be 1.68 mM. Spermidine and putrescine showed little or no effect. ICD1, the mitochondrial form of NADP-isocitrate dehydrogenase from rat and porcine heart was inhibited by spermine effectively, and by spermidine and putrescine to a lesser extent. Spermine inhibited the enzyme competitively with respect to NADP, and noncompetitively with respect to isocitrate. K(i) value of the enzyme for spermine was 1.3 mM. These results suggest that activation by spermine of cytosolic NADP-isocitrate dehydrogenase can enhance the antioxidant activity by regeneration of GSH, and further is responsible for the stimulation of lipid biosynthesis in cytosol. Spermine may contribute to NADPH supply by enhancing transhydrogenase (EC1.6.1.2) activity through the spermine-dependent activation of Ca(2+) -incorporation to mitochondria. PMID:22674798

  3. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    PubMed Central

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-01-01

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

  4. ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase.

    PubMed

    Mráček, Tomáš; Holzerová, Eliška; Drahota, Zdeněk; Kovářová, Nikola; Vrbacký, Marek; Ješina, Pavel; Houštěk, Josef

    2014-01-01

    Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production. PMID:23999537

  5. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

    PubMed Central

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F.; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  6. Isolation and characterization of an inducible NAD-dependent butyraldehyde dehydrogenase from clostridium acetobutylicum

    SciTech Connect

    Schreiber, W.; Duerre, P.

    1996-12-31

    A NAD-dependent butyraldehyde dehydrogenase (BAD) has been purified from C. acetobutylicum DSM 792 and DSM 173 1. This key enzyme of butanol production, catalyzing the conversion of butyryl-CoA to butyraldehyde, was induced shortly before the onset of butanol production and proved to be oxygen-sensitive. A one step purification procedure on reactive green 19 allowed to purify the enzyme to homogeneity. The purified protein was found to be extremely unstable and could only partially be stabilized by addition of mercaptoethanol and storage below -20{degrees}C. The enzyme subunit had a molecular mass of 39.5 kDa. In the reverse reaction (butyryl-CoA-forming) the apparent pH optimum was 9.75 and Vmax was significantly higher with butyraldehyde and propionaldehyde than with acetaldehyde. BAD could also use NADP+, but NAD+ was the preferred coenzyme for the reverse reaction. The N-terminal amino acid sequence of the C. acetobutylicurn DSM 792 protein showed high homology to glyceraldehyde-3-phosphate dehydrogenases (GAP), especially to the protein of C. pasteurianum. Genomic libraries of C. acetobutylicum DSM 792 were screened by hybridization using PCR-generated heterologous probes encoding the gap gene of C. pasteurianum. Sequence analysis of the positive clones revealed high homology, but no identity to the N-terminal amino acid sequence of the butyraldehyde dehydrogenase. Thus, BAD from C. acetobutylicum is distinctly different from other reported aldehyde dehydrogenases with butyraldehyde dehydrogenase activity.

  7. Ethylbenzene Dehydrogenase and Related Molybdenum Enzymes Involved in Oxygen-Independent Alkyl Chain Hydroxylation.

    PubMed

    Heider, Johann; Szaleniec, Maciej; Sünwoldt, Katharina; Boll, Matthias

    2016-01-01

    Ethylbenzene dehydrogenase initiates the anaerobic bacterial degradation of ethylbenzene and propylbenzene. Although the enzyme is currently only known from a few closely related denitrifying bacterial strains affiliated to the Rhodocyclaceae, it clearly marks a universally occurring mechanism used for attacking recalcitrant substrates in the absence of oxygen. Ethylbenzene dehydrogenase belongs to subfamily 2 of the DMSO reductase-type molybdenum enzymes together with paralogous enzymes involved in the oxygen-independent hydroxylation of p-cymene, the isoprenoid side chains of sterols and even possibly n-alkanes; the subfamily also extends to dimethylsulfide dehydrogenases, selenite, chlorate and perchlorate reductases and, most significantly, dissimilatory nitrate reductases. The biochemical, spectroscopic and structural properties of the oxygen-independent hydroxylases among these enzymes are summarized and compared. All of them consist of three subunits, contain a molybdenum-bis-molybdopterin guanine dinucleotide cofactor, five Fe-S clusters and a heme b cofactor of unusual ligation, and are localized in the periplasmic space as soluble enzymes. In the case of ethylbenzene dehydrogenase, it has been determined that the heme b cofactor has a rather high redox potential, which may also be inferred for the paralogous hydroxylases. The known structure of ethylbenzene dehydrogenase allowed the calculation of detailed models of the reaction mechanism based on the density function theory as well as QM-MM (quantum mechanics - molecular mechanics) methods, which yield predictions of mechanistic properties such as kinetic isotope effects that appeared consistent with experimental data. PMID:26960184

  8. Isolation and Characterization of Anaerobic Ethylbenzene Dehydrogenase, a Novel Mo-Fe-S Enzyme

    PubMed Central

    Johnson, Hope A.; Pelletier, Dale A.; Spormann, Alfred M.

    2001-01-01

    The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp. strain EB1 is the oxidation of ethylbenzene to (S)-(−)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (αβγ) with subunit masses of 100 kDa (α), 35 kDa (β), and 25 kDa (γ). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that ebdA encodes the α subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The ebdB gene encodes the β subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The ebdC gene encodes the γ subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes. PMID:11443088

  9. Kinetic studies of the uptake of aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro.

    PubMed Central

    Marra, E; Passarella, S; Casamassima, E; Perlino, E; Doonan, S; Quagliariello, E

    1985-01-01

    Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system. PMID:4015628

  10. Selective permeability of rat liver mitochondria to purified malate dehydrogenase isoenzymes in vitro.

    PubMed Central

    Passarella, S; Marra, E; Doonan, S; Quagliariello, E

    1980-01-01

    1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied. PMID:7236231

  11. Crystal structure of cod liver class I alcohol dehydrogenase: substrate pocket and structurally variable segments.

    PubMed Central

    Ramaswamy, S.; el Ahmad, M.; Danielsson, O.; Jörnvall, H.; Eklund, H.

    1996-01-01

    The structural framework of cod liver alcohol dehydrogenase is similar to that of horse and human alcohol dehydrogenases. In contrast, the substrate pocket differs significantly, and main differences are located in three loops. Nevertheless, the substrate pocket is hydrophobic like that of the mammalian class I enzymes and has a similar topography in spite of many main-chain and side-chain differences. The structural framework of alcohol dehydrogenase is also present in a number of related enzymes like glucose dehydrogenase and quinone oxidoreductase. These enzymes have completely different substrate specificity, but also for these enzymes, the corresponding loops of the substrate pocket have significantly different structures. The domains of the two subunits in the crystals of the cod enzyme further differ by a rotation of the catalytic domains by about 6 degrees. In one subunit, they close around the coenzyme similarly as in coenzyme complexes of the horse enzyme, but form a more open cleft in the other subunit, similar to the situation in coenzyme-free structures of the horse enzyme. The proton relay system differs from the mammalian class I alcohol dehydrogenases. His 51, which has been implicated in mammalian enzymes to be important for proton transfer from the buried active site to the surface is not present in the cod enzyme. A tyrosine in the corresponding position is turned into the substrate pocket and a water molecule occupies the same position in space as the His side chain, forming a shorter proton relay system. PMID:8845755

  12. Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa.

    PubMed Central

    Chow, C M; RajBhandary, U L

    1993-01-01

    We have isolated and characterized a gene, fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli. The gene is closely linked (less than 0.6 kb apart) to the leu-5 gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands. The expression patterns of these genes differ: fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while leu-5 mRNA appears during germination and mycelial growth. The structure of the fdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies. The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E. coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily. In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp. strain 101. All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N. crassa enzyme. We have further shown that expression of the N. crassa gene in E. coli leads to the production of formate dehydrogenase activity, indicating that the N. crassa gene specifies a functional polypeptide. Images PMID:8509325

  13. Structure of a highly NADP+-specific isocitrate dehydrogenase.

    PubMed

    Sidhu, Navdeep S; Delbaere, Louis T J; Sheldrick, George M

    2011-10-01

    Isocitrate dehydrogenase catalyzes the first oxidative and decarboxylation steps in the citric acid cycle. It also lies at a crucial bifurcation point between CO2-generating steps in the cycle and carbon-conserving steps in the glyoxylate bypass. Hence, the enzyme is a focus of regulation. The bacterial enzyme is typically dependent on the coenzyme nicotinamide adenine dinucleotide phosphate. The monomeric enzyme from Corynebacterium glutamicum is highly specific towards this coenzyme and the substrate isocitrate while retaining a high overall efficiency. Here, a 1.9 Å resolution crystal structure of the enzyme in complex with its coenzyme and the cofactor Mg2+ is reported. Coenzyme specificity is mediated by interactions with the negatively charged 2'-phosphate group, which is surrounded by the side chains of two arginines, one histidine and, via a water, one lysine residue, forming ion pairs and hydrogen bonds. Comparison with a previous apoenzyme structure indicates that the binding site is essentially preconfigured for coenzyme binding. In a second enzyme molecule in the asymmetric unit negatively charged aspartate and glutamate residues from a symmetry-related enzyme molecule interact with the positively charged arginines, abolishing coenzyme binding. The holoenzyme from C. glutamicum displays a 36° interdomain hinge-opening movement relative to the only previous holoenzyme structure of the monomeric enzyme: that from Azotobacter vinelandii. As a result, the active site is not blocked by the bound coenzyme as in the closed conformation of the latter, but is accessible to the substrate isocitrate. However, the substrate-binding site is disrupted in the open conformation. Hinge points could be pinpointed for the two molecules in the same crystal, which show a 13° hinge-bending movement relative to each other. One of the two pairs of hinge residues is intimately flanked on both sides by the isocitrate-binding site. This suggests that binding of a relatively

  14. Selective selC-Independent Selenocysteine Incorporation into Formate Dehydrogenases

    PubMed Central

    Zorn, Michael; Ihling, Christian H.; Golbik, Ralph; Sawers, R. Gary; Sinz, Andrea

    2013-01-01

    The formate dehydrogenases (Fdh) Fdh-O, Fdh-N, and Fdh-H, are the only proteins in Escherichia coli that incorporate selenocysteine at a specific position by decoding a UGA codon. However, an excess of selenium can lead to toxicity through misincorporation of selenocysteine into proteins. To determine whether selenocysteine substitutes for cysteine, we grew Escherichia coli in the presence of excess sodium selenite. The respiratory Fdh-N and Fdh-O enzymes, along with nitrate reductase (Nar) were co-purified from wild type strain MC4100 after anaerobic growth with nitrate and either 2 µM or 100 µM selenite. Mass spectrometric analysis of the catalytic subunits of both Fdhs identified the UGA-specified selenocysteine residue and revealed incorporation of additional, ‘non-specific’ selenocysteinyl residues, which always replaced particular cysteinyl residues. Although variable, their incorporation was not random and was independent of the selenite concentration used. Notably, these cysteines are likely to be non-essential for catalysis and they do not coordinate the iron-sulfur cluster. The remaining cysteinyl residues that could be identified were never substituted by selenocysteine. Selenomethionine was never observed in our analyses. Non-random substitution of particular cysteinyl residues was also noted in the electron-transferring subunit of both Fdhs as well as in the subunits of the Nar enzyme. Nar isolated from an E. coli selC mutant also showed a similar selenocysteine incorporation pattern to the wild-type indicating that non-specific selenocysteine incorporation was independent of the specific selenocysteine pathway. Thus, selenide replaces sulfide in the biosynthesis of cysteine and misacylated selenocysteyl-tRNACys decodes either UGU or UGC codons, which usually specify cysteine. Nevertheless, not every UGU or UGC codon was decoded as selenocysteine. Together, our results suggest that a degree of misincorporation of selenocysteine into enzymes

  15. Microbial and xanthine dehydrogenase inhibitory activity of some flavones.

    PubMed

    Khobragade, C N; Bodade, Ragini G; Shinde, M S; Jaju, Deepa R; Bhosle, R B; Dawane, B S

    2008-06-01

    Xanthine dehydrogenase (XDH) is responsible for the pathological condition called Gout. In the present study different flavones synthesized from chalcone were evaluated in vitro for their inhibitory activity. Inhibitory activity of flavones on XDH was determined in terms of inhibition of uric acid synthesis from Xanthine. The enzymatic activity was found maximum at pH 7.5 and temperature 40 degrees C. The flavones 6-chloro-2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(1)) and 6-chloro-7methyl-2-[3-(4-chloro-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one(F(2)),were noncompetitive and competitive inhibitor with Ki values 1.1 and 0.22 respectively. The flavones (F(1)), (F(2)), 6-chloro-2-[3-(4-chloro-phenyl)-1phenyl-1-H-pyrazol-4-yl]-chromen-4-one(F(3)), 8-bromo-6-chloro-2-[3-(4-chloro-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(4)), 2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(5)) and 6-methyl-2-[3-(4-hydroxy-phenyl)-1-phenyl-1-H-pyrazol-4-yl]-chromen-4-one (F(6)) were also screened for their antimicrobial activity, measured in terms of zone of inhibition. A broad spectrum antifungal activity was obtained against Trichoderma viridae, Candida albicans, Microsporum cannis, Penicillium chrysogenum and Fusarium moniliformae. In case of Aspergillus niger and Aspergillus flavous only spore formation was affected, while antibacterial activity was observed against Staphylococcus aureus, Bacillus subtilis and Serratia marsecens only. The flavones were further analyzed for quantitative structural activity relationship study (QSAR) by using PASS, online software to determine their Pa value. Toxicity and drug relevant properties were revealed by PALLAS software in terms of their molecular weight. Log P values were also studied. The result showed both the F(1) and F(2) flavones as antigout and therefore supports the development of novel drugs for the treatment of gout. PMID:18569337

  16. Glucose-6-phosphate dehydrogenase deficiency in Nigerian children.

    PubMed

    Williams, Olatundun; Gbadero, Daniel; Edowhorhu, Grace; Brearley, Ann; Slusher, Tina; Lund, Troy C

    2013-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy and in Sub-Saharan Africa, is a significant cause of infection- and drug-induced hemolysis and neonatal jaundice. Our goals were to determine the prevalence of G6PD deficiency among Nigerian children of different ethnic backgrounds and to identify predictors of G6PD deficiency by analyzing vital signs and hematocrit and by asking screening questions about symptoms of hemolysis. We studied 1,122 children (561 males and 561 females) aged 1 month to 15 years. The mean age was 7.4 ± 3.2 years. Children of Yoruba ethnicity made up the largest group (77.5%) followed by those Igbo descent (10.6%) and those of Igede (10.2%) and Tiv (1.8%) ethnicity. G6PD status was determined using the fluorescent spot method. We found that the overall prevalence of G6PD deficiency was 15.3% (24.1% in males, 6.6% in females). Yoruba children had a higher prevalence (16.9%) than Igede (10.5%), Igbo (10.1%) and Tiv (5.0%) children. The odds of G6PD deficiency were 0.38 times as high in Igbo children compared to Yoruba children (p=0.0500). The odds for Igede and Tiv children were not significantly different from Yoruba children (p=0.7528 and 0.9789 respectively). Mean oxygen saturation, heart rate and hematocrit were not significantly different in G6PD deficient and G6PD sufficient children. The odds of being G6PD deficient were 2.1 times higher in children with scleral icterus than those without (p=0.0351). In conclusion, we determined the prevalence of G6PD deficiency in Nigerian sub-populations. The odds of G6PD deficiency were decreased in Igbo children compared to Yoruba children. There was no association between vital parameters or hematocrit and G6PD deficiency. We found that a history of scleral icterus may increase the odds of G6PD deficiency, but we did not exclude other common causes of icterus such as sickle cell disease or malarial infection. PMID:23874768

  17. Prognostic values of aldehyde dehydrogenase 1 isoenzymes in ovarian cancer

    PubMed Central

    Ma, Yu-mei; Zhao, Shan

    2016-01-01

    Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate cancer stem cells in different cancer types, including ovarian cancer. However, which ALDH1’s isoenzymes are contributing to ALDH1 activity in ovarian cancer remains elusive. In addition, the prognostic value of an individual ALDH1 isoenzyme in ovarian cancer is not clear. Thus, we accessed the prognostic value of ALDH1 isoenzymes in ovarian cancer patients through the “Kaplan–Meier plotter” online database, which can be used to determine the effect of the genes on ovarian cancer prognosis. We found that high mRNA expression of five ALDH1 isoenzymes, such as ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, and ALDH1L1, was not correlated with overall survival (OS) for all 1,306 ovarian cancer patients. In addition, all five of the ALDH1 isoenzymes’ high mRNA expression was found to be uncorrelated with OS in serous cancer or endometrioid cancer patients. However, ALDH1A3’s high mRNA expression is associated with worse OS in grade II ovarian cancer patients, hazard ratio (HR) 1.53 (1.14–2.07), P=0.005. ALDH1A2’s high mRNA expression is significantly associated with worse OS in TP53 wild-type ovarian cancer patients, HR 2.86 (1.56–5.08), P=0.00036. In addition, ALDH1A3’s high mRNA expression is significantly associated with better OS in TP53 wild-type ovarian cancer patients, HR 0.56 (0.32–1.00), P=0.04. Our results indicate that although ALDH1 isoenzyme mRNA might not be a prognostic marker for overall ovarian cancer patients, some isoenzymes, such as ALDH1A2 and ALDH1A3, might be a good prognostic marker for some types of ovarian cancer patients. PMID:27110126

  18. Plasma Lactate Dehydrogenase Levels Predict Mortality in Acute Aortic Syndromes

    PubMed Central

    Morello, Fulvio; Ravetti, Anna; Nazerian, Peiman; Liedl, Giovanni; Veglio, Maria Grazia; Battista, Stefania; Vanni, Simone; Pivetta, Emanuele; Montrucchio, Giuseppe; Mengozzi, Giulio; Rinaldi, Mauro; Moiraghi, Corrado; Lupia, Enrico

    2016-01-01

    Abstract In acute aortic syndromes (AAS), organ malperfusion represents a key event impacting both on diagnosis and outcome. Increased levels of plasma lactate dehydrogenase (LDH), a biomarker of malperfusion, have been reported in AAS, but the performance of LDH for the diagnosis of AAS and the relation of LDH with outcome in AAS have not been evaluated so far. This was a bi-centric prospective diagnostic accuracy study and a cohort outcome study. From 2008 to 2014, patients from 2 Emergency Departments suspected of having AAS underwent LDH assay at presentation. A final diagnosis was obtained by aortic imaging. Patients diagnosed with AAS were followed-up for in-hospital mortality. One thousand five hundred seventy-eight consecutive patients were clinically eligible, and 999 patients were included in the study. The final diagnosis was AAS in 201 (20.1%) patients. Median LDH was 424 U/L (interquartile range [IQR] 367–557) in patients with AAS and 383 U/L (IQR 331–460) in patients with alternative diagnoses (P < 0.001). Using a cutoff of 450 U/L, the sensitivity of LDH for AAS was 44% (95% confidence interval [CI] 37–51) and the specificity was 73% (95% CI 69–76). Overall in-hospital mortality for AAS was 23.8%. Mortality was 32.6% in patients with LDH ≥ 450 U/L and 16.8% in patients with LDH < 450 U/L (P = 0.006). Following stratification according to LDH quartiles, in-hospital mortality was 12% in the first (lowest) quartile, 18.4% in the second quartile, 23.5% in the third quartile, and 38% in the fourth (highest) quartile (P = 0.01). LDH ≥ 450 U/L was further identified as an independent predictor of death in AAS both in univariate and in stepwise logistic regression analyses (odds ratio 2.28, 95% CI 1.11–4.66; P = 0.025), in addition to well-established risk markers such as advanced age and hypotension. Subgroup analysis showed excess mortality in association with LDH ≥ 450 U/L in elderly, hemodynamically stable

  19. Journal of the Society of Accelerative Learning and Teaching, 1993.

    ERIC Educational Resources Information Center

    Portes, Pedro R., Ed.

    1993-01-01

    Issue 1 and 2 is a special issue devoted to the cultural-historical school of thought about mental development based on the work of Lev Vygotsky. The four articles that discuss a cross section of issues from the Vygotskyan Paradigm are as follows: "Mind as a Cultural Achievement: Implications for IQ Testing" (Michael Cole); "Children's Social…

  20. Fermi National Acceleator Laboratory Annual Program Review 1992

    SciTech Connect

    Appel, Jeffrey A.; Jovanovic, Drasko; Pordes, Stephen

    1992-01-01

    This book is submitted as a written adjunct to the Annual DOE High Energy Physics Program Review of Fermilab, scheduled this year for March 31 - April 2, 1992. In it are described the functions and activities of the various Laboratory areas plus statements of plans and goals for the coming year.

  1. Journal of the Society for Accelerative Learning and Teaching, 1994.

    ERIC Educational Resources Information Center

    Journal of the Society for Accelerative Learning and Teaching, 1994

    1994-01-01

    Two issues of this serial include the following articles: "Editorial Note for Semiotics Issue" (Pedro Portes); "Qualitative Postmodernism and the Nature of Teaching and Learning" (Gary Shank); "Instructional Prescriptions Can Be Hazardous to Your Pedagogy!" (Donald J. Cunningham, Bruce Allen Knight, and Kathy K. Watson); "Toward a Mutual Interplay…

  2. The Journal of Suggestive-Accelerative Learning and Teaching.

    ERIC Educational Resources Information Center

    Schuster, Donald, Ed.

    1978-01-01

    The following articles are included in these proceedings: (1) "Teaching Remedial Reading with SALT," by Jean Taylor; (2) "Commentary to the 'Cinematographic Report,'" by Joseph Goldin; (3) "Interpretations of the Lozanov Method," by W. Jane Bancroft; (4) "Is a Little SALT a Dangerous Thing?" by Owen L. Caskey; (5) "Problems Related to the…

  3. Crystallization and initial X-ray diffraction analysis of human pyruvate dehydrogenase

    NASA Technical Reports Server (NTRS)

    Ciszak, E.; Korotchkina, L. G.; Hong, Y. S.; Joachimiak, A.; Patel, M. S.

    2001-01-01

    Human pyruvate dehydrogenase (E1) is a component enzyme of the pyruvate dehydrogenase complex. The enzyme catalyzes the irreversible decarboxylation of pyruvic acid and the rate-limiting reductive acetylation of the lipoyl moiety linked to the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. E1 is an alpha(2)beta(2) tetramer ( approximately 154 kDa). Crystals of this recombinant enzyme have been grown in polyethylene glycol 3350 using a vapor-diffusion method at 295 K. The crystals are characterized as orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 64.2, b = 126.9, c = 190.2 A. Crystals diffracted to a minimum d spacing of 2.5 A. The asymmetric unit contains one alpha(2)beta(2) tetrameric E1 assembly; self-rotation function analysis showed a pseudo-twofold symmetry relating the two alphabeta dimers.

  4. Relayed 13C magnetization transfer: Detection of malate dehydrogenase reaction in vivo

    NASA Astrophysics Data System (ADS)

    Yang, Jehoon; Shen, Jun

    2007-02-01

    Malate dehydrogenase catalyzes rapid interconversion between dilute metabolites oxaloacetate and malate. Both oxaloacetate and malate are below the detection threshold of in vivo MRS. Oxaloacetate is also in rapid exchange with aspartate catalyzed by aspartate aminotransferase, the latter metabolite is observable in vivo using 13C MRS. We hypothesized that the rapid turnover of oxaloacetate can effectively relay perturbation of magnetization between malate and aspartate. Here, we report indirect observation of the malate dehydrogenase reaction by saturating malate C2 resonance at 71.2 ppm and detecting a reduced aspartate C2 signal at 53.2 ppm due to relayed magnetization transfer via oxaloacetate C2 at 201.3 ppm. Using this strategy the rate of the cerebral malate dehydrogenase reaction was determined to be 9 ± 2 μmol/g wet weight/min (means ± SD, n = 5) at 11.7 Tesla in anesthetized adult rats infused with [1,6- 13C 2]glucose.

  5. Glutamate dehydrogenase: genetic mapping and isolation of regulatory mutants of Klebsiella aerogenes.

    PubMed Central

    Bender, R A; Macaluso, A; Magasanik, B

    1976-01-01

    The gene for glutamate dehydrogenase (gdhD) has been mapped in Klebsiella aerogenes by P1 transduction. It is linked to pyrF and trp with the order pyrF-trp-gdh. Complementation analysis using F' episomes from Escherichia coli suggests an analogous location in E. coli. Two mutants able to produce glutamate dehydrogenase in the presence of high levels of glutamine synthetase have been isolated. One, tightly linked to gdhD, shows normal repression control by glutamine synthetase but produces four times as much glutamate dehydrogenase activity as does the wild type under all conditions tested. The other revertant is not linked to gdhD or glnA. PMID:6429

  6. Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

    SciTech Connect

    Castro, Miguel E.; Molina, Roberto C.; Diaz, Waldo A.; Pradenas, Gonzalo A.; Vasquez, Claudio C.

    2009-02-27

    Potassium tellurite (K{sub 2}TeO{sub 3}) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.

  7. Cloning and expression of glucose 3-dehydrogenase from Halomonas sp. alpha-15 in Escherichia coli.

    PubMed

    Kojima, K; Tsugawa, W; Sode, K

    2001-03-23

    The gene encoding glucose 3-dehydrogenase (G3DH) from Halomonas sp. alpha-15 was cloned and expressed in Escherichia coli. An open reading frame of 1686 nucleotides was shown to encode G3DH. The flavine adenine dinucleotide binding motif was found in the N-terminal region of G3DH. The deduced primary structure of G3DH showed about 30% identity to sorbitol dehydrogenase from Gluconobacter oxydans and 2-keto-d-gluconate dehydrogenases from Erwinia herbicola and Pantoea citrea. The folding prediction of G3DH suggested that the 3D structure of G3DH was similar with cholesterol oxidase from Brevibacterium sterolicum or glucose oxidase from Aspergillus niger. PMID:11263965

  8. Inactivation of pyruvate dehydrogenase kinase 2 by mitochondrial reactive oxygen species.

    PubMed

    Hurd, Thomas R; Collins, Yvonne; Abakumova, Irina; Chouchani, Edward T; Baranowski, Bartlomiej; Fearnley, Ian M; Prime, Tracy A; Murphy, Michael P; James, Andrew M

    2012-10-12

    Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H(2)O(2)) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H(2)O(2) derives from superoxide (O(2)(.)), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O(2)(.) production, such as may occur under nutrient excess and low ATP demand, the increase in O(2)() and H(2)O(2) may provide feedback signals to modulate mitochondrial metabolism. PMID:22910903

  9. Inactivation of Pyruvate Dehydrogenase Kinase 2 by Mitochondrial Reactive Oxygen Species*

    PubMed Central

    Hurd, Thomas R.; Collins, Yvonne; Abakumova, Irina; Chouchani, Edward T.; Baranowski, Bartlomiej; Fearnley, Ian M.; Prime, Tracy A.; Murphy, Michael P.; James, Andrew M.

    2012-01-01

    Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H2O2) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H2O2 derives from superoxide (O2˙̄), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O2˙̄ production, such as may occur under nutrient excess and low ATP demand, the increase in O2˙̄ and H2O2 may provide feedback signals to modulate mitochondrial metabolism. PMID:22910903

  10. Structural and biochemical insights into 7β-hydroxysteroid dehydrogenase stereoselectivity.

    PubMed

    Savino, Simone; Ferrandi, Erica Elisa; Forneris, Federico; Rovida, Stefano; Riva, Sergio; Monti, Daniela; Mattevi, Andrea

    2016-06-01

    Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo- and regio-selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active-site accessibility, the bases of the specificity for NADP(+) , and the general architecture of the steroid binding site. Comparison with 7α-hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C-terminal extension reshapes the substrate site of the β-selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859-865. © 2016 Wiley Periodicals, Inc. PMID:27006087

  11. Effects of environmental conditions on xylose reductase and xylitol dehydrogenase production by Candida guilliermondii.

    PubMed

    Sene, L; Vitolo, M; Felipe, M G; Silva, S S

    2000-01-01

    The effects of environmental conditions, namely initial pH (2.5-7.0) and temperature (25 and 35 degrees C), on xylose reductase and xylitol dehydrogenase levels, as well as on xylitol production, were evaluated. Although the fermentative parameter values increased with an increase in pH and temperature (the maximum Yp/s and Qp were 0.75 g/g and 0.95 g/[L.h], respectively, both attained at pH 6.0, 35 degrees C), the highest xylose reductase activities (nearly 900 IU/mg of protein) were observed at an initial pH varying from 4.0 to 6.0. Xylitol dehydrogenase was favored by an increase in both initial pH and temperature of the medium. The highest xylitol dehydrogenase specific activity was attained at pH 6.5 and 35 degrees C (577 IU/mg of protein). PMID:10849803

  12. Control of glycolysis by glyceraldehyde-3-phosphate dehydrogenase in Streptococcus cremoris and Streptococcus lactis.

    PubMed Central

    Poolman, B; Bosman, B; Kiers, J; Konings, W N

    1987-01-01

    The decreased response of the energy metabolism of lactose-starved Streptococcus cremoris upon readdition of lactose is caused by a decrease of the glycolytic activity (B. Poolman, E. J. Smid, and W. N. Konings, J. Bacteriol. 169:1460-1468, 1987). The decrease in glycolysis is accompanied by a decrease in the activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase. The steady-state levels of pathway intermediates upon refeeding with lactose after various periods of starvation indicate that the decreased glycolysis is primarily due to diminished glyceraldehyde-3-phosphate dehydrogenase activity. Furthermore, quantification of the control strength exerted by glyceraldehyde-3-phosphate dehydrogenase on the overall activity of the glycolytic pathway shows that this enzyme can be significantly rate limiting in nongrowing cells. PMID:2824452

  13. Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

    PubMed Central

    Henningsen, Brooks M.; Hon, Shuen; Covalla, Sean F.; Sonu, Carolina; Argyros, D. Aaron; Barrett, Trisha F.; Wiswall, Erin; Froehlich, Allan C.

    2015-01-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter−1 acetate during fermentation of 114 g liter−1 glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter−1, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter−1 and raised the ethanol yield to 7% above the wild-type level. PMID:26386051

  14. Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

    PubMed

    Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. PMID:26386051

  15. NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenases from bacteroides fragilis.

    PubMed

    Macdonald, I A; Williams, C N; Mahony, D E; Christie, W M

    1975-03-28

    Twenty strains of Bacteroides fragilis were screened for hydroxysteroid oxidoreductase activity in cell-free preparations. Eighteen strains were shown to contain NAD-dependent 7alpha-hydroxysteroid dehydrogenase. Sixteen of the strains containing the NAD-dependent enzyme also contained NADP-depedent 7alpha-hydroxysteroid dehydrogenase, but invariably in lesser amounts. A strain particulary rich in both 7alpha-hydroxysteroid dehydrogenase activities was selected for further study. Measurement of activity as a function of pH revealed a fairly sharp optimal activity range of 9.5--10.0 for the NAD-dependent enzyme and a broad flat optimal range of 7.0--9.0 for the NADP-dependent enzyme. Michaelis constants for trihydroxy-bile acids for the NAD-dependent enzyme were in the range of 0.32--0.34 mM, whereas dihydroxy-bile acids gave a Km of 0.1 mM. Thin-layer chromatography studies on the oxidation product of 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid) by the dehydrogenase revealed a band corresponding to that of synthetic 3alpha-hydroxy, 7-keto-5beta-cholanoic acid. Similarly the oxidation product of chenodeoxycholic acid by both 7alpha-hydroxysteroid dehydrogenase and commercially available 3alpha-hy-droxysteroid dehydrogenase revealed a band corresponding to that of synthetic 3,7-diketo-5beta-cholanoic acid. Neither of these two oxidation products could be distinguished from those by the Escherichia coli dehydrogenase oxidation previously reported. Disc-gel electrophoresis of a cell-free lyophilized preparation indicated one active band for NAD-dependent activity of mobility similar to that for the NADP-dependent E. coli enzyme. The NADP-dependent dehydrogenase was unstable and rapidly lost activity after polyacylamide disc-gel electrophoresis, ultracentrifugation, freezing on refrigeration at 4 degrees C. No 3 alpha- or 12alpha-oriented oxidoreductase activity was demonstrated in any of the strains examined. PMID:236764

  16. Crystal structures of 11β-hydroxysteroid dehydrogenase type 1 and their use in drug discovery

    PubMed Central

    Thomas, Mark P; Potter, Barry VL

    2014-01-01

    Cortisol is synthesized by 11β-hydroxysteroid dehydrogenase type 1, inhibitors of which may treat disease associated with excessive cortisol levels. The crystal structures of 11β-hydroxysteroid dehydrogenase type 1 that have been released may aid drug discovery. The crystal structures have been analyzed in terms of the interactions between the protein and the ligands. Despite a variety of structurally different inhibitors the crystal structures of the proteins are quite similar. However, the differences are significant for drug discovery. The crystal structures can be of use in drug discovery, but care needs to be taken when selecting structures for use in virtual screening and ligand docking. PMID:21446847

  17. Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

    PubMed

    Baggetto, L G; Lehninger, A L

    1987-05-29

    Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented. PMID:3593337

  18. Affinity purifications of aldose reductase and xylitol dehydrogenase from the xylose-fermenting yeast Pachysolen tannophilus

    SciTech Connect

    Bolen, P.L.; Roth, K.A.; Freer, S.N.

    1986-10-01

    Although xylose is a major product of hydrolysis of lignocellulosic materials, few yeasts are able to convert it to ethanol. In Pachysolen tannophilus, one of the few xylose-fermenting yeasts found, aldose reductase and xylitol dehydrogenase were found to be key enzymes in the metabolic pathway for xylose fermentation. This paper presents a method for the rapid and simultaneous purification of both aldose reductase and xylitol dehydrogenase from P. tannophilus. Preliminary studies indicate that this method may be easily adapted to purify similar enzymes from other xylose-fermenting yeasts.

  19. [Physicochemical, catalytic, and regulatory properties of malate dehydrogenase from Rhodovulum steppense bacteria, strain A-20s].

    PubMed

    Eprintsev, A T; Falaleeva, M I; Parfenova, I V; Liashchenko, M S; Kompantseva, E I; Tret'iakova, A Iu

    2014-01-01

    The physicochemical, regulatory, and kinetic properties of malate dehydrogenase (EC 1.1.1.37) from haloalkaliphilic purple nonsulfur Rhodovulum steppense bacteria, strain A-20s, were studied. The malate dehydrogenase (MDH) preparation with a specific activity of 0.775 ± 0.113 U/mg protein was obtained in an electrophoretically homogeneous state using multistep purification. Using homogenous preparations, the molecular weight and the Michaelis constant of the enzyme were determined; the effects of metal ions, the temperature effect, and the thermal stability of the MDH were studied. The dimer structure of the enzyme was demonstrated by DS-Na-electrophoresis. PMID:25739304

  20. Excitotoxic increase of xanthine dehydrogenase and xanthine oxidase in the rat olfactory cortex.

    PubMed

    Battelli, M G; Buonamici, L; Abbondanza, A; Virgili, M; Contestabile, A; Stirpe, F

    1995-05-26

    Excitotoxic lesions induced by systemic injection of kainic acid, resulted in 2-3-fold increase of xanthine dehydrogenase and xanthine oxidase activities in the rat olfactory cortex 48-72 h after drug administration. A significant increase of the xanthine oxidase/dehydrogenase ratio was also observed at 4 and 48 h post-injection. No similar changes were noticed in the hippocampus. The enhancement of enzyme activity seems to be primarily a consequence of the altered cell composition in damaged area. Free radicals produced by the increased oxygen-dependent form of the enzyme could in turn aggravate the excitotoxic brain injury. PMID:7656426

  1. Simulated ischaemia-reperfusion conditions increase xanthine dehydrogenase and oxidase activities in rat brain slices.

    PubMed

    Battelli, M G; Buonamici, L; Virgili, M; Abbondanza, A; Contestabile, A

    1998-01-01

    Xanthine dehydrogenase and oxidase activities increased by 87% in rat brain slices after 30 min in vitro ischaemia. A further 41% increase was induced by 30 min simulated reperfusion of ischaemic slices. No conversion from the dehydrogenase to the oxidase activity was observed. The increment of enzyme activity was not due to neosynthesis of the enzyme, since it was not affected by the addition of cycloheximide during the ischaemic incubation. The increased oxygen-dependent form of the enzyme could aggravate the ischaemic brain injury by free radicals production, in particular after reperfusion. PMID:9460697

  2. Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases.

    PubMed

    Zhou, Meixia; Crawford, Yongping; Ng, Domingos; Tung, Jack; Pynn, Abigail F J; Meier, Angela; Yuk, Inn H; Vijayasankaran, Natarajan; Leach, Kimberly; Joly, John; Snedecor, Bradley; Shen, Amy

    2011-04-20

    Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors. PMID:21392546

  3. Functional Replacement of the Escherichia coli d-(−)-Lactate Dehydrogenase Gene (ldhA) with the l-(+)-Lactate Dehydrogenase Gene (ldhL) from Pediococcus acidilactici†

    PubMed Central

    Zhou, Shengde; Shanmugam, K. T.; Ingram, L. O.

    2003-01-01

    The microbial production of l-(+)-lactic acid is rapidly expanding to allow increased production of polylactic acid (PLA), a renewable, biodegradable plastic. The physical properties of PLA can be tailored for specific applications by controlling the ratio of l-(+) and d-(−) isomers. For most uses of PLA, the l-(+) isomer is more abundant. As an approach to reduce costs associated with biocatalysis (complex nutrients, antibiotics, aeration, product purification, and waste disposal), a recombinant derivative of Escherichia coli W3110 was developed that contains five chromosomal deletions (focA-pflB frdBC adhE ackA ldhA). This strain was constructed from a d-(−)-lactic acid-producing strain, SZ63 (focA-pflB frdBC adhE ackA), by replacing part of the chromosomal ldhA coding region with Pediococcus acidilactici ldhL encoding an l-lactate dehydrogenase. Although the initial strain (SZ79) grew and fermented poorly, a mutant (SZ85) was readily isolated by selecting for improved growth. SZ85 exhibited a 30-fold increase in l-lactate dehydrogenase activity in comparison to SZ79, functionally replacing the native d-lactate dehydrogenase activity. Sequencing revealed mutations in the upstream, coding, and terminator regions of ldhL in SZ85, which are presumed to be responsible for increased l-lactate dehydrogenase activity. SZ85 produced l-lactic acid in M9 mineral salts medium containing glucose or xylose with a yield of 93 to 95%, a purity of 98% (based on total fermentation products), and an optical purity greater than 99%. Unlike other recombinant biocatalysts for l-lactic acid, SZ85 remained prototrophic and is devoid of plasmids and antibiotic resistance genes. PMID:12676706

  4. Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal g...

  5. Analysis of Quaternary Structure of a [LDH-like] Malate Dehydrogenase of Plasmodium falciparum with Oligomeric Mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L-lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archea and other prokaryotes. Initial sequence a...

  6. Identification and analysis of the genes coding for the putative pyruvate dehydrogenase enzyme complex in Acholeplasma laidlawii.

    PubMed Central

    Wallbrandt, P; Tegman, V; Jonsson, B H; Wieslander, A

    1992-01-01

    A monospecific antibody recognizing two membrane proteins in Acholeplasma laidlawii identified a plasmid clone from a genomic library. The nucleotide sequence of the 4.6-kbp insert contained four sequential genes coding for proteins of 39 kDa (E1 alpha, N terminus not cloned), 36 kDa (E1 beta), 57 kDa (E2), and 36 kDa (E3; C terminus not cloned). The N termini of the cloned E2, E1 beta, and native A. laidlawii E2 proteins were verified by amino acid sequencing. Computer-aided searches showed that the translated DNA sequences were homologous to the four subenzymes of the pyruvate dehydrogenase complexes from gram-positive bacteria and humans. The plasmid-encoded 57-kDa (E2) protein was recognized by antibodies against the E2 subenzymes of the pyruvate and oxoglutarate dehydrogenase complexes from Bacillus subtilis. A substantial fraction of the E2 protein as well as part of the pyruvate dehydrogenase enzymatic activity was associated with the cytoplasmic membrane in A. laidlawii. In vivo complementation with three different Escherichia coli pyruvate dehydrogenase-defective mutants showed that the four plasmid-encoded proteins were able to restore pyruvate dehydrogenase enzyme activity in E. coli. Since A. laidlawii lacks oxoglutarate dehydrogenase and most likely branched-chain dehydrogenase enzyme complex activities, these results strongly suggest that the sequenced genes code for the pyruvate dehydrogenase complex. Images PMID:1735725

  7. Biochemical and Structural Studies of Uncharacterized Protein PA0743 from Pseudomonas aeruginosa Revealed NAD+-dependent l-Serine Dehydrogenase*

    PubMed Central

    Tchigvintsev, Anatoli; Singer, Alexander; Brown, Greg; Flick, Robert; Evdokimova, Elena; Tan, Kemin; Gonzalez, Claudio F.; Savchenko, Alexei; Yakunin, Alexander F.

    2012-01-01

    The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD+-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2–2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD+ complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases. PMID:22128181

  8. A potent specific inhibitor of 6-phosphogluconate dehydrogenase of Cryptococcus neoformans and of certain other fungal enzymes.

    PubMed

    Niehaus, W G; Flynn, T

    1993-09-01

    A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases from Cryptococcus neoformans and Schizophyllum commune and glucose-6-phosphate dehydrogenase from Schizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than the Cryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent. PMID:8302365

  9. Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum.

    PubMed Central

    Straub, P F; Reynolds, P H; Althomsons, S; Mett, V; Zhu, Y; Shearer, G; Kohl, D H

    1996-01-01

    We report here the cloning and sequencing of the gene for proline dehydrogenase (putA) of Bradyrhizobium japonicum. An open reading frame coding for 1,016 amino acids was identified. The B. japonicum gene codes for a bifunctional protein with proline dehydrogenase and pyrroline-5-carboxylate (P5C) dehydrogenase activities, as it does in Escherichia coli and Salmonella typhimurium. Comparison of the sequences of these proteins with other proline and P5C dehydrogenase sequences identified proline dehydrogenase and P5C dehydrogenase catalytic domains. Within the proline dehydrogenation domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Saccharomyces cerevisiae put1, and Drosophila melanogaster slgA. Within the P5C dehydrogenase domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Bacillus subtilis ipa76d, and S. cerevisiae put2. A consensus catalytic site for semialdehyde dehydrogenase was observed in the P5C dehydrogenase domain. This suggests that the substrate for this domain may be the open-chain gamma-glutamylsemialdehyde, not its cyclized form, P5C. Unlike the gene isolated from E. coli, S. typhimurium, and K. pneumoniae, the B. japonicum putA gene does not appear to be part of an operon with the proline porter gene (putP). Additionally, the B. japonicum gene lacks the putative C-terminal regulatory domain present in the E. coli and S. typhimurium genes. The gene was disrupted by insertion of antibiotic resistance gene cassettes, which were then recombined into the bacterial chromosome. Symbiotically active mutant strains that were devoid of putA activity were isolated. With this proline dehydrogenase clone, we will test the hypothesis that putA in symbiotic nitrogen-fixing B. japonicum bacteroids is transcriptionally regulated by drought and other stresses. PMID:8572700

  10. Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154→Cys forms of yeast xylitol dehydrogenase

    PubMed Central

    Klimacek, Mario; Hellmer, Heidemarie; Nidetzky, Bernd

    2007-01-01

    Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10–0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19±0.03 s−1 and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019±0.003% and 0.74±0.03% of wild-type catalytic efficiency (kcat/Ksorbitol=7800±700 M−1· s−1) and kcat (=161±4 s−1) for NAD+-dependent oxidation of sorbitol at 25 °C respectively. The pH profile of kcat/Ksorbitol for E154C decreased below an apparent pK of 9.1±0.3, reflecting a shift in pK by about +1.7–1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (≈+0.2 log units), suggesting that the observed pK in the binary enzyme–NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7±0.2 (E154C, 1.7±0.1) and 1.9±0.3 (E154C, 2.4±0.2) on kcat/Ksorbitol respectively. Transient kinetic studies of NAD+ reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687±12 s−1 in the pre-steady state, which features a turnover of 0.9±0.1 enzyme equivalents as NADH was produced with a rate constant of 409±3 s−1. The

  11. Structural aspects of the dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila.

    PubMed Central

    Bamforth, C W; Quayle, J R

    1979-01-01

    1. A dye-linked alcohol dehydrogenase was purified 60-fold from extracts of Rhodopseudomonas acidophila 10050 grown aerobically on ethanol. 2. The properties of this enzyme were identical with those of the alcohol dehydrogenase synthesized by this organism during growth on methanol anaerobically in the light, and they are judged to be the same enzyme. 3. The enzyme gave a single protein band, coincident with alcohol dehydrogenase activity, during electrophoresis on polyacrylamide gel. 4. The amino acid composition, ioselectric point, u.v. and visible absorption spectra of the enzyme were determined and compared with those of other similar enzymes. 5. The presence of 0.7--1.0 g-atom of non-haem, acidlabile iron/mol of enzyme was shown by atomic absorption spectrophotometry and colorimetric assay. The iron could not be dissociated from the enzyme by dialysis against chelating agents. 6. E.p.r. spectroscopy of the enzyme did not indicate any redox function for the iron during alcohol dehydrogenation, but showed a signal at g = 2.00 consistent with the presence of a protein-bound organic free radical. 8. Antisera were raised against alcohol (methanol) dehydrogenases purified from Rhodopseudomonas acidophila, Paracoccus denitrificans and Methylophilus methylotrophus. 9. The antiserum to the Rhodopseudomonas acidophila enzyme cross-reacted with neither of the two other antisera, nor with crude extracts of methanol-grown Hyphomicrobium X and Pseudomonas AM1, thus emphasizing its singular biochemical properties. PMID:229820

  12. Modification of Rhizopus lactate dehydrogenase for improved resistance to fructose 1,6-bisphosphate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizopus oryzae is frequently used for fermentative production of lactic acid. We determined that one of the key enzymes, lactate dehydrogenase (LDH), involved in synthesis of lactic acid by R. oryzae was significantly inhibited by fructose 1,6-bisphosphate (FBP) at physiological concentrations. Thi...

  13. Relationship of lactate dehydrogenase activity to body measurements of Angus x Charolais cows and calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives were to examine 1) relationships between lactate dehydrogenase (LDH) activity and body measurements of grazing beef cows, and 2) the association between maternal LDH activity in late gestation and subsequent calf birth weight (BRW), hip height (HH) at weaning, and adjusted weaning weight ...

  14. Role of Alanine Dehydrogenase of Mycobacterium tuberculosis during Recovery from Hypoxic Nonreplicating Persistence

    PubMed Central

    Giffin, Michelle M.; Shi, Lanbo; Gennaro, Maria L.; Sohaskey, Charles D.

    2016-01-01

    Mycobacterium tuberculosis can maintain a nonreplicating persistent state in the host for decades, but must maintain the ability to efficiently reactivate and produce active disease to survive and spread in a population. Among the enzymes expressed during this dormancy is alanine dehydrogenase, which converts pyruvate to alanine, and glyoxylate to glycine concurrent with the oxidation of NADH to NAD. It is involved in the metabolic remodeling of M. tuberculosis through its possible interactions with both the glyoxylate and methylcitrate cycle. Both mRNA levels and enzymatic activities of isocitrate lyase, the first enzyme of the glyoxylate cycle, and alanine dehydrogenase increased during entry into nonreplicating persistence, while the gene and activity for the second enzyme of the glyoxylate cycle, malate synthase were not. This could suggest a shift in carbon flow away from the glyoxylate cycle and instead through alanine dehydrogenase. Expression of ald was also induced in vitro by other persistence-inducing stresses such as nitric oxide, and was expressed at high levels in vivo during the initial lung infection in mice. Enzyme activity was maintained during extended hypoxia even after transcription levels decreased. An ald knockout mutant of M. tuberculosis showed no reduction in anaerobic survival in vitro, but resulted in a significant lag in the resumption of growth after reoxygenation. During reactivation the ald mutant had an altered NADH/NAD ratio, and alanine dehydrogenase is proposed to maintain the optimal NADH/NAD ratio during anaerobiosis in preparation of eventual regrowth, and during the initial response during reoxygenation. PMID:27203084

  15. Alcohol dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecanse and hexadecanol metabolism

    SciTech Connect

    Singer, M.E.; Finnerty, W.R.

    1985-12-01

    Multiple alcohol dehydrogenases (ADH) were demonstrated in Acinetobacter sp. strain HO1-N. ADH-A and ADH-B were distinguished on the basis of electrophoretic mobility, pyridine nucleotide cofactor requirement, and substrate specificity. ADH-A is a soluble, NAD-linked, inducible ethanol dehydrogenase (EDH). An ethanol-negative mutant (Eth1) was isolated which contained 6.5% of wild-type EDH activity and was deficient in ADH-A. Eth1 exhibited normal growth on hexadecane and hexadecanol. A second ethanol-negative mutant (Eth3) was acetaldehyde dehydrogenase (ALDH) deficient, having 12.5% of wild-type ALDH activity. Eth3 had threefold-higher EDH activity than the wild-type strain. ALDH is a soluble, NAD-linked, ethanol-inducible enzyme. Eth3 exhibited normal growth on hexadecane, hexadecanol, and fatty aldehyde. ADH-B is soluble, constitutive, NADP-linked ADH which was active with medium-chain-length alcohols. Hexadecanol dehydrogenase (HDH), a soluble and membrane-bound, NAD-linked ADH, was induced 5- to 11-fold by growth on hexadecane or hexadecanol. HDH was distinct from ADH-A and ADH-B. NAD-linked HDH appears to possess a functional role in hexadecane and hexadecanol dissimilation.

  16. Subcellular Localization and Biochemical Comparison of Cytosolic and Secreted Cytokinin Dehydrogenase Enzymes from Maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytokinin dehydrogenase (CKX, EC 1.5.99.12) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in cells of each plant. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a t...

  17. ISOZYME PROFILES OF LACTIC DEHYDROGENASE AND CREATINE PHOSPHOKINASE IN NEONATAL MOUSE HEARTS

    EPA Science Inventory

    Isozyme profiles of lactic dehydrogenase (LDH) and creatine phosphokinase (CPK) were determined in cardiac tissue of mice during postnatal development. LDH isozymes 1 and 5 showed a definite developmental change, achieving the adult values by 20 days of age, while the other three...

  18. Appearance of Novel Glucose-6-Phosphate Dehydrogenase Isoforms in Chlamydomonas reinhardtii during Growth on Nitrate.

    PubMed Central

    Huppe, H. C.; Turpin, D. H.

    1996-01-01

    Extractable glucose-6-phosphate dehydrogenase activity is higher from N-limited Chlamydomonas reinhardtii cells than from N-sufficient cells. Native gels reveal that the isoform complexity varies depending on the form of N supplied. The isoforms associated with NO3- growth appear within 2 h of switching cells from NH4+ to NO3-. PMID:12226271

  19. Relationship of lactate dehydrogenase activity with body measeurements of Angus x Charolais cows and calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Angus x Charolais cows (n = 87) and their Angus-sired, spring-born calves (n = 86) were utilized to examine relationships between lactate dehydrogenase (LDH) activity and body measurements of beef cows; and the relationship between maternal LDH activity in late gestation and subsequent calf birth we...

  20. EXPRESSION OF THE SPERMATOGENIC CELL-SPECIFIC GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDS) IN RAT TESTIS

    EPA Science Inventory

    The spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (GAPDS) has been cloned from a rat testis cDNA library and its pattern of expression determined. A 1417 nucleotide cDNA has been found to encode an enzyme with substantial homology to mouse GAPDS...

  1. Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignin is a significant recalcitrant in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired...

  2. INFLUENCE OF ENHANCED MALATE DEHYDROGENASE EXPRESSION BY ALFALFA ON DIVERSITY OF RHIZOBACTERIA AND SOIL NUTRIENT AVAILABILITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic alfalfa over-expressing a nodule-enhanced malate dehydrogenase (neMDH) cDNA and untransformed alfalfa plants were grown at the same field site and rhizosphere soils collected after 53 weeks of plant growth. These alfalfa lines differ in the amount and composition of root organic acids pro...

  3. Threonine-Insensitive Homoserine Dehydrogenase From Soybean: Genomic Organization, Kinetic Mechanism, and In vivo Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspartate kinase (AK) and homoserine dehydrogenase (HSD) functions as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback inhibited by threonine. In plants, the biochemical properties of AK and bifunctional AK-HSD enzymes have been characterized, but the mol...

  4. Multiple transcripts encode glucose 6-phosphate dehydrogenase in the southern cattle tick, Rhipicephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucose 6-phosphate dehydrogenase (G6PDH) is an enzyme that plays a critical role in the production of NADPH. Here we describe the identification of four transcripts (G6PDH-A, -B, -C, and -D) that putatively encode the enzyme in the southern cattle tick, Rhipicephalus (Boophilus) microplus. The geno...

  5. Direct Electrochemical Addressing of Immobilized Alcohol Dehydrogenase for the Heterogeneous Bioelectrocatalytic Reduction of Butyraldehyde to Butanol

    PubMed Central

    Schlager, S; Neugebauer, H; Haberbauer, M; Hinterberger, G; Sariciftci, N S

    2015-01-01

    Modified electrodes using immobilized alcohol dehydrogenase enzymes for the efficient electroreduction of butyraldehyde to butanol are presented as an important step for the utilization of CO2-reduction products. Alcohol dehydrogenase was immobilized, embedded in an alginate–silicate hybrid gel, on a carbon felt (CF) electrode. The application of this enzyme to the reduction of an aldehyde to an alcohol with the aid of the coenzyme nicotinamide adenine dinucleotide (NADH), in analogy to the final step in the natural reduction cascade of CO2 to alcohol, has been already reported. However, the use of such enzymatic reductions is limited because of the necessity of providing expensive NADH as a sacrificial electron and proton donor. Immobilization of such dehydrogenase enzymes on electrodes and direct pumping of electrons into the biocatalysts offers an easy and efficient way for the biochemical recycling of CO2 to valuable chemicals or alternative synthetic fuels. We report the direct electrochemical addressing of immobilized alcohol dehydrogenase for the reduction of butyraldehyde to butanol without consumption of NADH. The selective reduction of butyraldehyde to butanol occurs at room temperature, ambient pressure and neutral pH. Production of butanol was detected by using liquid-injection gas chromatography and was estimated to occur with Faradaic efficiencies of around 40 %. PMID:26113881

  6. Genetics Home Reference: short-chain acyl-CoA dehydrogenase deficiency

    MedlinePlus

    ... Download PDF Open All Close All Description Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is a condition that prevents the body from converting certain fats into energy, especially during periods without food (fasting). Signs and symptoms of SCAD deficiency may ...

  7. Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties, activity, and expression during tuber dormancy progression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in meristems isolated from field-g...

  8. Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation dr...

  9. Purification and Characterization of the Bifunctional Enzyme Lysine-Ketoglutarate Reductase-Saccharopine Dehydrogenase from Maize.

    PubMed Central

    Goncalves-Butruille, M.; Szajner, P.; Torigoi, E.; Leite, A.; Arruda, P.

    1996-01-01

    The first enzyme of the lysine degradation pathway in maize (Zea mays L.), lysine-ketoglutarate reductase, condenses lysine and [alpha]-ketoglutarate into saccharopine using NADPH as a cofactor, whereas the second, saccharopine dehydrogenase, converts saccharopine to [alpha]-aminoadipic-[delta]-semialdehyde and glutamic acid using NAD+ or NADP+ as a cofactor. The reductase and dehydrogenase activities are optimal at pH 7.0 and 9.0, respectively. Both enzyme activities, co-purified on diethylaminoethyl-cellulose and gel filtration columns, were detected on nondenaturing polyacrylamide gels as single bands with identical electrophoretic mobilities and share tissue specificity for the endosperm. The highly purified preparation containing the reductase and dehydrogenase activities showed a single polypeptide band of 125 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native form of the enzyme is a dimer of 260 kD. Limited proteolysis with elastase indicated that lysine-ketoglutarate reductase and saccharopine dehydrogenase from maize endosperm are located in two functionally independent domains of a bifunctional polypeptide. PMID:12226216

  10. Oxidation of an Adjacent Methionine Residue Inhibits Regulatory Seryl-phosphorylation of Pyruvate Dehydrogenase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A Met residue is located adjacent to phosphorylation site 1 in the sequences of mitochondrial pyruvate dehydrogenase E1alpha subunits. When synthetic peptides including site 1 were treated with Hydrogen peroxide, the Met residue was oxidized to methionine sulfoxide (MetSO), and the peptides were no...

  11. Effects of a 3β-hydroxysteroid Dehydrogenase Inhibitor, Trilostane, on the Fathead Minnow Reproductive Axis

    EPA Science Inventory

    A number of environmental contaminants and plant flavonoid compounds have been shown to inhibit the activity of 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD). Because 3β-HSD plays a critical role in steroid hormone synthesis, inhibition of 3β-HSD represents a potentia...

  12. Increasing the heme-dependent respiratory efficiency of Lactococcus lactis by inhibition of lactate dehydrogenase.

    PubMed

    Arioli, Stefania; Zambelli, Daniele; Guglielmetti, Simone; De Noni, Ivano; Pedersen, Martin B; Pedersen, Per Dedenroth; Dal Bello, Fabio; Mora, Diego

    2013-01-01

    The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism. PMID:23064338

  13. Often Ignored Facts about the Control of the 2-Oxoglutarate Dehydrogenase Complex

    ERIC Educational Resources Information Center

    Strumilo, Slawomir

    2005-01-01

    Information about the control of the activity of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme in the citric acid cycle, is not well covered in the biochemical education literature, especially as it concerns the allosteric regulation of OGDHC by adenine nucleotide and ortophosphate. From experimental work published during the last…

  14. EXPRESSION AND ASSEMBLY OF ARABIDOPSIS THALIANA PYRUVATE DEHYDROGENASE IN INSECT CELL CYTOPLASM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct, pDDR101, comprises the mature-E1alpha coding sequence under control of the polh promoter, plus the mature-E1beta coding sequence under ...

  15. Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum.

    PubMed Central

    Palosaari, N R; Rogers, P

    1988-01-01

    The coenzyme A (CoA)-linked butyraldehyde dehydrogenase (BAD) from Clostridium acetobutylicum was characterized and purified to homogeneity. The enzyme was induced over 200-fold, coincident with a shift from an acidogenic to a solventogenic fermentation, during batch culture growth. The increase in enzyme activity was found to require new protein synthesis since induction was blocked by the addition of rifampin and antibody against the purified enzyme showed the appearance of enzyme antigen beginning at the shift of the fermentation and increasing coordinately with the increase in enzyme specific activity. The CoA-linked acetaldehyde dehydrogenase was copurified with BAD during an 89-fold purification, indicating that one enzyme accounts for the synthesis of the two aldehyde intermediates for both butanol and ethanol production. Butanol dehydrogenase activity was clearly separate from the BAD enzyme activity on TEAE cellulose. A molecular weight of 115,000 was determined for the native enzyme, and the enzyme subunit had a molecular weight of 56,000 indicating that the active form is a homodimer. Kinetic constants were determined in both the forward and reverse directions. In the reverse direction both the Vmax and the apparent affinity for butyraldehyde and caproaldehyde were significantly greater than they were for acetaldehyde, while in the forward direction, the Vmax for butyryl-CoA was fivefold that for acetyl-CoA. These and other properties of BAD indicate that this enzyme is distinctly different from other reported CoA-dependent aldehyde dehydrogenases. Images PMID:3384801

  16. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and...

  17. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and...

  18. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and...

  19. The crystallographic structure of the mannitol 2-dehydrogenase NADP+ binary complex from Agaricus bisporus.

    PubMed

    Hörer, S; Stoop, J; Mooibroek, H; Baumann, U; Sassoon, J

    2001-07-20

    Mannitol, an acyclic six-carbon polyol, is one of the most abundant sugar alcohols occurring in nature. In the button mushroom, Agaricus bisporus, it is synthesized from fructose by the enzyme mannitol 2-dehydrogenase (MtDH; EC ) using NADPH as a cofactor. Mannitol serves as the main storage carbon (up to 50% of the fruit body dry weight) and plays a critical role in growth, fruit body development, osmoregulation, and salt tolerance. Furthermore, mannitol dehydrogenases are being evaluated for commercial mannitol production as alternatives to the less efficient chemical reduction of fructose. Given the importance of mannitol metabolism and mannitol dehydrogenases, MtDH was cloned into the pET28 expression system and overexpressed in Escherichia coli. Kinetic and physicochemical properties of the recombinant enzyme are indistinguishable from the natural enzyme. The crystal structure of its binary complex with NADP was solved at 1.5-A resolution and refined to an R value of 19.3%. It shows MtDH to be a tetramer and a member of the short chain dehydrogenase/reductase family of enzymes. The catalytic residues forming the so-called catalytic triad can be assigned to Ser(149), Tyr(169), and Lys(173). PMID:11335726

  20. Biochemical characterization of uronate dehydrogenases from three Pseudomonads, Chromohalobacter salixigens, and Polaromonas naphthalenivorans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzyme catalysts will be vital in the development of synthetic biology approaches for converting pectinic monosaccharides from citrus and beet processing waste streams to value-added materials. We describe here the biophysical and mechanistic characterization of uronate dehydrogenases from a wide va...

  1. Characterization of uronate dehydrogenases catalysing the initial step in an oxidative pathway

    PubMed Central

    Pick, André; Schmid, Jochen; Sieber, Volker

    2015-01-01

    Uronate dehydrogenases catalyse the oxidation of uronic acids to aldaric acids, which represent ‘top value-added chemicals’ that have the potential to substitute petroleum-derived chemicals. The identification and annotation of three uronate dehydrogenases derived from Fulvimarina pelagi HTCC2506, Streptomyces viridochromogenes DSM 40736 and Oceanicola granulosus DSM 15982 via sequence analysis is described. Characterization and comparison with two known uronate dehydrogenases in regard to substrate spectrum, catalytic activity and pH as well as temperature dependence was performed. The catalytic efficiency was investigated in two different buffer systems; potassium phosphate and Tris-HCl. In addition to the typical and well available substrates glucuronate and galacturonate also mannuronate as part of many structural polysaccharides were tested. The uronate dehydrogenase of Agrobacterium tumefaciens and Pseudomonas syringae showed catalytic dependency on the buffer system resulting in an increased Km especially for glucuronate in potassium phosphate compared with Tris-HCl buffer. Enzyme stability at 37°C of the different Udhs was in the order: P. syringae < S. viridochromogens < A. tumefaciens < F. pelagi < O. granulosus. All enzymes showed activity within a broad pH range from 7.0 to 9.5, only O. granulosus had a very narrow range around 7.0. PMID:25884328

  2. Role of Alanine Dehydrogenase of Mycobacterium tuberculosis during Recovery from Hypoxic Nonreplicating Persistence.

    PubMed

    Giffin, Michelle M; Shi, Lanbo; Gennaro, Maria L; Sohaskey, Charles D

    2016-01-01

    Mycobacterium tuberculosis can maintain a nonreplicating persistent state in the host for decades, but must maintain the ability to efficiently reactivate and produce active disease to survive and spread in a population. Among the enzymes expressed during this dormancy is alanine dehydrogenase, which converts pyruvate to alanine, and glyoxylate to glycine concurrent with the oxidation of NADH to NAD. It is involved in the metabolic remodeling of M. tuberculosis through its possible interactions with both the glyoxylate and methylcitrate cycle. Both mRNA levels and enzymatic activities of isocitrate lyase, the first enzyme of the glyoxylate cycle, and alanine dehydrogenase increased during entry into nonreplicating persistence, while the gene and activity for the second enzyme of the glyoxylate cycle, malate synthase were not. This could suggest a shift in carbon flow away from the glyoxylate cycle and instead through alanine dehydrogenase. Expression of ald was also induced in vitro by other persistence-inducing stresses such as nitric oxide, and was expressed at high levels in vivo during the initial lung infection in mice. Enzyme activity was maintained during extended hypoxia even after transcription levels decreased. An ald knockout mutant of M. tuberculosis showed no reduction in anaerobic survival in vitro, but resulted in a significant lag in the resumption of growth after reoxygenation. During reactivation the ald mutant had an altered NADH/NAD ratio, and alanine dehydrogenase is proposed to maintain the optimal NADH/NAD ratio during anaerobiosis in preparation of eventual regrowth, and during the initial response during reoxygenation. PMID:27203084

  3. Polyethylene glycol-induced heteroassociation of malate dehydrogenase and citrate synthase

    SciTech Connect

    Merz, J.M.; Webster, T.A.; Appleman, J.R.; Manley, E.R.; Yu, H.A.; Datta, A.; Ackerson, B.J.; Spivey, H.O.

    1987-10-01

    Studies by dynamic and total intensity light scattering, ultracentrifugation, electron microscopy, and chemical crosslinking on solutions of the pig heart mitochondrial enzymes, malate dehydrogenase and citrate synthase (separately and together) demonstrate that polyethylene glycol induces very large homoassociations of each enzyme, and still larger heteroenzyme complexes between these two enzymes in the solution phase. Specificity of this heteroassociation is indicated by the facts that heteroassociations with bovine serum albumin were not observed for either the mitochondrial dehydrogenase or the synthase or between cytosolic malate dehydrogenase and citrate synthase. The weight fraction of the enzymes in the mitochondrial dehydrogenase-synthase associated particles in the solution phase was less than 0.03% with the dilute conditions used in the dynamic light scattering measurements. Neither palmitoyl-CoA nor other solution conditions tested significantly increased this weight fraction of associated enzymes in the solution phase. Because of the extremely low solubility of the associated species, however, the majority of the enzymes can be precipitated as the heteroenzyme complex. This precipitation is a classical first-order transition in spite of the large particle sizes and broad size distribution. Ionic effects on the solubility of the heteroenzyme complex appear to be of general electrostatic nature. Polyethylene glycol was found to be more potent in precipitating this complex than dextrans, polyvinylpyrrolidones, ficoll, and beta-lactoglobulin.

  4. Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Morgan, Chad; Moir, Neil

    1996-11-01

    A rapid microscale procedure has been developed for the isolation and purification of yeast alcohol dehydrogenase. Glass beads are used for cytolysis, PEG precipitation for partial purification and a cibacron blue affinity column for the final step. A 27.5 fold purification can be achieved in 2 - 3 hours.

  5. Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

    ERIC Educational Resources Information Center

    Nash, Barbara T.

    2007-01-01

    SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

  6. Expression of Cellobiose Dehydrogenase from Neurospora crassa in Pichia pastoris and its purification and characterization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A gene encoding cellobiose dehydrogenase (CDH) from Neurospora crassa strain FGSC 2489 has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. Recombinant CDH without the native signal sequence and fused with a his6-tag (rNC-...

  7. Structural and transcriptional analysis of plant genes encoding the bifunctional lysine ketoglutarate reductase saccharopine dehydrogenase enzyme

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The analysis of a wheat lysine ketoglutarate reductase – saccharopine dehydrogenase (LKR/SDH) gene and comparative structural and functional analyses among available plant genes provides new information on this important gene. Both the structure of the LKR/SDH gene and the immediately adjacent genes...

  8. Amino ketone formation and aminopropanol-dehydrogenase activity in rat-liver preparations

    PubMed Central

    Turner, J. M.; Willetts, A. J.

    1967-01-01

    1. Rat tissue homogenates convert dl-1-aminopropan-2-ol into aminoacetone. Liver homogenates have relatively high aminopropanol-dehydrogenase activity compared with kidney, heart, spleen and muscle preparations. 2. Maximum activity of liver homogenates is exhibited at pH9·8. The Km for aminopropanol is approx. 15mm, calculated for a single enantiomorph, and the maximum activity is approx. 9mμmoles of aminoacetone formed/mg. wet wt. of liver/hr.at 37°. Aminoacetone is also formed from l-threonine, but less rapidly. An unidentified amino ketone is formed from dl-4-amino-3-hydroxybutyrate, the Km for which is approx. 200mm at pH9·8. 3. Aminopropanol-dehydrogenase activity in homogenates is inhibited non-competitively by dl-3-hydroxybutyrate, the Ki being approx. 200mm. EDTA and other chelating agents are weakly inhibitory, and whereas potassium chloride activates slightly at low concentrations, inhibition occurs at 50–100mm. 4. It is concluded that aminopropanol-dehydrogenase is located in mitochondria, and in contrast with l-threonine dehydrogenase can be readily solubilized from mitochondrial preparations by ultrasonic treatment. 5. Soluble extracts of disintegrated mitochondria exhibit maximum aminopropanol-dehydrogenase activity at pH9·1 At this pH, Km values for the amino alcohol and NAD+ are approx. 200 and 1·3mm respectively. Under optimum conditions the maximum velocity is approx. 70mμmoles of aminoacetone formed/mg. of protein/hr. at 37°. Chelating agents and thiol reagents appear to have little effect on enzyme activity, but potassium chloride inhibits at all concentrations tested up to 80mm. dl-3-Hydroxybutyrate is only slightly inhibitory. 6. Dehydrogenase activities for l-threonine and dl-4-amino-3-hydroxybutyrate appear to be distinct from that for aminopropanol. 7. Intraperitoneal injection of aminopropanol into rats leads to excretion of aminoacetone in the urine. Aminoacetone excretion proportional to the amount of the amino alcohol

  9. Biochemical and molecular characterization of the Clostridium magnum acetoin dehydrogenase enzyme system.

    PubMed Central

    Krüger, N; Oppermann, F B; Lorenzl, H; Steinbüchel, A

    1994-01-01

    E2 (dihydrolipoamide acetyltransferase) and E3 (dihydrolipoamide dehydrogenase) of the Clostridium magnum acetoin dehydrogenase enzyme system were copurified in a three-step procedure from acetoin-grown cells. The denatured E2-E3 preparation comprised two polypeptides with M(r)s of 49,000 and 67,000, respectively. Microsequencing of both proteins revealed identical amino acid sequences. By use of oligonucleotide probes based on the N-terminal sequences of the alpha and beta subunits of E1 (acetoin dehydrogenase, thymine PPi dependent), which were purified recently (H. Lorenzl, F.B. Oppermann, B. Schmidt, and A. Steinbüchel, Antonie van Leeuwenhoek 63:219-225, 1993), and of E2-E3, structural genes acoA (encoding E1 alpha), acoB (encoding E1 beta), acoC (encoding E2), and acoL (encoding E3) were identified on a single ClaI restriction fragment and expressed in Escherichia coli. The nucleotide sequences of acoA (978 bp), acoB (999 bp), acoC (1,332 bp), and acoL (1,734 bp), as well as those of acoX (996 bp) and acoR (1,956 bp), were determined. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 35,532), E1 beta (M(r), 35,541), E2 (M(r), 48,149), and E3 (M(r), 61,255) exhibited striking similarities to the amino acid sequences of the corresponding components of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system and the Alcaligenes eutrophus acetoin-cleaving system, respectively. Significant homologies to the enzyme components of various 2-oxo acid dehydrogenase complexes were also found, indicating a close relationship between the two enzyme systems. As a result of the partial repetition of the 5' coding region of acoC into the corresponding part of acoL, the E3 component of the C. magnum acetoin dehydrogenase enzyme system contains an N-terminal lipoyl domain, which is unique among dihydrolipoamide dehydrogenases. We found strong similarities between the AcoR and AcoX sequences and the A. eutrophus acoR gene product

  10. Re-design of Saccharomyces cerevisiae flavocytochrome b2: introduction of L-mandelate dehydrogenase activity.

    PubMed

    Sinclair, R; Reid, G A; Chapman, S K

    1998-07-01

    Flavocytochrome b2 from Saccharomyces cerevisiae is an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate. The double mutation of Ala-198-->Gly and Leu-230-->Ala results in an enzyme with a kcat value (25 degrees C) with L-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with L-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of L-mandelate at the active site of the enzyme. To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme. In addition, the double mutation of Ala-198-->Gly and Leu-230-->Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with L-mandelate than with l-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase. PMID:9639570

  11. Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

    PubMed

    Broadbent, Jeffery R; Gummalla, Sanjay; Hughes, Joanne E; Johnson, Mark E; Rankin, Scott A; Drake, Mary Anne

    2004-08-01

    Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that alpha-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development. PMID:15294819

  12. Chemical modification of aldehyde dehydrogenase by a vinyl ketone analogue of an insect pheromone.

    PubMed

    Blatter, E E; Tasayco, M L; Prestwich, G; Pietruszko, R

    1990-12-01

    A major component of the sex pheromone from the tobacco budworm moth Heliothis virescens is a C16 straight-chain aldehyde with a single unsaturation at the eleventh position. The sex pheromones are inactivated when metabolized to their corresponding acids by insect aldehyde dehydrogenase. During this investigation it was demonstrated that the C16 aldehyde is a good substrate for human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 with Km and Kcat. values at pH 7.0 of 2 microM and 0.4 mumol of NADH/min per mg and of 0.6 microM and 0.24 mumol of NADH/min per mg respectively. A vinyl ketone analogue of the pheromone inhibited insect pheromone metabolism; it also inactivated human aldehyde dehydrogenase. Total inactivation of both isoenzymes was achieved at stoichiometric (equal or less than the subunit number) concentrations of vinyl ketone, incorporating 2.1-2.6 molecules/molecule of enzyme. Substrate protection was observed in the presence of the parent aldehyde and 5'-AMP. Peptide maps of tryptic digests of the E2 isoenzyme modified with 3H-labelled vinyl ketone showed that incorporation occurred into a single peptide peak. The labelled peptide of E2 isoenzyme was further purified on h.p.l.c. and sequenced. The label was incorporated into cysteine-302 in the primary structure of E2 isoenzyme, thus indicating that cysteine-302 is located in the aldehyde substrate area of the active site of aldehyde dehydrogenase. Affinity labelling of aldehyde dehydrogenase with vinyl ketones may prove to be of general utility in biochemical studies of these enzymes. PMID:2268265

  13. Relation between Neonatal Icter and Gilbert Syndrome in Gloucose-6-Phosphate Dehydrogenase Deficient Subjects

    PubMed Central

    Zahedpasha, Yadollah; Ahmadpour, Mousa; Niaki, Haleh Akhavan; Alaee, Ehsan

    2014-01-01

    Background and Aim: The pathogenesis of neonatal hyperbilirubinemia hasn’t been completely defined in Gloucose-6-Phosphate Dehydrogenase (G6PD) deficient newborns. The aim of this study was to detect the relationship between Gilbert’s syndrome and hyperbilirubinemia in Gloucose-6-Phosphate Dehydrogenase (G6PD) deficient neonates. Materials and Methods: This case-control study was conducted in Amirkola pediatrics teaching hospital, Babol, Iran. A total number of one hundred four infants were included in the study (51 infants with neonatal jaundice and Gloucose-6-Phosphate Dehydrogenase (G6PD) deficiency admitted to phototherapy or transfusion were selected as the case group and 53 infants with Gloucose-6-Phosphate Dehydrogenase (G6PD) deficiency admitted for other reasons than jaundice were selected as the control group). Exclusion criteria were ABO or Rh incompatibility or other reasons that made Coombs test positive, sepsis, hepatosplenomegaly, metabolic diseases, medical treatment and phototherapy. The promoter and coding regions of Uridine diphosphate Glucuronosyl Transferase 1A1 (UGT1A1) of genomic DNA were amplified by polymerase chain reaction (PCR) isolated from leukocytes. We used chi-square test and t-test to compare cases and controls. Results: Distribution of Gilbert genome was not significantly different between the two groups; among cases, 33.3% were homozygote, 35.3% heterozygote, and 31.4% normal. Among controls, 22.6% were homozygote, 34% heterozygote, and 43.4% normal (p-value=xxx). Hyperbilirubinemia family history didn’t differ significantly between these two groups. Conclusions: We showed that in Gloucose-6-Phosphate Dehydrogenase (G6PD) deficient neonates, there was no significant association between Gilbert’s syndrome (promoter polymorphism) and hyperbilirubinemia. PMID:24783083

  14. Slow-binding inhibition of the Escherichia coli pyruvate dehydrogenase multienzyme complex by acetylphosphinate.

    PubMed

    Schönbrunn-Hanebeck, E; Laber, B; Amrhein, N

    1990-05-22

    The pyruvate analogue acetylphosphinate (CH3-CO-PO2H2) inhibits the pyruvate dehydrogenase component (E1) of the Escherichia coli pyruvate dehydrogenase multienzyme complex in a time-dependent process with biphasic reaction kinetics. The formation of an initial, rapidly reversible enzyme-inhibitor complex (EI) with an apparent Ki of 0.12 +/- 0.025 microM is followed by the conversion to a tighter complex (EI) at a maximal rate of k3 = 0.87 +/- 0.34 min-1. The inhibition is reversible (dissociation rate constant k4 = 0.038 +/- 0.002 min-1), requires the presence of the cofactors thiamin pyrophosphate and Mg2+, and is competitive with regard to pyruvate. The microscopic rate constants give a value of 5 nM for the overall dissociation constant [Ki = [E] [I]/[( EI] + [EI]) = Kik4/(k3 + k4)] compared with values of 10 and 3.5 nM obtained by steady-state methods. Thus acetylphosphinate binds by 5 orders of magnitude more tightly to pyruvate dehydrogenase than does pyruvate (Km = 0.35 mM). Acetylphosphinate also affects the pyruvate dehydrogenase complex fluorescence when excited at 290 nm in a time-dependent manner with a maximal rate constant of 0.99 min-1, suggesting a conformational change in the enzyme complex as the slow step in conversion of EI to EI (k3). All these features taken together suggest that the interaction of the pyruvate dehydrogenase with acetylphosphinate involves the formation of a thiamin pyrophosphate-acetylphosphinate adduct that resembles the normal reaction intermediate, 2-(1-carboxy-1-hydroxyethyl)thiamin pyrophosphate (alpha-lactylthiamin pyrophosphate). PMID:2194562

  15. Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases.

    PubMed Central

    Arimitsu, E; Aoki, S; Ishikura, S; Nakanishi, K; Matsuura, K; Hara, A

    1999-01-01

    Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins. PMID:10477285

  16. Assessment of freshness and freeze-thawing of sea bream fillets (Sparus aurata) by a cytosolic enzyme: Lactate dehydrogenase.

    PubMed

    Diop, Mamadou; Watier, Denis; Masson, Pierre-Yves; Diouf, Amadou; Amara, Rachid; Grard, Thierry; Lencel, Philippe

    2016-11-01

    The evaluation of freshness and freeze-thawing of fish fillets was carried out by assessment of autolysis of cells using a cytosolic enzyme lactate dehydrogenase. Autolysis plays an important role in spoilage of fish and postmortem changes in fish tissue are due to the breakdown of the cellular structures and release of cytoplasmic contents. The outflow of a cytosolic enzyme, lactate dehydrogenase, was studied in sea bream fillets and the Sparus aurata fibroblasts (SAF-1) cell-line during an 8day storage period at +4°C. A significant increase of lactate dehydrogenase release was observed, especially after 5days of storage. The ratio between the free and the total lactate dehydrogenase activity is a promising predictive marker to measure the quality of fresh fish fillets. The effect of freeze-thawing on cytosolic lactate dehydrogenase and lysosomal α-d-glucosidase activities was also tested. Despite the protecting effect of the tissue compared to the cell-line, a loss of lactate dehydrogenase activity, but not of α-d-glucosidase, was observed. In conclusion, lactate dehydrogenase may be used as a marker to both assess freshness of fish and distinguish between fresh and frozen-thawed fish fillets. PMID:27211667

  17. Suppressing glioblastoma stem cell function by aldehyde dehydrogenase inhibition with chloramphenicol or disulfiram as a new treatment adjunct: an hypothesis.

    PubMed

    Kast, Richard E; Belda-Iniesta, Cristobal

    2009-12-01

    Strong expression of aldehyde dehydrogenase is a prominent feature of both normal and cancer stem cells, including the stem cell sub-population of glioblastoma. Aldehyde dehydrogenase function is used by cancer stem cells to repopulate a tumor mass after chemotherapy cytoreduction. Cancer stem cells tend to be chemotherapy compared to the non-stem cell majority cell population in several common human cancers. Such has been demonstrated specifically in glioblastoma. In normal hematopoietic stem cells with unimpaired high levels of aldehyde dehydrogenase, stem cells divide rarely and then asymmetrically to a daughter stem cell and a daughter cell on a path of differentiation or symmetrically with both daughter cells on a differentiated path. If a parallel situation obtains in glioblastoma stem cells, the migrating, far flung paucicellular extensions will be stem cell rich and use aldehyde dehydrogenase to generate the characteristic multiple metastases made up of mostly non-stem cells. With inhibition of aldehyde dehydrogenase, stem cell division to non-stem daughter cells tends to become blocked. We have three old yet potent aldehyde dehydrogenase inhibitors on the market- chloral hydrate, chloramphenicol, and disulfiram- they should be investigated as adjuncts in glioblastoma chemotherapy. If GBM stem cell function can be thwarted by potent aldehyde dehydrogenase inhibition, they will be less able to regenerate a stem cell derived tumor mass after primary resection or chemotherapy. PMID:19500061

  18. Fermentation and alternative respiration compensate for NADH dehydrogenase deficiency in a prokaryotic model of DJ-1-associated Parkinsonism.

    PubMed

    Messaoudi, Nadia; Gautier, Valérie; Dairou, Julien; Mihoub, Mouhad; Lelandais, Gaëlle; Bouloc, Philippe; Landoulsi, Ahmed; Richarme, Gilbert

    2015-11-01

    YajL is the closest prokaryotic homologue of Parkinson's disease-associated DJ-1, a protein of undefined function involved in the oxidative stress response. We reported recently that YajL and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as covalent chaperones for the thiol proteome, including the NuoG subunit of NADH dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant. We report here that this mutant compensates for low NADH dehydrogenase activity by utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB and d-amino acid dehydrogenase DadA, and mixed acid aerobic fermentations characterized by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino acids over Krebs cycle-related amino acids, and thus the yajL mutant utilizes pyruvate-centred respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect and constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies, especially those associated with Alzheimer's and Parkinson's diseases. PMID:26377309

  19. Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production

    PubMed Central

    Maleki, Susan; Mærk, Mali; Valla, Svein

    2015-01-01

    The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell. PMID:25746989

  20. The pyruvate dehydrogenase complex in cancer: An old metabolic gatekeeper regulated by new pathways and pharmacological agents.

    PubMed

    Saunier, Elise; Benelli, Chantal; Bortoli, Sylvie

    2016-02-15

    Cancer cells exhibit an altered metabolism which is characterized by a preference for aerobic glycolysis more than mitochondrial oxidation of pyruvate. This provides anabolic support and selective growth advantage for cancer cells. Recently, a new concept has arisen suggesting that these metabolic changes may be due, in part, to an attenuated mitochondrial function which results from the inhibition of the pyruvate dehydrogenase complex (PDC). This mitochondrial complex links glycolysis to the Krebs cycle and the current understanding of its regulation involves the cyclic phosphorylation and dephosphorylation by specific pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs). PMID:25868605

  1. Major Role of NAD-Dependent Lactate Dehydrogenases in Aerobic Lactate Utilization in Lactobacillus plantarum during Early Stationary Phase

    PubMed Central

    Goffin, Philippe; Lorquet, Frédérique; Kleerebezem, Michiel; Hols, Pascal

    2004-01-01

    NAD-independent lactate dehydrogenases are commonly thought to be responsible for lactate utilization during the stationary phase of aerobic growth in Lactobacillus plantarum. To substantiate this view, we constructed single and double knockout mutants for the corresponding genes, loxD and loxL. Lactate-to-acetate conversion was not impaired in these strains, while it was completely blocked in mutants deficient in NAD-dependent lactate dehydrogenase activities, encoded by the ldhD and ldhL genes. We conclude that NAD-dependent but not NAD-independent lactate dehydrogenases are involved in this process. PMID:15375150

  2. Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain [alpha]-Ketoacid Dehydrogenase Complex

    SciTech Connect

    Brautigam, Chad A.; Wynn, R. Max; Chuang, Jacinta L.; Naik, Mandar T.; Young, Brittany B.; Huang, Tai-huang; Chuang, David T.

    2012-02-27

    The purified mammalian branched-chain {alpha}-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain {alpha}-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-{angstrom} resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other {alpha}-ketoacid dehydrogenase complexes.

  3. Purification and Characterization of a Novel NAD(P)+-Farnesol Dehydrogenase from Polygonum minus Leaves.

    PubMed

    Ahmad-Sohdi, Nor-Ain-Shahajar; Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura; Hassan, Maizom

    2015-01-01

    Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with Km values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The Km values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control. PMID:26600471

  4. Purification and Characterization of a Novel NAD(P)+-Farnesol Dehydrogenase from Polygonum minus Leaves

    PubMed Central

    Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura

    2015-01-01

    Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with Km values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The Km values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control. PMID:26600471

  5. [Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

    PubMed

    Voloshchuk, O N; Kopylchuk, G P

    2016-01-01

    Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage. PMID:27143375

  6. Response surface methodology to optimize partition and purification of two recombinant oxidoreductase enzymes, glucose dehydrogenase and d-galactose dehydrogenase in aqueous two-phase systems.

    PubMed

    Shahbaz Mohammadi, Hamid; Mostafavi, Seyede Samaneh; Soleimani, Saeideh; Bozorgian, Sajad; Pooraskari, Maryam; Kianmehr, Anvarsadat

    2015-04-01

    Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25°C, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family. PMID:25591389

  7. Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.

    PubMed

    Yip, Shirley S M; Zhou, Meixia; Joly, John; Snedecor, Bradley; Shen, Amy; Crawford, Yongping

    2014-09-01

    Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3. PMID:24841241

  8. Plants Possess a Cyclic Mitochondrial Metabolic Pathway similar to the Mammalian Metabolic Repair Mechanism Involving Malate Dehydrogenase and l-2-Hydroxyglutarate Dehydrogenase.

    PubMed

    Hüdig, Meike; Maier, Alexander; Scherrers, Isabell; Seidel, Laura; Jansen, Erwin E W; Mettler-Altmann, Tabea; Engqvist, Martin K M; Maurino, Veronica G

    2015-09-01

    Enzymatic side reactions can give rise to the formation of wasteful and toxic products that are removed by metabolite repair pathways. In this work, we identify and characterize a mitochondrial metabolic repair mechanism in Arabidopsis thaliana involving malate dehydrogenase (mMDH) and l-2-hydroxyglutarate dehydrogenase (l-2HGDH). We analyze the kinetic properties of both A. thaliana mMDH isoforms, and show that they produce l-2-hydroxyglutarate (l-2HG) from 2-ketoglutarate (2-KG) at low rates in side reactions. We identify A. thaliana l-2HGDH as a mitochondrial FAD-containing oxidase that converts l-2HG back to 2-KG. Using loss-of-function mutants, we show that the electrons produced in the l-2HGDH reaction are transferred to the mitochondrial electron transport chain through the electron transfer protein (ETF). Thus, plants possess the biochemical components of an l-2HG metabolic repair system identical to that found in mammals. While deficiencies in the metabolism of l-2HG result in fatal disorders in mammals, accumulation of l-2HG in plants does not adversely affect their development under a range of tested conditions. However, orthologs of l-2HGDH are found in all examined genomes of viridiplantae, indicating that the repair reaction we identified makes an essential contribution to plant fitness in as yet unidentified conditions in the wild. PMID:26203119

  9. Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia.

    PubMed Central

    Jonas, S. K.; Benedetto, C.; Flatman, A.; Hammond, R. H.; Micheletti, L.; Riley, C.; Riley, P. A.; Spargo, D. J.; Zonca, M.; Slater, T. F.

    1992-01-01

    The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected]. Images Figure 1 PMID:1637668

  10. Acetylphosphinate is the most potent mechanism-based substrate-like inhibitor of both the human and E. coli pyruvate dehydrogenase components of the pyruvate dehydrogenase complex

    PubMed Central

    Nemeria, Natalia S.; Korotchkina, Lioubov G.; Chakraborty, Sumit; Patel, Mulchand S.; Jordan, Frank

    2006-01-01

    Two analogues of pyruvate, acetylphosphinate and acetylmethylphosphinate were tested as inhibitors of the E1 (pyruvate dehydrogenase) component of the human and Escherichia coli pyruvate dehydrogenase complexes. This is the first instance of such studies on the human enzyme. The acetylphosphinate is a stronger inhibitor of both enzymes (Ki< 1 μM) than acetylmethylphosphinate. Both inhibitors are found to be reversible tight-binding inhibitors. With both inhibitors and with both enzymes, the inhibition apparently takes place by formation of a C2α-phosphinolactylthiamin diphosphate derivative, a covalent adduct of the inhibitor and the coenzyme, mimicking the behavior of substrate and forming a stable analogue of the C2α-lactylthiamin diphosphate. Formation of the intermediate analogue in each case is confirmed by the appearance of a positive circular dichroism signal in the 305-306 nm range, attributed to the 1',4'-iminopyrimidine tautomeric form of the coenzyme. It is further shown that the αHis63 residue of the human E1 has a role in the formation of C2α-lactylthiamin diphosphate since the αHis63Ala variant is only modestly inhibited by either inhibitor, nor did either compound generate the circular dichroism bands assigned to different tautomeric forms of the 4'-aminopyrimidine ring of the coenzyme seen with the wild type enzyme. Interestingly, opposite enantiomers of the carboligase side product acetoin are produced by the human and bacterial enzymes. PMID:17070897

  11. Acetylphosphinate is the most potent mechanism-based substrate-like inhibitor of both the human and Escherichia coli pyruvate dehydrogenase components of the pyruvate dehydrogenase complex.

    PubMed

    Nemeria, Natalia S; Korotchkina, Lioubov G; Chakraborty, Sumit; Patel, Mulchand S; Jordan, Frank

    2006-12-01

    Two analogues of pyruvate, acetylphosphinate and acetylmethylphosphinate were tested as inhibitors of the E1 (pyruvate dehydrogenase) component of the human and Escherichia coli pyruvate dehydrogenase complexes. This is the first instance of such studies on the human enzyme. The acetylphosphinate is a stronger inhibitor of both enzymes (Ki < 1 microM) than acetylmethylphosphinate. Both inhibitors are found to be reversible tight-binding inhibitors. With both inhibitors and with both enzymes, the inhibition apparently takes place by formation of a C2alpha-phosphinolactylthiamin diphosphate derivative, a covalent adduct of the inhibitor and the coenzyme, mimicking the behavior of substrate and forming a stable analogue of the C2alpha-lactylthiamin diphosphate. Formation of the intermediate analogue in each case is confirmed by the appearance of a positive circular dichroism band in the 305-306 nm range, attributed to the 1',4'-iminopyrimidine tautomeric form of the coenzyme. It is further shown that the alphaHis63 residue of the human E1 has a role in the formation of C2alpha-lactylthiamin diphosphate since the alphaHis63Ala variant is only modestly inhibited by either inhibitor, nor did either compound generate the circular dichroism bands assigned to different tautomeric forms of the 4'-aminopyrimidine ring of the coenzyme seen with the wild-type enzyme. Interestingly, opposite enantiomers of the carboligase side product acetoin are produced by the human and bacterial enzymes. PMID:17070897

  12. Cloning, expression, purification and preliminary crystallographic characterization of a shikimate dehydrogenase from Corynebacterium glutamicum

    SciTech Connect

    Schoepe, Jan Niefind, Karsten; Chatterjee, Shivani; Schomburg, Dietmar

    2006-07-01

    The crystallization and preliminary X-ray characterization of a shikimate dehydrogenase from C. glutamicum is presented. The shikimate dehydrogenase from Corynebacterium glutamicum has been cloned into an Escherichia coli expression vector, overexpressed and purified. Native crystals were obtained by the vapour-diffusion technique using 2-methyl-2,4-pentanediol as a precipitant. The crystals belong to the centred monoclinic space group C2, with unit-cell parameters a = 118.77, b = 63.17, c = 35.67 Å, β = 92.26° (at 100 K), and diffract to 1.64 Å on a synchrotron X-ray source. The asymmetric unit is likely to contain one molecule, corresponding to a packing density of 2.08 Å{sup 3} Da{sup −1} and a solvent content of about 41%.

  13. Molecular Analysis of the Gene Encoding F420-Dependent Glucose-6-Phosphate Dehydrogenase from Mycobacterium smegmatis

    PubMed Central

    Purwantini, Endang; Daniels, Lacy

    1998-01-01

    The gene fgd, which codes for F420-dependent glucose-6-phosphate dehydrogenase (FGD), was cloned from Mycobacterium smegmatis, and its sequence was determined and analyzed. A homolog of FGD which has a very high similarity to the M. smegmatis FGD-derived amino acid sequence was identified in Mycobacterium tuberculosis. FGD showed significant homology with F420-dependent N5,N10-methylene-tetrahydromethanopterin reductase (MER) from methanogenic archaea and with several hypothetical proteins from M. tuberculosis and Archaeoglobus fulgidus, but FGD showed no significant homology with NADP-dependent glucose-6-phosphate dehydrogenases. Multiple alignment of FGD and MER proteins revealed four conserved consensus sequences. Multiple alignment of FGD with the hypothetical proteins also revealed portions of the same conserved sequences. Moderately high levels of FGD were expressed in Escherichia coli BL21(DE3) carrying fgd in pBluescript. PMID:9555906

  14. Electron paramagnetic resonance studies of the tungsten-containing formate dehydrogenase from Clostridium thermoaceticum.

    PubMed

    Deaton, J C; Solomon, E I; Watt, G D; Wetherbee, P J; Durfor, C N

    1987-12-16

    The redox centers in the tungsten-containing formate dehydrogenase from Clostridium thermoaceticum were examined by potentiometric titration and electron paramagnetic resonance spectroscopy. At low temperature two overlapping iron-sulfur signals which correlated with enzymatic activity were observed with formal potentials near -400 mV vs. SHE. Based on their temperature dependences, one signal is assigned to a reduced Fe2S2 cluster and one to a reduced Fe4S4 cluster. Quantitation of signal intensity suggests two Fe2S2 and two Fe4S4 clusters per formate dehydrogenase molecule. Another signal (g = 2.101, 1.980, 1.950) present in low concentrations at more negative potentials was observable up to 200 degrees K and is not attributed to any iron-sulfur cluster. The possible origin of this signal is analyzed using ligand field theory, and the redox behavior is considered with respect to possible ligation at the active site. PMID:2827642

  15. Activation of hepatic branched-chain 2-oxoacid dehydrogenase by rat liver cytosolic supernatant.

    PubMed

    Hauschildt, S

    1986-10-29

    Hepatic branched-chain 2-oxoacid dehydrogenase is inactivated by nutritional alterations. Reactivation occurs during preincubation of intact mitochondria in the presence of rat liver cytosolic supernatant. Cytosolic supernatant contains two factors capable of reactivating the enzyme. On gel-filtration (Sephadex G-100), one factor (AF1) elutes in the molecular range of 35,000-40,000 and the other factor (AF2) elutes slightly later than inorganic phosphate. AF2 is stable against heat denaturation and treatment with proteinases. It is destroyed by alkaline phosphatase and in the presence of Ap5A, atractyloside, CaCl2 and NaF its stimulatory effect on branched-chain 2-oxoacid dehydrogenase activity is abolished. Inhibition of activation by NaF suggests that a phosphatase might be involved in the activation process. PMID:3768411

  16. Screening of Aspergillus-derived FAD-glucose dehydrogenases from fungal genome database.

    PubMed

    Mori, Kazushige; Nakajima, Mitsuharu; Kojima, Katsuhiro; Murakami, Koudai; Ferri, Stefano; Sode, Koji

    2011-11-01

    Aspergillus-derived FAD-dependent glucose dehydrogenases (FADGDHs) were screened from fungal genomic databases, primarily by searching for putative homologues of the Aspergillus niger-derived glucose oxidase (GOD). Focusing on a GOD active-site motif, putative proteins annotated as belonging to the glucose methanol choline (GMC) oxidoreductase family were selected. Phylogenetic analysis of these putative proteins produced a GOD clade, which includes the A. niger and Penicillium amagasakiens GODs, and a second clade made up of putative proteins showing 30-40% homology with GOD. The genes encoding the proteins from the second clade were functionally expressed in Escherichia coli, resulting in dye-mediated glucose dehydrogenase (GDH) activity but not GOD activity. These results suggest that the putative proteins belonging to the second clade are FADGDHs. The 3D structure models of these FADGDHs were compared with the 3D structure of GOD. PMID:21748361

  17. Crystallization and preliminary X-ray crystallographic analysis of rabbit l-gulonate 3-dehydrogenase

    SciTech Connect

    Asada, Yukuhiko; Kuroishi, Chizu; Ukita, Yoko; Sumii, Rie; Endo, Satoshi; Matsunaga, Toshiyuki; Hara, Akira; Kunishima, Naoki

    2008-03-01

    The preliminary X-ray crystallographic study of rabbit l-gulonate 3-dehydrogenase is described. Rabbit l-gulonate 3-dehydrogenase was crystallized using the oil-microbatch method at 295 K. X-ray diffraction data were collected to 1.70 Å resolution from a crystal at 100 K using synchrotron radiation. The crystal belongs to the C-centred monoclinic space group C2, with unit-cell parameters a = 71.81, b = 69.08, c = 65.64 Å, β = 102.7°. Assuming the presence of a monomeric protomer in the asymmetric unit gives a V{sub M} value of 2.21 Å{sup 3} Da{sup −1} and a solvent content of 44.4%. A cocrystal with NADH, which was isomorphous to the apo form, was also prepared and diffraction data were collected to 1.85 Å resolution using Cu Kα radiation at 100 K.

  18. Tea triterpenoidal saponins from the roots of Camellia sinensis have inhibitory effects against alcohol dehydrogenase.

    PubMed

    Varughese, Titto; Manir, Md Maniruzzaman; Rahaman, Mozahidur; Kim, Jeong Kee; Lee, Byeong-Gon; Moon, Surk-Sik

    2011-12-01

    Ten new polyhydroxyolean-12-ene pentacyclic triterpenoidal saponins, named rogchaponins 1-10, were isolated from the methanolic extract of the roots of Camellia sinensis by a series of chromatographic methods (silica gel flash column and C18 MPLC followed by C18 HPLC). Their structures were established by 1D and 2D-NMR techniques along with IR and HR-TOF-MS. Rogchaponins R4 ( 4) and R5 (5) showed inhibitory activities against yeast alcohol dehydrogenase (ADH) with IC (50) values of 16.1 ± 3.2 and 15.4 ± 3.3 µM, respectively. A 4-methylpyrazole positive control exhibited an IC (50) of 2750 ± 50 µM. However, the saponins showed no inhibitory activity against yeast aldehyde dehydrogenase (ALDH). PMID:21786220

  19. Expression, Purification, Crystallization And Preliminary X-Ray Studies of Histamine Dehydrogenase From Nocardioides Simplex

    SciTech Connect

    Reed, T.M.; Hirakawa, H.; Mure, M.; Scott, E.E.; Limburg, J.

    2009-05-21

    Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and an ammonium ion. HADH is functionally related to trimethylamine dehydrogenase (TMADH), but HADH has strict substrate specificity towards histamine. HADH is a homodimer, with each 76 kDa subunit containing two redox cofactors: a [4Fe-4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, it was sought to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 {angstrom} resolution at the SSRL synchrotron with 99.7% completeness. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 101.14, b = 107.03, c = 153.35 {angstrom}.

  20. Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor

    SciTech Connect

    Savkur, Rajesh S.; Bramlett, Kelli S.; Michael, Laura F.; Burris, Thomas P. . E-mail: burris_thomas_p@lilly.com

    2005-04-01

    The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression.

  1. Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

    PubMed Central

    Hitosugi, Taro; Fan, Jun; Chung, Tae-Wook; Lythgoe, Katherine; Wang, Xu; Xie, Jianxin; Ge, Qingyuan; Gu, Ting-Lei; Polakiewicz, Roberto D.; Roesel, Johannes L.; Chen, Zhuo (Georgia); Boggon, Titus J.; Lonial, Sagar; Fu, Haian; Khuri, Fadlo R.; Kang, Sumin; Chen, Jing

    2011-01-01

    SUMMARY Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate, and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth. PMID:22195962

  2. The preparation and kinetics of lactate dehydrogenase attached to water-insoluble particles and sheets

    PubMed Central

    Wilson, R. J. H.; Kay, G.; Lilly, M. D.

    1968-01-01

    1. The preparation of lactate dehydrogenase covalently attached to anion-exchange cellulose particles and sheets by use of a dichloro-sym-triazinyl dyestuff, Procion brilliant orange MGS, is described. 2. The stability and kinetic properties of these preparations were investigated. 3. An equation is derived to describe the change in concentration of a substrate when passed through a uniform bed of a substrate-inhibited enzyme. A number of theoretical curves are shown to illustrate the system. 4. A titrimetric assay for lactate dehydrogenase is described, and shown to be stoicheiometric over the range pH5·0–9·2. 5. The results are discussed in relation to previous work, and the effects of charged groups on the support, and of the diffusion film surrounding any particle in suspension, are treated qualitatively. PMID:5673529

  3. Purification, crystallization and preliminary crystallographic study of a recombinant plant aminoaldehyde dehydrogenase from Pisum sativum

    PubMed Central

    Tylichová, Martina; Briozzo, Pierre; Kopečný, David; Ferrero, Julien; Moréra, Solange; Joly, Nathalie; Snégaroff, Jacques; Šebela, Marek

    2008-01-01

    Aminoaldehydes are products of polyamine degradation and are known to be reactive metabolites that are toxic to living cells at high concentrations. These compounds are catabolized by aminoaldehyde dehydrogenases, which are enzymes that contain a nicotinamide adenine dinucleotide coenzyme. Amino­aldehyde dehydrogenase from Pisum sativum was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop method. A complete data set was collected to 2.8 Å resolution at 100 K. Crystals belong to the monoclinic space group P21, with unit-cell parameters a = 86.4, b = 216.6, c = 205.4 Å, β = 98.1°. Molecular replacement was performed and led to the identification of six dimers per asymmetric unit. PMID:18259056

  4. Peroxisomal NADP-isocitrate dehydrogenase is required for Arabidopsis stomatal movement.

    PubMed

    Leterrier, Marina; Barroso, Juan B; Valderrama, Raquel; Begara-Morales, Juan C; Sánchez-Calvo, Beatriz; Chaki, Mounira; Luque, Francisco; Viñegla, Benjamin; Palma, José M; Corpas, Francisco J

    2016-03-01

    Peroxisomes are subcellular organelles characterized by a simple morphological structure but have a complex biochemical machinery involved in signaling processes through molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). Nicotinamide adenine dinucleotide phosphate (NADPH) is an essential component in cell redox homeostasis, and its regeneration is critical for reductive biosynthesis and detoxification pathways. Plants have several NADPH-generating dehydrogenases, with NADP-isocitrate dehydrogenase (NADP-ICDH) being one of these enzymes. Arabidopsis contains three genes that encode for cytosolic, mitochondrial/chloroplastic, and peroxisomal NADP-ICDH isozymes although the specific function of each of these remains largely unknown. Using two T-DNA insertion lines of the peroxisomal NADP-ICDH designated as picdh-1 and picdh-2, the data show that the peroxisomal NADP-ICDH is involved in stomatal movements, suggesting that peroxisomes are a new element in the signaling network of guard cells. PMID:25894616

  5. Aldehyde dehydrogenases: From eye crystallins to metabolic disease and cancer stem cells

    PubMed Central

    Vasiliou, Vasilis; Thompson, David C.; Smith, Clay; Fujita, Mayumi; Chen, Ying

    2014-01-01

    The aldehyde dehydrogenase (ALDH) superfamily is composed of nicotinamide adenine dinucleotide (phosphate) (NAD(P)+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. To date, 24 ALDH gene families have been identified in the eukaryotic genome. In addition to aldehyde metabolizing capacity, ALDHs have additional catalytic (e.g. esterase and reductase) and non-catalytic activities. The latter include functioning as structural elements in the eye (crystallins) and as binding molecules to endobiotics and xenobiotics. Mutations in human ALDH genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases. Most recently ALDH polymorphisms have been associated with gout and osteoporosis. Aldehyde dehydrogenase enzymes also play important roles in embryogenesis and development, neurotransmission, oxidative stress and cancer. This article serves as a comprehensive review of the current state of knowledge regarding the ALDH superfamily and the contribution of ALDHs to various physiological and pathophysiological processes. PMID:23159885

  6. Structural basis of substrate specificity of bifunctional isocitrate dehydrogenase kinase/phosphatase

    PubMed Central

    Yates, Susan P.; Edwards, Thomas E.; Bryan, Cassie M.; Stein, Adam J.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.; Zheng, Jimin; Jia, Zongchao

    2012-01-01

    Isocitrate dehydrogenase kinase/phosphatase (AceK) regulates entry into the glyoxylate bypass by reversibly phosphorylating isocitrate dehydrogenase (ICDH). Based on the recent complex structure of AceK-ICDH from E. coli, we have classified the structures of homodimeric NADP+-ICDHs to rationalize and predict which organisms likely contain substrates for AceK. One example is Burkholderia pseudomallei (Bp). Here we report a crystal structure of Bp-ICDH which exhibits the necessary structural elements required for AceK recognition. Kinetic analyses provided further confirmation that Bp-ICDH is a substrate for AceK. We conclude that the highly stringent AceK binding sites on ICDH are maintained only in Gram-negative bacteria. PMID:21870819

  7. A Novel 3-Hydroxysteroid Dehydrogenase That Regulates Reproductive Development and Longevity

    PubMed Central

    Wollam, Joshua; Magner, Daniel B.; Magomedova, Lilia; Rass, Elisabeth; Shen, Yidong; Rottiers, Veerle; Habermann, Bianca; Cummins, Carolyn L.; Antebi, Adam

    2012-01-01

    Endogenous small molecule metabolites that regulate animal longevity are emerging as a novel means to influence health and life span. In C. elegans, bile acid-like steroids called the dafachronic acids (DAs) regulate developmental timing and longevity through the conserved nuclear hormone receptor DAF-12, a homolog of mammalian sterol-regulated receptors LXR and FXR. Using metabolic genetics, mass spectrometry, and biochemical approaches, we identify new activities in DA biosynthesis and characterize an evolutionarily conserved short chain dehydrogenase, DHS-16, as a novel 3-hydroxysteroid dehydrogenase. Through regulation of DA production, DHS-16 controls DAF-12 activity governing longevity in response to signals from the gonad. Our elucidation of C. elegans bile acid biosynthetic pathways reveals the possibility of novel ligands as well as striking biochemical conservation to other animals, which could illuminate new targets for manipulating longevity in metazoans. PMID:22505847

  8. Chemical cross-linking of mitochondrial NADH dehydrogenase from bovine heart.

    PubMed Central

    Cleeter, M W; Banister, S H; Ragan, C I

    1985-01-01

    The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by using two cleavable cross-linking agents, disuccinimidyl tartrate and (ethylene glycol)yl bis-(succinimidyl succinate). Cross-linking was analysed primarily by immunoblotting to detect products containing subunits of the iron-protein fraction from chaotropic resolution of the enzyme, namely those of 75, 49, 30 and 13 kDa. By using both the isolated iron-protein fraction and the intact dehydrogenase, cross-links were identified between these four subunits, from these subunits to the largest subunit of the flavoprotein fraction, which contains the active site for NADH, and from these subunits to polypeptides in the hydrophobic shell, which surrounds the hydrophilic iron-protein and flavoprotein fractions. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:4004775

  9. Metabolism of 1-aminoethylphosphinate generates acetylphosphinate, a potent inhibitor of pyruvate dehydrogenase.

    PubMed Central

    Laber, B; Amrhein, N

    1987-01-01

    The alanine analogue 1-aminoethylphosphinate [H3C-CH(NH2)-PO2H2] effectively inhibited anthocyanin synthesis in buckwheat hypocotyls and caused an increase in the concentrations of alanine and alanine-derived metabolites. Aminotransferase inhibitors partially alleviated the effects of the analogue. 1-Aminoethylphosphinate did not affect the growth of Klebsiella pneumoniae under anaerobic conditions, but under aerobic conditions it inhibited growth and caused the massive excretion of pyruvate. The analogue inhibited the pyruvate dehydrogenase complex in vitro in the presence of an aminotransferase activity. The transamination product of 1-aminoethylphosphinate, acetylphosphinate (H3C-CO-PO2H2), was found to inhibit the pyruvate dehydrogenase complex in a time-dependent reaction that followed first-order and saturation kinetics and required the presence of thiamin pyrophosphate. PMID:3325039

  10. Metabolism of 1-aminoethylphosphinate generates acetylphosphinate, a potent inhibitor of pyruvate dehydrogenase.

    PubMed

    Laber, B; Amrhein, N

    1987-12-01

    The alanine analogue 1-aminoethylphosphinate [H3C-CH(NH2)-PO2H2] effectively inhibited anthocyanin synthesis in buckwheat hypocotyls and caused an increase in the concentrations of alanine and alanine-derived metabolites. Aminotransferase inhibitors partially alleviated the effects of the analogue. 1-Aminoethylphosphinate did not affect the growth of Klebsiella pneumoniae under anaerobic conditions, but under aerobic conditions it inhibited growth and caused the massive excretion of pyruvate. The analogue inhibited the pyruvate dehydrogenase complex in vitro in the presence of an aminotransferase activity. The transamination product of 1-aminoethylphosphinate, acetylphosphinate (H3C-CO-PO2H2), was found to inhibit the pyruvate dehydrogenase complex in a time-dependent reaction that followed first-order and saturation kinetics and required the presence of thiamin pyrophosphate. PMID:3325039

  11. Pro-haloacetate Nanoparticles for Efficient Cancer Therapy via Pyruvate Dehydrogenase Kinase Modulation

    NASA Astrophysics Data System (ADS)

    Misra, Santosh K.; Ye, Mao; Ostadhossein, Fatemeh; Pan, Dipanjan

    2016-06-01

    Anticancer agents based on haloacetic acids are developed for inhibition of pyruvate dehydrogenase kinase (PDK), an enzyme responsible for reversing the suppression of mitochondria-dependent apoptosis. Through molecular docking studies mono- and dihaloacetates are identified as potent PDK2 binders and matched their efficiency with dichloroacetic acid. In silico screening directed their conversion to phospholipid prodrugs, which were subsequently self-assembled to pro-haloacetate nanoparticles. Following a thorough physico-chemical characterization, the functional activity of these novel agents was established in wide ranges of human cancer cell lines in vitro and in vivo in rodents. Results indicated that the newly explored PDK modulators can act as efficient agent for cancer regression. A Pyruvate dehydrogenase (PDH) assay mechanistically confirmed that these agents trigger their activity through the mitochondria-dependent apoptosis.

  12. Pro-haloacetate Nanoparticles for Efficient Cancer Therapy via Pyruvate Dehydrogenase Kinase Modulation

    PubMed Central

    Misra, Santosh K.; Ye, Mao; Ostadhossein, Fatemeh; Pan, Dipanjan

    2016-01-01

    Anticancer agents based on haloacetic acids are developed for inhibition of pyruvate dehydrogenase kinase (PDK), an enzyme responsible for reversing the suppression of mitochondria-dependent apoptosis. Through molecular docking studies mono- and dihaloacetates are identified as potent PDK2 binders and matched their efficiency with dichloroacetic acid. In silico screening directed their conversion to phospholipid prodrugs, which were subsequently self-assembled to pro-haloacetate nanoparticles. Following a thorough physico-chemical characterization, the functional activity of these novel agents was established in wide ranges of human cancer cell lines in vitro and in vivo in rodents. Results indicated that the newly explored PDK modulators can act as efficient agent for cancer regression. A Pyruvate dehydrogenase (PDH) assay mechanistically confirmed that these agents trigger their activity through the mitochondria-dependent apoptosis. PMID:27323896

  13. A new role for α-ketoglutarate dehydrogenase complex: regulating metabolism through post-translational modification of other enzymes.

    PubMed

    McKenna, Mary C; Rae, Caroline D

    2015-07-01

    This Editorial highlights a study by Gibson et al. published in this issue of JNeurochem, in which the authors reveal a novel role for the α-ketoglutarate dehydrogenase complex (KGDHC) in post-translational modification of proteins. KGDHC may catalyze post-translational modification of itself as well as several other proteins by succinylation of lysine residues. The authors' report of an enzyme responsible for succinylation of key mitochondrial enzymes represents a major step toward our understanding of the complex functional metabolome. TCA, tricarboxylic acid; KG, α-ketoglutarate; KGDHC, α-ketoglutarate dehydrogenase complex; FUM, fumarase; MDH, malate dehydrogenase; ME, malic enzyme; GDH, glutamate dehydrogenase; AAT, aspartate aminotransferase; GS, glutamine synthetase; PAG, phosphate-activated glutaminase; SIRT3, silent information regulator 3; SIRT5, silent information regulator 5. PMID:26052752

  14. Engineering a domain-locking disulfide into a bacterial malate dehydrogenase produces a redox-sensitive enzyme.

    SciTech Connect

    Muslin, E. H.; Li, D.; Stevens, F. J.; Donnelly, M.; Schiffer, M.; Anderson, L. E.; Univ. of Illinois at Chicago

    1995-06-01

    Light-dependent reduction of cystine disulfide bonds results in activation of several of the enzymes of photosynthetic carbon metabolism within the chloroplast. We have modeled the tertiary structure of four of these light-activated enzymes, namely NADP-linked malate dehydrogenase, glyceraldehyde-3-P dehydrogenase, fructosebisphosphatase, and sedoheptulosebisphosphatase, and identified cysteines in each enzyme that be expected to form inactivating disulfide bonds (Li, D., F. J. Stevens, M. Schiffer, and L. E. Anderson, 1994. Biophys. J. 67:29-35). We have now converted two residues in the Escherichia coli NAD-linked malate dehydrogenase to cysteines and produced a redox-sensitive enzyme. Oxidation of domain-locking cysteine residues in the mutant enzyme clearly mimics dark inactivation of the redox-sensitive chloroplast dehydrogenase. This result is completely consistent with our proposed mechanism.

  15. Regulation of carbon monoxide dehydrogenase and hydrogenase in Rhodospirillum rubrum: Effects of CO and oxygen on synthesis and activity

    SciTech Connect

    Bonam, D.; Lehman, L.; Roberts, G.P.; Ludden, P.W.

    1989-06-01

    Exposure of the photosynthetic bacterium Rhodospirillum rubrum to carbon monoxide led to increased carbon monoxide dehydrogenase and hydrogenase activities due to de novo protein synthesis of both enzymes. Two-dimensional gels of (/sup 35/S)methionine-pulse-labeled cells showed that induction of CO dehydrogenase synthesis was rapidly initiated (less than 5 min upon exposure to CO) and was inhibited by oxygen. Both CO dehydrogenase and the CO-induced hydrogenase were inactivated by oxygen in vivo and in vitro. In contrast to CO dehydrogenase, the CO-induced hydrogenase was 95% inactivated by heating at 70 degrees C for 5 min. Unlike other hydrogenases, this CO-induced hydrogenase was inhibited only 60% by a 100% CO gas phase.

  16. Structure of glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae at 2.45 Å resolution

    PubMed Central

    Alarcon, David Aparicio; Nandi, Munmun; Carpena, Xavi; Fita, Ignacio; Loewen, Peter C.

    2012-01-01

    The interconversion of glycerol 3-phosphate and dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenases provides a link between carbohydrate and lipid metabolism and provides Saccharomyces cerevisiae with protection against osmotic and anoxic stress. The first structure of a glycerol-3-phosphate dehydrogenase from S. cerevisiae, GPD1, is reported at 2.45 Å resolution. The asymmetric unit contains two monomers, each of which is organized with N- and C-terminal domains. The N-terminal domain contains a classic Rossmann fold with the (β-α-β-α-β)2 motif typical of many NAD+-dependent enzymes, while the C-terminal domain is mainly α-helical. Structural and phylogenetic comparisons reveal four main structure types among the five families of glycerol-3-phosphate and glycerol-1-phosphate dehydrogenases and reveal that the Clostridium acetobutylican protein with PDB code 3ce9 is a glycerol-1-­phosphate dehydrogenase. PMID:23143232

  17. Stenotrophomonas Infection in a Patient with Glucose-6-Phosphate Dehydrogenase Deficiency

    PubMed Central

    Harthan, Aaron A.; Heger, Margaret L

    2013-01-01

    The drug of choice for treatment of Stenotrophomonas maltophilia is sulfamethoxazole/trimethoprim, and second-line therapy usually consists of a fluoroquinolone. However, in patients with glucose-6-phosphate dehydrogenase deficiency, neither sulfamethoxazole/trimethoprim nor a fluoroquinolone is a preferred option as it may result in hemolysis. Currently, there is a paucity of data regarding treatment of S maltophilia infection in these patients. This case report presents a patient who was successfully treated with doxycycline and inhaled colistimethate. PMID:23798908

  18. Purification and Interconversion of Homoserine Dehydrogenase from Daucus carota Cell Suspension Cultures 1

    PubMed Central

    Matthews, Benjamin F.; Farrar, Margaret J.; Gray, Ann C.

    1989-01-01

    Homoserine dehydrogenase from cell suspension cultures of carrot (Daucus carota L.) has been purified to apparent homogeneity by a combination of selective heat denaturation, ion exchange and gel filtration chromatographies, and preparative gel electrophoresis. Carrot homoserine dehydrogenase is composed of subunits of equal molecular weight (85,000 ± 5,000). During purification, the enzyme exists predominantly in two molecular weight forms, 180,000 and 240,000. The enzyme can be reversibly converted from one form to the other, and each has different regulatory properties. When the enzyme is dialyzed in the presence of 5 millimolar threonine, the purified enzyme is converted into its trimeric form (240,000), which is completely inhibited by 5 millimolar threonine and is stimulated 2.6-fold by K+. When the enzyme is dialyzed in the presence of K+ and absence of threonine, the purified enzyme is converted into a dimer (180,000), which is not inhibited by threonine and is only stimulated 1.5-fold by K+. The enzyme also can polymerize under certain conditions to form higher molecular weight aggregates ranging in size up to 720,000, which also are catalytically active. This interconversion of homoserine dehydrogenase conformations may reflect the daily stream of events occurring in vivo. When light stimulates protein synthesis, the threonine pool decreases in the chloroplast, while K+ concentrations increase. The change in threonine and K+ concentrations shift the homoserine dehydrogenase from the threonine-sensitive to the threonine-insensitive conformation resulting in increased production of threonine, which would meet the demands of protein synthesis. The reverse process would occur in the dark. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:16667218

  19. Formate dehydrogenase from the methane oxidizer Methylosinus trichosporium OB3b.

    PubMed Central

    Yoch, D C; Chen, Y P; Hardin, M G

    1990-01-01

    Formate dehydrogenase (NAD+ dependent) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b. When the enzyme was isolated anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 cellulose and Sephacryl S-300 columns; they were approximately 315,000 and 155,000 daltons. The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels. The Mr of the alpha-subunit was 53,800 +/- 2,800, and that of the beta-subunit was 102,600 +/- 3,900. The enzyme (Mr 315,000) was composed of these subunits in an apparent alpha 2 beta 2 arrangement. Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000). Similar levels of acid-labile sulfide were detected. No other metals were found in stoichiometric amounts. When the enzyme was isolated aerobically, there was no cofactor requirement for NAD reduction; however, when isolated anaerobically, activity was 80 to 90% dependent on the addition of flavin mononucleotide (FMN) to the reaction mixture. Furthermore, the addition of formate to an active, anoxic solution of formate dehydrogenase rapidly inactivated it in the absence of an electron acceptor; this activity could be reconstituted approximately 85% by 50 nM FMN. Flavin adenine dinucleotide could not replace FMN in reconstituting enzyme activity. The Kms of formate dehydrogenase for formate, NAD, and FMN were 146, 200, and 0.02 microM, respectively. "Pseudomonas oxalaticus" formate dehydrogenase, which has physical characteristics nearly identical to those of the M. trichosporium enzyme, was also shown to be inactivated under anoxic conditions by formate and reactivated by FMN. The evolutionary significance of this similarity is discussed. Images PMID:2376564

  20. Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer

    PubMed Central

    Cheung, Yee-Wai; Kwok, Jane; Law, Alan W. L.; Watt, Rory M.; Kotaka, Masayo; Tanner, Julian A.

    2013-01-01

    DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson–Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics. PMID:24043813