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Sample records for active-site cysteine residue

  1. Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site.

    PubMed

    Zhu, Wen-Jing; Li, Miao; Wang, Xiao-Yun

    2007-12-01

    Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK. PMID:17765964

  2. Role of a cysteine residue in the active site of ERK and the MAPKK family

    SciTech Connect

    Ohori, Makoto; Kinoshita, Takayoshi; Yoshimura, Seiji; Warizaya, Masaichi; Nakajima, Hidenori . E-mail: hidenori.nakajima@jp.astellas.com; Miyake, Hiroshi

    2007-02-16

    Kinases of mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated protein kinase (ERK), represent likely targets for pharmacological intervention in proliferative diseases. Here, we report that FR148083 inhibits ERK2 enzyme activity and TGF{beta}-induced AP-1-dependent luciferase expression with respective IC{sub 50} values of 0.08 and 0.05 {mu}M. FR265083 (1'-2' dihydro form) and FR263574 (1'-2' and 7'-8' tetrahydro form) exhibited 5.5-fold less and no activity, respectively, indicating that both the {alpha},{beta}-unsaturated ketone and the conformation of the lactone ring contribute to this inhibitory activity. The X-ray crystal structure of the ERK2/FR148083 complex revealed that the compound binds to the ATP binding site of ERK2, involving a covalent bond to S{gamma} of ERK2 Cys166, hydrogen bonds with the backbone NH of Met108, N{zeta} of Lys114, backbone C=O of Ser153, N{delta}2 of Asn154, and hydrophobic interactions with the side chains of Ile31, Val39, Ala52, and Leu156. The covalent bond motif in the ERK2/FR148083 complex assures that the inhibitor has high activity for ERK2 and no activity for other MAPKs such as JNK1 and p38MAPK{alpha}/{beta}/{gamma}/{delta} which have leucine residues at the site corresponding to Cys166 in ERK2. On the other hand, MEK1 and MKK7, kinases of the MAPKK family which also can be inhibited by FR148083, contain a cysteine residue corresponding to Cys166 of ERK2. The covalent binding to the common cysteine residue in the ATP-binding site is therefore likely to play a crucial role in the inhibitory activity for these MAP kinases. These findings on the molecular recognition mechanisms of FR148083 for kinases with Cys166 should provide a novel strategy for the pharmacological intervention of MAPK cascades.

  3. Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site

    SciTech Connect

    Bednar, R.A.; Fried, W.B.; Lock, Y.W.; Pramanik, B. )

    1989-08-25

    Chalcone isomerase from soybean is inactivated by stoichiometric amounts of p-mercuribenzoate or HgCl{sub 2}. Spectral titration of the enzyme with p-mercuribenzoate indicates that a single thiol group is modified. Treatment of modified enzyme with KCN or thiols results in a complete restoration of enzyme activity demonstrating that the inactivation is not due to irreversible protein denaturation. A product of the enzymatic reaction, naringenin, provides complete kinetic protection against inactivation by both mercurials. The binding constant (33 microM) for naringenin determined from the concentration dependence of the protection agrees with the inhibition constant (34 microM) for naringenin as a competitive inhibitor of the catalytic reaction. This agreement demonstrates that the observed kinetic protection results from the specific binding of naringenin to the active site. Incubation of native chalcone isomerase with sodium tetrathionate (0.1 M) results in a slow time-dependent loss of enzymatic activity. The inactivation of chalcone isomerase by tetrathionate and N-ethylmaleimide becomes very rapid in the presence of 6 M urea, indicating that the native tertiary structure is responsible for the low reactivity of the enzymatic thiol. The stoichiometric modification of reduced and denatured chalcone isomerase by ({sup 3}H) N-ethylmaleimide indicates that the enzyme contains only a single cysteine residue and does not contain any disulfides. The evidence presented suggests that the only half-cystine residue in chalcone isomerase is located in the active site and thereby provides the first clue to the location of the active site in chalcone isomerase.

  4. Evidence for proximal cysteine and lysine residues at or near the active site of arginine kinase of Stichopus japonicus.

    PubMed

    Guo, Qin; Chen, Baoyu; Wang, Xicheng

    2004-12-01

    Inactivation of arginine kinase (AK) of Stichopus japonicus by o-phthalaldehyde (OPTA) was investigated. The modified enzyme showed an absorption peak at 337 nm and a fluorescent emission peak at 410 nm, which are characteristic of an isoindole derivative formed by OPTA binding to a thiol and an amine group in proximity within the enzyme. Loss of enzymatic activity was concomitant with an increase in fluorescence intensity at 410 nm. Stoichiometry studies by Tsou's method showed that among the cysteine residues available for OPTA modification in the enzyme, only one was essential for the enzyme activity. This cysteine residue is located in a highly hydrophobic environment, presumably near ATP and ADP binding region. This conclusion was verified by 5,5 -dithiobis(2-nitrobenzoic acid) modification. In addition, these results were supported by means of electrophoresis and ultraviolet, fluorescence, circular dichroism spectroscopy and fast performance liquid chromatography. Sequence comparison suggested that this essential cysteine residue maybe the conservative Cys274. PMID:15627388

  5. Single Residue Mutation in Active Site of Serine Acetyltransferase Isoform 3 from Entamoeba histolytica Assists in Partial Regaining of Feedback Inhibition by Cysteine

    PubMed Central

    Kumar, Sudhir; Mazumder, Mohit; Dharavath, Sudhaker; Gourinath, S.

    2013-01-01

    The cysteine biosynthetic pathway is essential for survival of the protist pathogen Entamoeba histolytica, and functions by producing cysteine for countering oxidative attack during infection in human hosts. Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS) are involved in cysteine biosynthesis and are present in three isoforms each. While EhSAT1 and EhSAT2 are feedback inhibited by end product cysteine, EhSAT3 is nearly insensitive to such inhibition. The active site residues of EhSAT1 and of EhSAT3 are identical except for position 208, which is a histidine residue in EhSAT1 and a serine residue in EhSAT3. A combination of comparative modeling, multiple molecular dynamics simulations and free energy calculation studies showed a difference in binding energies of native EhSAT3 and of a S208H-EhSAT3 mutant for cysteine. Mutants have also been generated in vitro, replacing serine with histidine at position 208 in EhSAT3 and replacing histidine 208 with serine in EhSAT1. These mutants showed decreased affinity for substrate serine, as indicated by Km, compared to the native enzymes. Inhibition kinetics in the presence of physiological concentrations of serine show that IC50 of EhSAT1 increases by about 18 folds from 9.59 µM for native to 169.88 µM for H208S-EhSAT1 mutant. Similar measurements with EhSAT3 confirm it to be insensitive to cysteine inhibition while its mutant (S208H-EhSAT3) shows a gain of cysteine inhibition by 36% and the IC50 of 3.5 mM. Histidine 208 appears to be one of the important residues that distinguish the serine substrate from the cysteine inhibitor. PMID:23437075

  6. Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue.

    PubMed

    Sigal, I S; Harwood, B G; Arentzen, R

    1982-12-01

    We describe a procedure by which the codon (AGC) for the active-site serine-70 of pBR322 beta-lactamase (penicillinase, penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is altered to that for cysteine (TGC). The pertinent nucleotide bases, A-G-C-A, positions 410-413, of pBR322 are excised by treating a limited HgiAI digest of pBR322 with the 3' leads to 5' exonuclease of T4 DNA polymerase. The new sequence, T-G-C-A, is inserted in two steps. First, the Kpn I molecular linker d(T-G-G-T-A-C-C-A) is ligated into the gap described above. The internal sequence G-T-A-C is then excised enzymatically with Kpn I and T4 DNA polymerase and the molecule is recircularized. This mutant gene, which codes for a thiol-beta-lactamase, confers on Escherichia coli K-12 hosts an ampicillin resistance that is reduced compared with that given by pBR322 yet is greater than that of E. coli lacking any intact beta-lactamase gene. Cell-free extracts of E. coli strains hosting the thiol-beta-lactamase gene possess a p-chloromercuribenzoate-sensitive beta-lactamase activity.

  7. Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site.

    PubMed

    Keskar, S S; Srinivasan, M C; Deshpande, V V

    1989-07-01

    Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.

  8. Characterization and sequencing of an active-site cysteine-containing peptide from the xylanase of a thermotolerant Streptomyces.

    PubMed

    Keskar, S S; Rao, M B; Deshpande, V V

    1992-02-01

    The kinetics of chemical modification of the xylanase from a thermotolerant Streptomyces T7 indicated the involvement of 1 mol of cysteine residue/mol of enzyme [Keskar, Srinivasan & Deshpande (1989) Biochem. J. 261, 49-55]. The chromophoric reagent N-(2,4-dinitroanilino)maleimide (DAM) reacts covalently with thiol groups of xylanase with complete inactivation. Protection against inactivation was provided by the substrate (xylan). The purified xylanase that had been modified with DAM was digested with pepsin and the peptides were purified by gel filtration followed by peptide mapping. The active-site peptide was distinguished from the other thiol-containing peptides by comparison of the peptides generated by labelling the enzyme in the presence and in the absence of the substrate. The peptide mapping of the modified enzyme in the absence of xylan showed three yellow peptides, whereas in the presence of xylan only two yellow peptides were detected. The active-site peptide protected by the substrate failed to form the complex with DAM. The modified active-site peptide was isolated and sequenced. Gas-phase sequencing provided the following sequence: Ser-Val-Ile-Met-Xaa-Ile-Asp-His-Ile-Arg-Phe. This is the first report on the isolation and sequencing of the active-site peptide from a xylanase. The comparison of reactive cysteine-containing peptide sequence with the catalytic regions of other glucanases revealed the presence of a conserved aspartic acid residue.

  9. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

  10. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

  11. The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site

    PubMed Central

    Aurelius, Oskar; Johansson, Renzo; Bågenholm, Viktoria; Lundin, Daniel; Tholander, Fredrik; Balhuizen, Alexander; Beck, Tobias; Sahlin, Margareta; Sjöberg, Britt-Marie; Mulliez, Etienne; Logan, Derek T.

    2015-01-01

    Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga

  12. Fluorescent labeling of specific cysteine residues using CyMPL

    PubMed Central

    Puljung, Michael C.

    2012-01-01

    The unique reactivity and relative scarcity of cysteine among amino acids makes it a convenient target for the site-specific chemical modification of proteins. Commercially available fluorophores and modifiers react with cysteine through a variety of electrophilic functional groups. However, it can be difficult to obtain specific labeling of a desired cysteine residue in a protein with multiple cysteines, in a mixture of proteins, or in a protein's native environment. CyMPL (Cysteine Metal Protection and Labeling) enables specific labeling by incorporating a cysteine of interest into a minimal binding site for group 12 metal ions (e.g. Cd2+ and Zn2+). These sites can be inserted into any region of known secondary structure in virtually any protein and cause minimal structural perturbation. Bound metal ions protect the cysteine from reaction while background cysteines are blocked with non-fluorescent modifiers. The metal ions are subsequently removed and the deprotected cysteine is labeled specifically. PMID:23151742

  13. Flexibility of active-site gorge aromatic residues and non-gorge aromatic residues in acetylcholinesterase

    SciTech Connect

    Ghattyvenkatakrishna, Pavan K; Uberbacher, Edward C

    2013-01-01

    The presence of an unusually large number of aromatic residues in the active site gorge of acetylcholinesterase has been a topic of great interest. Flexibility of these residues has been suspected to be a key player in controlling ligand traversal in the gorge. This raises the question of whether the over representation of aromatic residues in the gorge implies higher than normal flexibility of those residues. The current study suggests that it does not. Large changes in the hydrophobic cross sectional area due to dihedral oscillations are probably the reason behind their presence in the gorge.

  14. A Methionine Residue Promotes Hyperoxidation of the Catalytic Cysteine of Mouse Methionine Sulfoxide Reductase A.

    PubMed

    Kim, Geumsoo; Levine, Rodney L

    2016-06-28

    Methionine sulfoxide reductase A (msrA) reduces methionine sulfoxide in proteins back to methionine. Its catalytic cysteine (Cys72-SH) has a low pKa that facilitates oxidation by methionine sulfoxide to cysteine sulfenic acid. If the catalytic cycle proceeds efficiently, the sulfenic acid is reduced back to cysteine at the expense of thioredoxin. However, the sulfenic acid is vulnerable to "irreversible" oxidation to cysteine sulfinic acid that inactivates msrA (hyperoxidation). We observed that human msrA is resistant to hyperoxidation while mouse msrA is readily hyperoxidized by micromolar concentrations of hydrogen peroxide. We investigated the basis of this difference in susceptibility to hyperoxidation and established that it is controlled by the presence or absence of a Met residue in the carboxyl-terminal domain of the enzyme, Met229. This residue is Val in human msrA, and when it was mutated to Met, human msrA became sensitive to hyperoxidation. Conversely, mouse msrA was rendered insensitive to hyperoxidation when Met229 was mutated to Val or one of five other residues. Positioning of the methionine at residue 229 is not critical, as hyperoxidation occurred as long as the methionine was located within the group of 14 carboxyl-terminal residues. The carboxyl domain of msrA is known to be flexible and to have access to the active site, and Met residues are known to form stable, noncovalent bonds with aromatic residues through interaction of the sulfur atom with the aromatic ring. We propose that Met229 forms such a bond with Trp74 at the active site, preventing formation of a protective sulfenylamide with Cys72 sulfenic acid. As a consequence, the sulfenic acid is available for facile, irreversible oxidation to cysteine sulfinic acid. PMID:27259041

  15. Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

    PubMed Central

    O'Farrell, Paul A.; Joshua-Tor, Leemor

    2006-01-01

    Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes. PMID:17007609

  16. The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida.

    PubMed

    Fukumoto, Mitsuki; Kudou, Daizou; Murano, Shouko; Shiba, Tomoo; Sato, Dan; Tamura, Takashi; Harada, Shigeharu; Inagaki, Kenji

    2012-01-01

    Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

  17. Targeted reengineering of protein geranylgeranyltransferase type I selectivity functionally implicates active-site residues in protein-substrate recognition.

    PubMed

    Gangopadhyay, Soumyashree A; Losito, Erica L; Hougland, James L

    2014-01-21

    Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins. PMID:24344934

  18. Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

    PubMed

    Cane, D E; Shim, J H; Xue, Q; Fitzsimons, B C; Hohn, T M

    1995-02-28

    Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7873527

  19. The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

    NASA Astrophysics Data System (ADS)

    Qi, Jian-Xun; Jiang, Fan

    2011-05-01

    The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting.

  20. Involvement of the Cys-Tyr cofactor on iron binding in the active site of human cysteine dioxygenase.

    PubMed

    Arjune, Sita; Schwarz, Guenter; Belaidi, Abdel A

    2015-01-01

    Sulfur metabolism has gained increasing medical interest over the last years. In particular, cysteine dioxygenase (CDO) has been recognized as a potential marker in oncology due to its altered gene expression in various cancer types. Human CDO is a non-heme iron-dependent enzyme, which catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid, which is further metabolized to taurine or pyruvate and sulfate. Several studies have reported a unique post-translational modification of human CDO consisting of a cross-link between cysteine 93 and tyrosine 157 (Cys-Tyr), which increases catalytic efficiency in a substrate-dependent manner. However, the reaction mechanism by which the Cys-Tyr cofactor increases catalytic efficiency remains unclear. In this study, steady-state kinetics were determined for wild type CDO and two different variants being either impaired or saturated with the Cys-Tyr cofactor. Cofactor formation in CDO resulted in an approximately fivefold increase in k cat and tenfold increase in k cat/K m over the cofactor-free CDO variant. Furthermore, iron titration experiments revealed an 18-fold decrease in K d of iron upon cross-link formation. This finding suggests a structural role of the Cys-Tyr cofactor in coordinating the ferrous iron in the active site of CDO in accordance with the previously postulated reaction mechanism of human CDO. Finally, we identified product-based inhibition and α-ketoglutarate and glutarate as CDO inhibitors using a simplified well plate-based activity assay. This assay can be used for high-throughput identification of additional inhibitors, which may contribute to understand the functional importance of CDO in sulfur amino acid metabolism and related diseases.

  1. IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE

    EPA Science Inventory

    Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes methylation of inorganic arsenic to mono, di, and trimethylated arsenicals. Orthologous AS3MT genes in genomes ranging from simple echinoderm to human predict a protein with five conserved cysteine (C) residues. In ...

  2. Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense.

    PubMed

    Caffrey, C R; Hansell, E; Lucas, K D; Brinen, L S; Alvarez Hernandez, A; Cheng, J; Gwaltney, S L; Roush, W R; Stierhof, Y D; Bogyo, M; Steverding, D; McKerrow, J H

    2001-11-01

    Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine. PMID:11704274

  3. Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles.

    PubMed

    Ang, Swee Kim; Zhang, Mengqi; Lodi, Tiziana; Lu, Hui

    2014-06-01

    Erv1 (essential for respiration and viability 1), is an essential component of the MIA (mitochondrial import and assembly) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a thiol oxidoreductase with a CPC motif at its active site, oxidizes newly imported substrate proteins. Erv1 a FAD-dependent thiol oxidase, in turn reoxidizes Mia40 via its N-terminal Cys30-Cys33 shuttle disulfide. However, it is unclear how the two shuttle cysteine residues of Erv1 relay electrons from the Mia40 CPC motif to the Erv1 active-site Cys130-Cys133 disulfide. In the present study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteine residues were indeed required for import of MIA pathway substrates and Erv1 enzyme function to oxidize Mia40. Furthermore, our results revealed that the two shuttle cysteine residues of Erv1 are functionally distinct. Although Cys33 is essential for forming the intermediate disulfide Cys33-Cys130' and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from the Mia40 CPC motif; and (ii) resolves the Erv1 Cys33-Cys130 intermediate disulfide. Taken together, we conclude that both shuttle cysteine residues are required for Erv1 function, and play complementary, but distinct, roles to ensure rapid turnover of active Erv1.

  4. Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles.

    PubMed

    Ang, Swee Kim; Zhang, Mengqi; Lodi, Tiziana; Lu, Hui

    2014-06-01

    Erv1 (essential for respiration and viability 1), is an essential component of the MIA (mitochondrial import and assembly) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a thiol oxidoreductase with a CPC motif at its active site, oxidizes newly imported substrate proteins. Erv1 a FAD-dependent thiol oxidase, in turn reoxidizes Mia40 via its N-terminal Cys30-Cys33 shuttle disulfide. However, it is unclear how the two shuttle cysteine residues of Erv1 relay electrons from the Mia40 CPC motif to the Erv1 active-site Cys130-Cys133 disulfide. In the present study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteine residues were indeed required for import of MIA pathway substrates and Erv1 enzyme function to oxidize Mia40. Furthermore, our results revealed that the two shuttle cysteine residues of Erv1 are functionally distinct. Although Cys33 is essential for forming the intermediate disulfide Cys33-Cys130' and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from the Mia40 CPC motif; and (ii) resolves the Erv1 Cys33-Cys130 intermediate disulfide. Taken together, we conclude that both shuttle cysteine residues are required for Erv1 function, and play complementary, but distinct, roles to ensure rapid turnover of active Erv1. PMID:24625320

  5. Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase.

    PubMed

    Tinguely, J N; Wermuth, B

    1999-02-01

    Carbonyl reductase is highly susceptible to inactivation by organomercurials suggesting the presence of a reactive cysteine residue in, or close to, the active site. This residue is also close to a site which binds glutathione. Structurally, carbonyl reductase belongs to the short-chain dehydrogenase/reductase family and contains five cysteine residues, none of which is conserved within the family. In order to identify the reactive residue and investigate its possible role in glutathione binding, alanine was substituted for each cysteine residue of human carbonyl reductase by site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli and purified to homogeneity. Four of the five mutants (C26A, C122A C150A and C226A) exhibited wild-type-like enzyme activity, although K(m) values of C226A for three structurally different substrates were increased threefold to 10-fold. The fifth mutant, C227A, showed a 10-15-fold decrease in kcat and a threefold to 40-fold increase in K(m), resulting in a 30-500-fold drop in kcat/K(m). NaCl (300 mM) increased the activity of C227A 16-fold, whereas the activity of the wild-type enzyme was only doubled. Substitution of serine rather than alanine for Cys227 similarly affected the kinetic constants with the exception that NaCl did not activate the enzyme. Both C227A and C227S mutants were insensitive to inactivation by 4-hydroxymercuribenzoate. Unlike the parent carbonyl compounds, the glutathione adducts of menadione and prostaglandin A1 were better substrates for the C227A and C227S mutants than the wild-type enzyme. Conversely, the binding of free glutathione to both mutants was reduced. Our findings indicate that Cys227 is the reactive residue and suggest that it is involved in the binding of both substrate and glutathione. PMID:10091578

  6. Role of cysteine residues in pseudouridine synthases of different families.

    PubMed

    Ramamurthy, V; Swann, S L; Spedaliere, C J; Mueller, E G

    1999-10-01

    The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

  7. Bromopyruvate, an active site-directed inactivator of E. coli 2-keto-4-hydroxyglutarate(KHG) aldolase, modifies glutamic acid residue-45

    SciTech Connect

    Vlahos, C.J.; Dekker, E.E.

    1987-05-01

    E. coli KHG-aldolase (2-keto-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate), a novel trimeric Class I aldolase, requires one active-site lysine residue (Lys 133)/subunit for Schiff-base formation as well as one arginine residue (Arg 49)/subunit for catalytic activity. The substrate analog, 3-bromopyruvate (BRPY), causes a time- and concentration-dependent loss of KHG-aldolase activity. This inactivation is regarded as active site-directed since: (a) BRPY modification results in complete loss of enzymatic activity; (b) saturation kinetics are exhibited, suggesting that a reversible complex is formed between the aldolase and BRPY prior to the rate-limiting inactivation step; (c) over 90% of the initial aldolase activity is protected by either substrate, pyruvate or KHG; (d) 1.1 mol of /sup 14/C-BRPY is bound/enzyme subunit. Peptide isolation and sequencing show that the incorporated radioactivity is associated with residue Glu-45. Denaturation of the enzyme with guanidine x HCl following treatment with excess /sup 14/C-BRPY allows for the incorporation of carbon-14 at Cys-159 and Cys-180 as well. The presence of pyruvate protects Glu-45 from being esterified but does not prevent the alkylation of the two cysteine residues. These results suggest that Glu-45 is essential for the catalytic activity of E. coli KHG-aldolase, most likely functioning as the active-site amphoteric proton donor/acceptor moiety that is involved in the overall mechanism of the reaction catalyzed by this enzyme.

  8. Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase.

    PubMed

    Batt, C A; Jamieson, A C; Vandeyar, M A

    1990-01-01

    Two conserved histidine residues (His-101 and His-271) appear to be essential components in the active site of the enzyme xylose (glucose) isomerase (EC 5.3.1.5). These amino acid residues were targeted for mutagenesis on the basis of sequence homology among xylose isomerases isolated from Escherichia coli, Bacillus subtilis, Ampullariella sp. strain 3876, and Streptomyces violaceus-niger. Each residue was selectively replaced by site-directed mutagenesis and shown to be essential for activity. No measurable activity was observed for any mutations replacing either His-101 or His-271. Circular dichroism measurements revealed no significant change in the overall conformation of the mutant enzymes, and all formed dimers similar to the wild-type enzyme. Mutations at His-271 could be distinguished from those at His-101, since the former resulted in a thermolabile protein whereas no significant change in heat stability was observed for the latter. Based upon these results and structural data recently reported, we speculate that His-101 is the catalytic base mediating the reaction. Replacement of His-271 may render the enzyme thermolabile, since this residue appears to be a ligand for one of the metal ions in the active site of the enzyme. PMID:2405386

  9. Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death.

    PubMed

    Kubo, Takeya; Nakajima, Hidemitsu; Nakatsuji, Masatoshi; Itakura, Masanori; Kaneshige, Akihiro; Azuma, Yasu-Taka; Inui, Takashi; Takeuchi, Tadayoshi

    2016-02-29

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a homotetrameric enzyme involved in a key step of glycolysis, also has a role in mediating cell death under nitrosative stress. Our previous reports suggest that nitric oxide-induced intramolecular disulfide-bonding GAPDH aggregation, which occurs through oxidation of the active site cysteine (Cys-152), participates in a mechanism to account for nitric oxide-induced death signaling in some neurodegenerative/neuropsychiatric disorders. Here, we demonstrate a rescue strategy for nitric oxide-induced cell death accompanied by GAPDH aggregation in a mutant with a substitution of Cys-152 to alanine (C152A-GAPDH). Pre-incubation of purified wild-type GAPDH with C152A-GAPDH under exposure to nitric oxide inhibited wild-type GAPDH aggregation in a concentration-dependent manner in vitro. Several lines of structural analysis revealed that C152A-GAPDH extensively interfered with nitric oxide-induced GAPDH-amyloidogenesis. Overexpression of doxycycline-inducible C152A-GAPDH in SH-SY5Y neuroblastoma significantly rescued nitric oxide-induced death, concomitant with the decreased formation of GAPDH aggregates. Further, both co-immunoprecipitation assays and simulation models revealed a heterotetramer composed of one dimer each of wild-type GAPDH and C152A-GAPDH. These results suggest that the C152A-GAPDH mutant acts as a dominant-negative molecule against GAPDH aggregation via the formation of this GAPDH heterotetramer. This study may contribute to a new therapeutic approach utilizing C152A-GAPDH against brain damage in nitrosative stress-related disorders.

  10. Salvage of the thiamin pyrimidine moiety by plant TenA proteins lacking an active-site cysteine.

    PubMed

    Zallot, Rémi; Yazdani, Mohammad; Goyer, Aymeric; Ziemak, Michael J; Guan, Jiahn-Chou; McCarty, Donald R; de Crécy-Lagard, Valérie; Gerdes, Svetlana; Garrett, Timothy J; Benach, Jordi; Hunt, John F; Shintani, David K; Hanson, Andrew D

    2014-10-01

    The TenA protein family occurs in prokaryotes, plants and fungi; it has two subfamilies, one (TenA_C) having an active-site cysteine, the other (TenA_E) not. TenA_C proteins participate in thiamin salvage by hydrolysing the thiamin breakdown product amino-HMP (4-amino-5-aminomethyl-2-methylpyrimidine) to HMP (4-amino-5-hydroxymethyl-2-methylpyrimidine); the function of TenA_E proteins is unknown. Comparative analysis of prokaryote and plant genomes predicted that (i) TenA_E has a salvage role similar to, but not identical with, that of TenA_C and (ii) that TenA_E and TenA_C also have non-salvage roles since they occur in organisms that cannot make thiamin. Recombinant Arabidopsis and maize TenA_E proteins (At3g16990, GRMZM2G080501) hydrolysed amino-HMP to HMP and, far more actively, hydrolysed the N-formyl derivative of amino-HMP to amino-HMP. Ablating the At3g16990 gene in a line with a null mutation in the HMP biosynthesis gene ThiC prevented its rescue by amino-HMP. Ablating At3g16990 in the wild-type increased sensitivity to paraquat-induced oxidative stress; HMP overcame this increased sensitivity. Furthermore, the expression of TenA_E and ThiC genes in Arabidopsis and maize was inversely correlated. These results indicate that TenA_E proteins mediate amidohydrolase and aminohydrolase steps in the salvage of thiamin breakdown products. As such products can be toxic, TenA_E proteins may also pre-empt toxicity.

  11. Active site-directed plasmin inhibitors: Extension on the P2 residue.

    PubMed

    Hidaka, Koushi; Gohda, Keigo; Teno, Naoki; Wanaka, Keiko; Tsuda, Yuko

    2016-02-15

    Based on the structure of YO-2 [N-(trans-4-aminomethylcyclohexanecarbonyl)-l-Tyr(O-picolyl)-NH-octyl], active site-directed plasmin (Plm) inhibitors were explored. The picolyl moiety in the Tyr(O-picolyl) residue (namely, the P2 residue) was replaced with smaller or larger groups, such as hydrogen, tert-butyl, benzyl, (2-naphthyl)methyl, and (quinolin-2-yl)methyl. Those efforts produced compound 17 {N-(trans-4-aminomethylcyclohexanecarbonyl)-l-Tyr[O-(quinolin-2-yl)methyl]-NH-octyl} [IC50=0.22 and 77μM for Plm and urokinase (UK), respectively], which showed not only 2.4-fold greater Plm inhibition than YO-2, but also an improvement in selectivity (Plm/UK) by 35-fold. The docking experiments of the Plm-17 complexes disclosed that the amino group of the tranexamyl moiety interacted with the side-chain of Asp753 which formed S1 site.

  12. Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster.

    PubMed

    Menger, Katja E; James, Andrew M; Cochemé, Helena M; Harbour, Michael E; Chouchani, Edward T; Ding, Shujing; Fearnley, Ian M; Partridge, Linda; Murphy, Michael P

    2015-06-30

    Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. Although aging and fasting alter many redox processes, the role of cysteine residues is uncertain. To address this, we used a redox proteomic technique, oxidative isotope-coded affinity tags (OxICAT), to assess cysteine-residue redox changes in Drosophila melanogaster during aging and fasting. This approach enabled us to simultaneously identify and quantify the redox state of several hundred cysteine residues in vivo. Cysteine residues within young flies had a bimodal distribution with peaks at ∼10% and ∼85% reversibly oxidized. Surprisingly, these cysteine residues did not become more oxidized with age. In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized. We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster.

  13. The ribotoxin restrictocin recognizes its RNA substrate by selective engagement of active site residues.

    PubMed

    Plantinga, Matthew J; Korennykh, Alexei V; Piccirilli, Joseph A; Correll, Carl C

    2011-04-12

    Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of ∼1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein-RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes. PMID:21417210

  14. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

    Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

    2015-02-24

    [FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

  15. Electrostatics of cysteine residues in proteins: Parameterization and validation of a simple model

    PubMed Central

    Salsbury, Freddie R.; Poole, Leslie B.; Fetrow, Jacquelyn S.

    2013-01-01

    One of the most popular and simple models for the calculation of pKas from a protein structure is the semi-macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pKas. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pKas; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pKas. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pKa values (where the calculation should reproduce the pKa within experimental error). Both the general behavior of cysteines in proteins and the perturbed pKa in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pKa should be shifted, and validation of force field parameters for cysteine residues. PMID:22777874

  16. The bifunctional active site of S-adenosylmethionine synthetase. Roles of the basic residues.

    PubMed

    Taylor, J C; Markham, G D

    2000-02-11

    S-adenosylmethionine (AdoMet) synthetase catalyzes a unique two-step enzymatic reaction leading to formation of the primary biological alkylating agent. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site, which lies between two subunits, contains four lysines and one histidine as basic residues. In order to test the proposed charge and hydrogen bonding roles in catalytic function, each lysine has been changed to an uncharged methionine or alanine, and the histidine has been altered to asparagine. The resultant enzyme variants are all tetramers like the wild type enzyme; however, circular dichroism spectra show reductions in helix content for the K245*M and K269M mutants. (The asterisk denotes that the residue is in the second subunit.) Four mutants have k(cat) reductions of approximately 10(3)-10(4)-fold in AdoMet synthesis; however, the k(cat) of K165*M variant is only reduced 2-fold. In each mutant, there is a smaller catalytic impairment in the partial reaction of tripolyphosphate hydrolysis. The K165*A enzyme has a 100-fold greater k(cat) for tripolyphosphate hydrolysis than the wild type enzyme, but this mutant is not activated by AdoMet in contrast to the wild type enzyme. The properties of these mutants require reassessment of the catalytic roles of these residues. PMID:10660564

  17. The pepsin residue glycine-76 contributes to active-site loop flexibility and participates in catalysis.

    PubMed Central

    Okoniewska, M; Tanaka, T; Yada, R Y

    2000-01-01

    Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71--80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes. PMID:10861225

  18. Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

    PubMed

    Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

    2016-09-01

    A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

  19. Role of cysteine residues in the redox-regulated oligomerization and nucleotide binding to EhRabX3.

    PubMed

    Chandra, Mintu; Datta, Sunando

    2016-08-01

    The enteric protozoan parasite, Entamoeba histolytica, an etiological agent of amebiasis, is involved in the adhesion and destruction of human tissues. Worldwide, the parasite causes about 50 million cases of amebiasis and 100,000 deaths annually. EhRabX3, a unique amoebic Rab GTPase with tandem G-domains, possesses an unusually large number of cysteine residues in its N-terminal domain. Crystal structure of EhRabX3 revealed an intra-molecular disulfide bond between C39 and C163 which is critical for maintaining the 3-dimensional architecture and biochemical function of this protein. The remaining six cysteine residues were found to be surface exposed and predicted to be involved in inter-molecular disulfide bonds. In the current study, using biophysical and mutational approaches, we have investigated the role of the cysteine residues in the assembly of EhRabX3 oligomer. The self-association of EhRabX3 is found to be redox sensitive, in vitro. Furthermore, the oligomeric conformation of EhRabX3 failed to bind and exchange the guanine nucleotide, indicating structural re-organization of the active site. Altogether, our results provide valuable insights into the redox-dependent oligomerization of EhRabX3 and its implication on nucleotide binding. PMID:27485554

  20. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  1. A Mutational Analysis of the Active Site Loop Residues in cis-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Schroeder, Gottfried K.; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 share 34% sequence identity (54% similarity). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis-isomer. Although the basis for these differences is unknown, a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A 6-residue active site loop in cis-CaaD shows a strikingly different conformation from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. In order to examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to the wild type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover due to the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to

  2. The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of Escherichia coli Are Important but Not Essential for Cytochrome c Maturation In Vivo

    PubMed Central

    Fabianek, Renata A.; Hennecke, Hauke; Thöny-Meyer, Linda

    1998-01-01

    A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome c maturation in Escherichia coli. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in ccmG resulted in the complete absence of all c-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels of Bradyrhizobium japonicum holocytochrome c550 expressed in E. coli. This defect, but not that of the ccmG null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG. PMID:9537397

  3. Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase.

    PubMed

    Robbins, Thomas; Kapilivsky, Joshuah; Cane, David E; Khosla, Chaitan

    2016-08-16

    Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C-C bond formation. S315 played a role in coupling decarboxylation to C-C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs.

  4. Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase.

    PubMed

    Robbins, Thomas; Kapilivsky, Joshuah; Cane, David E; Khosla, Chaitan

    2016-08-16

    Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C-C bond formation. S315 played a role in coupling decarboxylation to C-C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs. PMID:27441852

  5. SufE D74R Substitution Alters Active Site Loop Dynamics To Further Enhance SufE Interaction with the SufS Cysteine Desulfurase

    PubMed Central

    Dai, Yuyuan; Kim, Dokyong; Dong, Guangchao; Busenlehner, Laura S.; Frantom, Patrick A.; Outten, F. Wayne

    2015-01-01

    Many essential metalloproteins require iron–sulfur (Fe–S) cluster cofactors for their function. In vivo persulfide formation from L-cysteine is a key step in the biogenesis of Fe–S clusters in most organisms. In Escherichia coli, the SufS cysteine desulfurase mobilizes persulfide from L-cysteine via a PLP-dependent ping-pong reaction. SufS requires the SufE partner protein to transfer the persulfide to the SufB Fe–S cluster scaffold. Without SufE, the SufS enzyme fails to efficiently turn over and remains locked in the persulfide-bound state. Coordinated protein–protein interactions mediate sulfur transfer from SufS to SufE. Multiple studies have suggested that SufE must undergo a conformational change to extend its active site Cys loop during sulfur transfer from SufS. To test this putative model, we mutated SufE Asp74 to Arg (D74R) to increase the dynamics of the SufE Cys51 loop. Amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of SufE D74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Our results indicate that the mutant protein has a stronger binding affinity for SufS than that of wild-type SufE. In addition, SufE D74R can still enhance SufS desulfurase activity and did not show saturation at higher SufE D74R concentrations, unlike wild-type SufE. These results show that dynamic changes may shift SufE to a sulfur-acceptor state that interacts more strongly with SufS. PMID:26171726

  6. SufE D74R Substitution Alters Active Site Loop Dynamics To Further Enhance SufE Interaction with the SufS Cysteine Desulfurase.

    PubMed

    Dai, Yuyuan; Kim, Dokyong; Dong, Guangchao; Busenlehner, Laura S; Frantom, Patrick A; Outten, F Wayne

    2015-08-11

    Many essential metalloproteins require iron-sulfur (Fe-S) cluster cofactors for their function. In vivo persulfide formation from l-cysteine is a key step in the biogenesis of Fe-S clusters in most organisms. In Escherichia coli, the SufS cysteine desulfurase mobilizes persulfide from l-cysteine via a PLP-dependent ping-pong reaction. SufS requires the SufE partner protein to transfer the persulfide to the SufB Fe-S cluster scaffold. Without SufE, the SufS enzyme fails to efficiently turn over and remains locked in the persulfide-bound state. Coordinated protein-protein interactions mediate sulfur transfer from SufS to SufE. Multiple studies have suggested that SufE must undergo a conformational change to extend its active site Cys loop during sulfur transfer from SufS. To test this putative model, we mutated SufE Asp74 to Arg (D74R) to increase the dynamics of the SufE Cys51 loop. Amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of SufE D74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Our results indicate that the mutant protein has a stronger binding affinity for SufS than that of wild-type SufE. In addition, SufE D74R can still enhance SufS desulfurase activity and did not show saturation at higher SufE D74R concentrations, unlike wild-type SufE. These results show that dynamic changes may shift SufE to a sulfur-acceptor state that interacts more strongly with SufS.

  7. Electrostatics of cysteine residues in proteins: parameterization and validation of a simple model.

    PubMed

    Salsbury, Freddie R; Poole, Leslie B; Fetrow, Jacquelyn S

    2012-11-01

    One of the most popular and simple models for the calculation of pK(a) s from a protein structure is the semi-macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pK(a) s. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pK(a) s; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pK(a) s. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pK(a) values (where the calculation should reproduce the pK(a) within experimental error). Both the general behavior of cysteines in proteins and the perturbed pK(a) in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pK(a) should be shifted, and validation of force field parameters for cysteine residues.

  8. Conserved tyrosine 182 residue in hyperthermophilic esterase EstE1 plays a critical role in stabilizing the active site.

    PubMed

    Truongvan, Ngoc; Chung, Hye-Shin; Jang, Sei-Heon; Lee, ChangWoo

    2016-03-01

    An aromatic amino acid, Tyr or Trp, located in the esterase active site wall, is highly conserved, with hyperthermophilic esterases showing preference for Tyr and lower temperature esterases showing preference for Trp. In this study, we investigated the role of Tyr(182) in the active site wall of hyperthermophilic esterase EstE1. Mutation of Tyr to Phe or Ala had a moderate effect on EstE1 thermal stability. However, a small-to-large mutation such as Tyr to His or Trp had a devastating effect on thermal stability. All mutant EstE1 enzymes showed reduced catalytic rates and enhanced substrate affinities as compared with wild-type EstE1. Hydrogen bond formation involving Tyr(182) was unimportant for maintaining EstE1 thermal stability, as the EstE1 structure is already adapted to high temperatures via increased intramolecular interactions. However, removal of hydrogen bond from Tyr(182) significantly decreased EstE1 catalytic activity, suggesting its role in stabilization of the active site. These results suggest that Tyr is preferred over a similarly sized Phe residue or bulky His or Trp residue in the active site walls of hyperthermophilic esterases for stabilizing the active site and regulating catalytic activity at high temperatures. PMID:26838013

  9. Conserved Active Site Residues Limit Inhibition of a Copper-Containing Nitrite By Small Molecules

    SciTech Connect

    Tocheva, E.I.; Eltis, L.D.; Murphy, M.E.P.

    2009-05-26

    The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.

  10. Zinc cysteine active sites of metalloproteins: a density functional theory and x-ray absorption fine structure study.

    PubMed

    Dimakis, Nicholas; Farooqi, Mohammed Junaid; Garza, Emily Sofia; Bunker, Grant

    2008-03-21

    Density functional theory (DFT) and x-ray absorption fine structure (XAFS) spectroscopy are complementary tools for the biophysical study of active sites in metalloproteins. DFT is used to compute XAFS multiple scattering Debye Waller factors, which are then employed in genetic algorithm-based fitting process to obtain a global fit to the XAFS in the space of fitting parameters. Zn-Cys sites, which serve important functions as transcriptional switches in Zn finger proteins and matrix metalloproteinases, previously have proven intractable by this method; here these limitations are removed. In this work we evaluate optimal DFT nonlocal functionals and basis sets for determining optimal geometries and vibrational densities of states of mixed ligation Zn(His)(4-n)(Cys)(n) sites. Theoretical results are compared to experimental XAFS measurements and Raman spectra from the literature and tabulated for use.

  11. Selective Loss of Cysteine Residues and Disulphide Bonds in a Potato Proteinase Inhibitor II Family

    PubMed Central

    Li, Xiu-Qing; Zhang, Tieling; Donnelly, Danielle

    2011-01-01

    Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short) superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C), and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks) ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution. PMID:21494600

  12. Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

    PubMed Central

    Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I.; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity. PMID:24278284

  13. Crystal structure of a papain-fold protein without the catalytic residue: a novel member in the cysteine proteinase family.

    PubMed

    Zhang, Min; Wei, Zhiyi; Chang, Shaojie; Teng, Maikun; Gong, Weimin

    2006-04-21

    A 31kDa cysteine protease, SPE31, was isolated from the seeds of a legume plant, Pachyrizhus erosus. The protein was purified, crystallized and the 3D structure solved using molecular replacement. The cDNA was obtained by RT PCR followed by amplification using mRNA isolated from the seeds of the legume plant as a template. Analysis of the cDNA sequence and the 3D structure indicated the protein to belong to the papain family. Detailed analysis of the structure revealed an unusual replacement of the conserved catalytic Cys with Gly. Replacement of another conserved residue Ala/Gly by a Phe sterically blocks the access of the substrate to the active site. A polyethyleneglycol molecule and a natural peptide fragment were bound to the surface of the active site. Asn159 was found to be glycosylated. The SPE31 cDNA sequence shares several features with P34, a protein found in soybeans, that is implicated in plant defense mechanisms as an elicitor receptor binding to syringolide. P34 has also been shown to interact with vegetative storage proteins and NADH-dependent hydroxypyruvate reductase. These roles suggest that SPE31 and P34 form a unique subfamily within the papain family. The crystal structure of SPE31 complexed with a natural peptide ligand reveals a unique active site architecture. In addition, the clear evidence of glycosylated Asn159 provides useful information towards understanding the functional mechanism of SPE31/P34. PMID:16497323

  14. Computational study on the roles of amino acid residues in the active site formation mechanism of blue-light photoreceptors

    NASA Astrophysics Data System (ADS)

    Sato, Ryuma; Kitoh-Nishioka, Hirotaka; Ando, Koji; Yamato, Takahisa

    2015-07-01

    To examine the functional roles of the active site methionine (M-site) and glutamic acid (E-site) residues of blue-light photoreceptors, we performed in silico mutation at the M-site in a systematic manner and focused on the hydrogen bonding between the E-site and the substrate: the cyclobutane-pyrimidine dimer (CPD). Fragment molecular orbital calculations with electron correlations demonstrated that substitution of the M-site methionine with either alanine or glutamine always destabilizes the interaction energy between the E-site and the CPD by more than 12.0 kcal/mol, indicating that the methionine and glutamic acid residues cooperatively facilitate the enzymatic reaction in the active site.

  15. Cysteine 532 and cysteine 545 are the N-ethylmaleimide-reactive residues of the Neurospora plasma membrane H+-ATPase.

    PubMed

    Pardo, J P; Slayman, C W

    1989-06-01

    Previous studies from this laboratory (Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 222-226; Davenport, J. W., and Slayman, C. W. (1988) J. Biol. Chem. 263, 16007-16013) have used the sulfhydryl reagent N-ethylmaleimide (NEM) to define two sites on the Neurospora plasma membrane H+-ATPase: a "fast" site which reacts in several minutes with no loss of enzymatic activity and a "slow" site which reacts in tens of minutes to produce complete inactivation of the enzyme. The slow site is protected when MgATP or MgADP is bound to the catalytic site of the ATPase. The present study demonstrates that the fluorescent reagent 5-[2-iodoacetamido)ethyl)-1-aminonaphthalenesulfonic acid (IAEDANS) can be used to label five of the eight cysteine residues of the Neurospora ATPase (Cys376, Cys409, Cys472, Cys532, Cys545). Tryptic peptides bearing those residues have been purified by high performance liquid chromatography and located within the known primary structure of the ATPase by amino acid analysis and/or sequencing. By pretreating the enzyme with NEM in the presence or absence of MgADP before incubation with IAEDANS, it has been possible to identify the fast NEM site as Cys545 and the slow MgADP-protectable NEM site as Cys532. Both residues lie within the central hydrophilic domain of the protein, close to a highly conserved stretch of amino acids that may be involved in nucleotide binding. However, all five IAEDANS-reactive cysteines can be nearly completely modified by the less bulky sulfhydryl reagent methyl methanethiosulfonate with less than 20% inhibition of enzyme activity; thus, none of the five cysteines can be considered to play a direct role in the reaction cycle of the ATPase.

  16. Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila.

    PubMed

    Gorrell, Andrea; Lawrence, Sarah H; Ferry, James G

    2005-03-18

    Acetate kinase catalyzes transfer of the gamma-phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680-686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5'-[gamma-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al(3+), and F(-) contained a linear array of ADP-AlF(3)-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF(3) mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His(180) was in close proximity to AlF(3) is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg(241) was also found adjacent to AlF(3), consistent with a role for stabilization of the transition state. Kinetic analyses of Arg(241) and Arg(91) replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate. PMID:15647264

  17. Two Japanese CADASIL families exhibiting Notch3 mutation R75P not involving cysteine residue.

    PubMed

    Mizuno, Toshiki; Muranishi, Manabu; Torugun, Torusunjian; Tango, Hiromi; Nagakane, Yoshinari; Kudeken, Tukasa; Kawase, Yuji; Kawabe, Kiyokazu; Oshima, Fumiko; Yaoi, Takeshi; Itoh, Kyoko; Fushiki, Shinji; Nakagawa, Masanori

    2008-01-01

    Most previously reported mutations in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) result in an odd number of cysteine residues within the epidermal growth factor (EGF)-like repeats in Notch3. We report here R75P mutation in two Japanese CADASIL families not directly involving cysteine residues located within the first EGF-like repeats. Probands in both families had repeated episodes of stroke, depression, dementia as well as T2 high-intensity lesions in the basal ganglia and periventricular white matter, but fewer white matter lesions in the temporal pole on MRI. These families provide new insights into the diagnosis and pathomechanisms of CADASIL. PMID:19043263

  18. Evaluation of Methods for the Calculation of the pKa of Cysteine Residues in Proteins.

    PubMed

    Awoonor-Williams, Ernest; Rowley, Christopher N

    2016-09-13

    Methods for the calculation of the pKa ionizable amino acids are valuable tools for understanding pH-dependent properties of proteins. Cysteine is unique among the amino acids because of the chemical reactivity of its thiol group (S-H), which plays an instrumental role in several biochemical and regulatory functions. The acidity of noncatalytic cysteine residues is a factor in their susceptibility to chemical modification. Despite the plethora of existing pKa computing methods, no definitive protocol exists for accurately calculating the pKa's of cysteine residues in proteins. A cysteine pKa test set was developed, which is comprised of 18 cysteine residues in 12 proteins where the pKa's have been determined experimentally and an experimental structure is available. The pKa's of these residues were calculated using three methods that use an implicit solvent model (H++, MCCE, and PROPKA) and an all-atom replica-exchange thermodynamic integration approach with the CHARMM36 and AMBER ff99SB-ILDNP force fields. The models that use implicit solvation methods were generally unreliable in predicting cysteine residue pKa's, with RMSDs between 3.41 and 4.72 pKa units. On average, the explicit solvent methods performed better than the implicit solvent methods. RMSD values of 2.40 and 3.20 were obtained for simulations with the CHARMM36 and AMBER ff99SB-ILDNP force fields, respectively. Further development of these methods is necessary because the performance of the best method is similar to that of the null-model (RMSD = 2.74) and these differences in RMSD are of limited statistical significance given the small size of our test set. PMID:27541839

  19. Engineering of cysteine residues leads to improved production of a human dipeptidase enzyme in E. coli.

    PubMed

    O'Dwyer, Ronan; Razzaque, Rafia; Hu, Xuejun; Hollingshead, Susan K; Wall, J Gerard

    2009-10-01

    Low yields, poor folding efficiencies and improper disulfide bridge formation limit large-scale production of cysteine-rich proteins in Escherichia coli. Human renal dipeptidase (MDP), the only human beta-lactamase known to date, is a homodimeric enzyme, which contains six cysteine residues per monomer. It hydrolyses penem and carbapenem beta-lactam antibiotics and can cleave dipeptides containing amino acids in both D: - and L: -configurations. In this study, MDP accumulated in inactive form in high molecular weight, disulfide-linked aggregates when produced in the E. coli periplasm. Mutagenesis of Cys361 that mediates dimer formation and Cys93 that is unpaired in the native MDP led to production of soluble recombinant enzyme, with no change in activity compared with the wild-type enzyme. The removal of unpaired or structurally inessential cysteine residues in this manner may allow functional production of many multiply disulfide-linked recombinant proteins in E. coli.

  20. Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both L-ornithine and L-lysine.

    PubMed

    Lee, Y S; Cho, Y D

    2001-12-15

    The cDNA encoding ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90% identity with Datura stramonium ODC, and 44% identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native M(r) of 92000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both l-ornithine and l-lysine with K(m) values of 562 microM and 1592 microM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by alpha-difluoromethylornithine (K(i) 1.15 microM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys(95)) and cysteine (Cys(96), Cys(338) and Cys(377)) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity. Except for Cys(96), each mutation caused a substantial loss in enzyme activity. Most notably, Lys(95) increased the K(m) for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the k(cat) for ODC and lysine decarboxylase (LDC) activity respectively. The Cys(377)-->Ala mutant possessed a k(cat) that was lowered by 23-fold, and the K(m) value was decreased by 1.4-fold for l-ornithine. The three-dimensional model of ODC protein constructed on the basis of the crystal structure of Trypanosoma brucei, mouse and human ODCs localized the four residues in the active-site cleft. This is the first work carried out on active-site residues of plant ODC, where ODC and LDC

  1. HIGH-THROUGHPUT IDENTIFICATION OF CATALYTIC REDOX-ACTIVE CYSTEINE RESIDUES

    EPA Science Inventory

    Cysteine (Cys) residues often play critical roles in proteins; however, identification of their specific functions has been limited to case-by-case experimental approaches. We developed a procedure for high-throughput identification of catalytic redox-active Cys in proteins by se...

  2. Subsite-specific contributions of different aromatic residues in the active site architecture of glycoside hydrolase family 12

    PubMed Central

    Zhang, Xiaomei; Wang, Shuai; Wu, Xiuyun; Liu, Shijia; Li, Dandan; Xu, Hao; Gao, Peiji; Chen, Guanjun; Wang, Lushan

    2015-01-01

    The active site architecture of glycoside hydrolase (GH) is a contiguous subregion of the enzyme constituted by residues clustered in the three-dimensional space, recognizing the monomeric unit of ligand through hydrogen bonds and hydrophobic interactions. Mutations of the key residues in the active site architecture of the GH12 family exerted different impacts on catalytic efficiency. Binding affinities between the aromatic amino acids and carbohydrate rings were quantitatively determined by isothermal titration calorimetry (ITC) and the quantum mechanical (QM) method, showing that the binding capacity order of Tyr>Trp>His (and Phe) was determined by their side-chain properties. The results also revealed that the binding constant of a certain residue remained unchanged when altering its location, while the catalytic efficiency changed dramatically. Increased binding affinity at a relatively distant subsite, such as the mutant of W7Y at the −4 subsite, resulted in a marked increase in the intermediate product of cellotetraose and enhanced the reactivity of endoglucanase by 144%; while tighter binding near the catalytic center, i.e. W22Y at the −2 subsite, enabled the enzyme to bind and hydrolyze smaller oligosaccharides. Clarification of the specific roles of the aromatics at different subsites may pave the way for a more rational design of GHs. PMID:26670009

  3. Key active site residues in the inhibition of acetylcholinesterases by soman.

    PubMed

    Qian, N; Kovach, I M

    1993-12-27

    Molecular modeling (GEMM 7.3) and molecular mechanics calculations (YETI V 5.3) using the X-ray coordinates for acetylcholinesterase (AChE) from Torpedo californica indicate electrostatic stabilization by the active site, Glu-199, of the developing positive charge on the incipient carbonium ion in the dealkylation in the adducts of AChE with PSCR and PSCS diastereomers of 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman). His-440 is indispensable as a general acid catalyst of C-O bond breaking in the dealkylation reaction and that of bond breaking to the Ser gamma-O in reactivation. This demand for catalysis seems to be satisfied for the reactivation of enzyme from the PSCS diastereomer of soman, but not from the P(S)C(R) diastereomer.

  4. Divergent contributions of conserved active site residues to transcription by eukaryotic RNA polymerases I and II.

    PubMed

    Viktorovskaya, Olga V; Engel, Krysta L; French, Sarah L; Cui, Ping; Vandeventer, Paul J; Pavlovic, Emily M; Beyer, Ann L; Kaplan, Craig D; Schneider, David A

    2013-09-12

    Multisubunit RNA polymerases (msRNAPs) exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL) in the largest subunit of RNA polymerase I (Pol I) yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss of function in Pol I (Pol II: rpb1- E1103G; Pol I: rpa190-E1224G). Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase activity, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps.

  5. Divergent contributions of conserved active site residues to transcription by eukaryotic RNA polymerases I and II

    PubMed Central

    Viktorovskaya, Olga V.; Engel, Krysta L.; French, Sarah L.; Cui, Ping; Vandeventer, Paul J.; Pavlovic, Emily M.; Beyer, Ann L.; Kaplan, Craig D.; Schneider, David A.

    2013-01-01

    SUMMARY Multisubunit RNA polymerases (msRNAPs) exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL) in the largest subunit of RNA polymerase I (Pol I) yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss-of-function in Pol I [Pol II: rpb1- E1103G; Pol I: rpa190-E1224G]. Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase function, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps. PMID:23994471

  6. Mutational analysis of the active site residues of a D: -psicose 3-epimerase from Agrobacterium tumefaciens.

    PubMed

    Kim, Hye-Jung; Yeom, Soo-Jin; Kim, Kwangsoo; Rhee, Sangkee; Kim, Dooil; Oh, Deok-Kun

    2010-02-01

    D-Psicose 3-epimerase from Agrobacterium tumefacience catalyzes the conversion of D: -fructose to D-psicose. According to mutational analysis, the ring at position 112, the negative charge at position 156, and the positive charge at position 215 were essential components for enzyme activity and for binding fructose and psicose. The surface contact area and distance to the bound substrate by molecular modeling suggest that the positive charge of Arg215 was involved in stabilization of cis-endiol intermediate. The distances between the catalytic residues (Glu150 and Glu244) and Mn(2+) are critical to the catalysis, and the negative charges of the metal-binding residues are important for interaction with metal ion. The kinetic parameters of the D183E and H209A mutants for metal-binding residues with substrate and the near-UV circular dichroism spectra indicate that the metal ion bound to Asp183 and His209 is involved not only in catalysis but also in substrate binding.

  7. Redox Sensitivities of Global Cellular Cysteine Residues under Reductive and Oxidative Stress.

    PubMed

    Araki, Kazutaka; Kusano, Hidewo; Sasaki, Naoyuki; Tanaka, Riko; Hatta, Tomohisa; Fukui, Kazuhiko; Natsume, Tohru

    2016-08-01

    The protein cysteine residue is one of the amino acids most susceptible to oxidative modifications, frequently caused by oxidative stress. Several applications have enabled cysteine-targeted proteomics analysis with simultaneous detection and quantitation. In this study, we employed a quantitative approach using a set of iodoacetyl-based cysteine reactive isobaric tags (iodoTMT) and evaluated the transient cellular oxidation ratio of free and reversibly modified cysteine thiols under DTT and hydrogen peroxide (H2O2) treatments. DTT treatment (1 mM for 5 min) reduced most cysteine thiols, irrespective of their cellular localizations. It also caused some unique oxidative shifts, including for peroxiredoxin 2 (PRDX2), uroporphyrinogen decarboxylase (UROD), and thioredoxin (TXN), proteins reportedly affected by cellular reactive oxygen species production. Modest H2O2 treatment (50 μM for 5 min) did not cause global oxidations but instead had apparently reductive effects. Moreover, with H2O2, significant oxidative shifts were observed only in redox active proteins, like PRDX2, peroxiredoxin 1 (PRDX1), TXN, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Overall, our quantitative data illustrated both H2O2- and reduction-mediated cellular responses, whereby while redox homeostasis is maintained, highly reactive thiols can potentiate the specific, rapid cellular signaling to counteract acute redox stress.

  8. Bovine inositol monophosphatase. Modification, identification and mutagenesis of reactive cysteine residues.

    PubMed Central

    Knowles, M R; Gee, N; McAllister, G; Ragan, C I; Greasley, P J; Gore, M G

    1992-01-01

    1. Bovine inositol monophosphatase reacts with thiol reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM) and iodoacetic acid (IAA). 2. Modification by NEM results in nearly total loss of enzyme activity, whereas modification by IAA causes a slight increase in activity. 3. The loss of activity caused by NEM can be prevented by the inclusion of Ins1P, or better Ins1P and LiCl in the reaction mixture. 4. Two equivalents of p-nitrothiobenzoate (NTB2-) are released from the native enzyme on reaction with DTNB, and six equivalents of NTB2- are released from the SDS-denatured enzyme, suggesting that none of the six cysteine residues per molecule of enzyme is involved in intra- or inter-molecular disulphide bridges. 5. Both NEM and IAA react with two cysteine residues (residues 141 and 184 in the sequence) in a mutually exclusive manner. 6. NEM also reacts stoichiometrically with residue 218. 7. The NEM-induced loss of enzyme activity is accompanied by a 15% decrease in protein fluorescence. 8. A mutant of the enzyme which has an Ala-218 replacement for Cys-218 has full activity and is not sensitive to NEM, showing that the modification of this cysteine by NEM causes inhibition of the native protein by steric effects and that Cys-218 is not essential for activity. PMID:1322134

  9. A Mutational Analysis of Active Site Residues in trans-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Poelarends, Gerrit J.; Serrano, Hector; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    trans -3-Chloroacrylic acid dehalogenase (CaaD) catalyzes the hydrolytic dehalogenation of trans-3-haloacrylates to yield malonate semialdehyde by a mechanism utilizing βPro-1, αArg-8, αArg-11, and αGlu-52. These residues are implicated in a promiscuous hydratase activity where 2-oxo-3-pentynoate is processed to acetopyruvate. The roles of three nearby residues (βAsn-39, αPhe-39, and αPhe-50) are unexplored. Mutants were constructed at these positions (βN39A, αF39A, αF39T, αF50A and αF50Y) and kinetic parameters determined along with those of the αR8K and αR11K mutants. Analysis indicates that αArg-8, αArg-11, and βAsn-39 are critical for dehalogenase activity whereas αArg-11 and αPhe-50 are critical for hydratase activity. Docking studies suggest structural bases for these observations. PMID:23851010

  10. Chemical Modification of Papain and Subtilisin: An Active Site Comparison

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

    An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

  11. Toxic tau oligomer formation blocked by capping of cysteine residues with 1,2-dihydroxybenzene groups

    PubMed Central

    Soeda, Yoshiyuki; Yoshikawa, Misato; Almeida, Osborne F. X.; Sumioka, Akio; Maeda, Sumihiro; Osada, Hiroyuki; Kondoh, Yasumitsu; Saito, Akiko; Miyasaka, Tomohiro; Kimura, Tetsuya; Suzuki, Masaaki; Koyama, Hiroko; Yoshiike, Yuji; Sugimoto, Hachiro; Ihara, Yasuo; Takashima, Akihiko

    2015-01-01

    Neurofibrillary tangles, composed of hyperphosphorylated tau fibrils, are a pathological hallmark of Alzheimer's disease; the neurofibrillary tangle load correlates strongly with clinical progression of the disease. A growing body of evidence indicates that tau oligomer formation precedes the appearance of neurofibrillary tangles and contributes to neuronal loss. Here we show that tau oligomer formation can be inhibited by compounds whose chemical backbone includes 1,2-dihydroxybenzene. Specifically, we demonstrate that 1,2-dihydroxybenzene-containing compounds bind to and cap cysteine residues of tau and prevent its aggregation by hindering interactions between tau molecules. Further, we show that orally administered DL-isoproterenol, an adrenergic receptor agonist whose skeleton includes 1,2-dihydroxybenzene and which penetrates the brain, reduces the levels of detergent-insoluble tau, neuronal loss and reverses neurofibrillary tangle-associated brain dysfunction. Thus, compounds that target the cysteine residues of tau may prove useful in halting the progression of Alzheimer's disease and other tauopathies. PMID:26671725

  12. Probing impact of active site residue mutations on stability and activity of Neisseria polysaccharea amylosucrase.

    PubMed

    Daudé, David; Topham, Christopher M; Remaud-Siméon, Magali; André, Isabelle

    2013-12-01

    The amylosucrase from Neisseria polysaccharea is a transglucosidase from the GH13 family of glycoside-hydrolases that naturally catalyzes the synthesis of α-glucans from the widely available donor sucrose. Interestingly, natural molecular evolution has modeled a dense hydrogen bond network at subsite -1 responsible for the specific recognition of sucrose and conversely, it has loosened interactions at the subsite +1 creating a highly promiscuous subsite +1. The residues forming these subsites are considered to be likely involved in the activity as well as the overall stability of the enzyme. To assess their role, a structure-based approach was followed to reshape the subsite -1. A strategy based on stability change predictions, using the FoldX algorithm, was considered to identify the best candidates for site-directed mutagenesis and guide the construction of a small targeted library. A miniaturized purification protocol was developed and both mutant stability and substrate promiscuity were explored. A range of 8 °C between extreme melting temperature values was observed and some variants were able to synthesize series of oligosaccharides with distributions differing from that of the parental enzyme. The crucial role of subsite -1 was thus highlighted and the biocatalysts generated can now be considered as starting points for further engineering purposes.

  13. Identification of putative active-site residues in the DNase domain of colicin E9 by random mutagenesis.

    PubMed

    Garinot-Schneider, C; Pommer, A J; Moore, G R; Kleanthous, C; James, R

    1996-08-01

    We have used random mutagenesis to identify putative active-site residues in the C-terminal cytotoxic endonuclease domain of the bacterial toxin colicin E9. Six single-site mutations in the DNase domain were isolated which destroyed the toxic action of the colicin. DNA sequencing identified the mutations as Gly460Asp, Arg544Gly, Glu548Gly, Thr571Ile, His575Tyr and His579Tyr. All six wild-type residues are highly conserved in the DNase domains of both the E group colicins and the closely related pyocins. Site-directed mutagenesis was then used to substitute the wild-type amino acid residue at each of these positions for an alanine residue in order to distinguish important from unimportant sites. Two of the six alanine-mutant colicins (Gly460Ala and His579Ala) exhibited significant in vivo activity, unlike the original mutation of these residues, and were therefore not characterised further. The Thr571Ala mutant colicin, although not inactive, was significantly less active than the control. The other three alanine mutants (Arg544Ala, Glu548Ala and His575Ala remained completely inactive in the in vivo tests. Each 15 kDa alanine-mutant DNase domain was overexpressed and purified using a tandem-expression strategy which relies on the enzyme being able to bind to the natural inhibitor, Im9. Tryptophan emission spectra of the alanine mutants showed significant alterations in the emission maxima of all but the His575Ala mutant, suggesting changes in the tertiary structure of these mutant proteins. Activity measurements, using the spectrophotometric Kunitz assay, indicated that the Thr571Ala mutant was partially active as an endonuclease but the remaining alanine mutants were all completely inactive. All four mutant proteins, however, retained their ability to bind DNA in a gel shift assay, suggesting the mutations affect catalytic rather than substrate-binding residues. Searching the sequence databases for possible homology to other DNA-binding proteins revealed a

  14. Solution oxygen-17 NMR application for observing a peroxidized cysteine residue in oxidized human SOD1

    NASA Astrophysics Data System (ADS)

    Fujiwara, Noriko; Yoshihara, Daisaku; Sakiyama, Haruhiko; Eguchi, Hironobu; Suzuki, Keiichiro

    2016-12-01

    NMR active nuclei, 1H, 13C and 15N, are usually used for determination of protein structure. However, solution 17O-NMR application to proteins is extremely limited although oxygen is an essential element in biomolecules. Proteins are oxidized through cysteine residues by two types of oxidation. One is reversible oxidation such as disulphide bonding (Cys-S-S-Cys) and the other is irreversible oxidation to cysteine sulfinic acid (Cys-SO 2H) and cysteine sulfonic acid (Cys-SO 3H). Copper,Zinc-superoxide dismutase (SOD1) is a key enzyme in the protection of cells from the superoxide anion radical. The SH group at Cys 111 residue in human SOD1 is selectively oxidized to -SO 2H and -SO 3H with atmospheric oxygen, and this oxidized human SOD1 is also suggested to play an important role in the pathophysiology of various neurodegenerative diseases, probably mainly via protein aggregation. Therefore, information on the structural and the dynamics of the oxidized cysteine residue would be crucial for the understanding of protein aggregation mechanism. Although the -SO 3H group on proteins cannot be directly detected by conventional NMR techniques, we successfully performed the site-specific 17O-labeling of Cys 111 in SOD1 using ^{17}it {O}2 gas and the 17O-NMR analysis for the first time. We observed clear 17O signal derived from a protein molecule and show that 17O-NMR is a sensitive probe for studying the structure and dynamics of the 17O-labeled protein molecule. This novel and unique strategy can have great impact on many research fields in biology and chemistry.

  15. Signal transduction in light–oxygen–voltage receptors lacking the adduct-forming cysteine residue

    PubMed Central

    Yee, Estella F.; Diensthuber, Ralph P.; Vaidya, Anand T.; Borbat, Peter P.; Engelhard, Christopher; Freed, Jack H.; Bittl, Robert; Möglich, Andreas; Crane, Brian R.

    2015-01-01

    Light–oxygen–voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications. PMID:26648256

  16. Signal transduction in light-oxygen-voltage receptors lacking the adduct-forming cysteine residue.

    PubMed

    Yee, Estella F; Diensthuber, Ralph P; Vaidya, Anand T; Borbat, Peter P; Engelhard, Christopher; Freed, Jack H; Bittl, Robert; Möglich, Andreas; Crane, Brian R

    2015-12-09

    Light-oxygen-voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications.

  17. Myeloperoxidase Inactivates TIMP-1 by Oxidizing Its N-terminal Cysteine Residue

    PubMed Central

    Wang, Yi; Rosen, Henry; Madtes, David K.; Shao, Baohai; Martin, Thomas R.; Heinecke, Jay W.; Fu, Xiaoyun

    2016-01-01

    An imbalance between the proteolytic activity of matrix metalloproteinases (MMPs) and the activity of tissue inhibitors of metalloproteinases (TIMPs) is implicated in tissue injury during inflammation. The N-terminal cysteine of TIMP-1 plays a key role in the inhibitory activity of the protein because it coordinates the essential catalytic Zn2+ of the MMP, preventing the metal ion from functioning. An important mechanism for controlling the interaction of TIMPs with MMPs might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase (MPO) system of phagocytes. Here, we show that HOCl generated by the MPO-H2O2-chloride system inactivates TIMP-1 by oxidizing its N-terminal cysteine. The product is a novel 2-oxo acid. Liquid chromatography-mass spectrometry and tandem mass spectrometry analyses demonstrated that methionine and N-terminal cysteine residues were rapidly oxidized by MPO-derived HOCl but only oxidation of the N-terminal cysteine of TIMP-1 correlated well with loss of inhibitory activity. Importantly, we detected the signature 2-oxo-acid N-terminal peptide in tryptic digests of bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome, demonstrating that TIMP-1 oxidation occurs in vivo. Loss of the N-terminal amino group and disulfide structure are crucial for preventing TIMP-1 from inhibiting MMPs. Our findings suggest that pericellular production of HOCl by phagocytes is a pathogenic mechanism for impairing TIMP-1 activity during inflammation. PMID:17726014

  18. Evidence for the Role of Active Site Residues in the Hairpin Ribozyme from Molecular Simulations along the Reaction Path

    PubMed Central

    2015-01-01

    The hairpin ribozyme accelerates a phosphoryl transfer reaction without catalytic participation of divalent metal ions. Residues A38 and G8 have been implicated as playing roles in general acid and base catalysis, respectively. Here we explore the structure and dynamics of key active site residues using more than 1 μs of molecular dynamics simulations of the hairpin ribozyme at different stages along the catalytic pathway. Analysis of results indicates hydrogen bond interactions between the nucleophile and proR nonbridging oxygen are correlated with active inline attack conformations. Further, the simulation results suggest a possible alternative role for G8 to promote inline fitness and facilitate activation of the nucleophile by hydrogen bonding, although this does not necessarily exclude an additional role as a general base. Finally, we suggest that substitution of G8 with N7- or N3-deazaguanosine which have elevated pKa values, both with and without thio modifications at the 5′ leaving group position, would provide valuable insight into the specific role of G8 in catalysis. PMID:24842535

  19. Determination of lysine residues affinity labeled in the active site of yeast RNA polymerase II(B) by mutagenesis.

    PubMed Central

    Treich, I; Carles, C; Sentenac, A; Riva, M

    1992-01-01

    In a previous study, yeast RNA polymerase II(B) was affinity labeled with two nucleotide derivatives (III and VIII) (1). In both cases, the labeled site was localized to the C-terminal part of the B150 subunit. The potential target lysyl residues of derivative III were mapped to the conserved domain H, between Asn946 and Met999. In the present work, we have mutagenized to arginine the five lysines present in domain H. Three lysines can be replaced, individually or simultaneously, without affecting cell growth, and each mutated enzyme can still be affinity labeled. Hence one or both of the other two lysyl residues, Lys979 and Lys987, is the target of the affinity reagent. These two lysines were each found to be essential for cell viability. Derivative VIII labeled another domain in addition to domain H. Supported by analogous results obtained for E. coli RNA polymerase using derivative VIII (2), we hypothesized that the second domain labeled by this derivative in the B150 subunit was domain I. Mutagenesis of the unique lysine present in domain I demonstrated that Lys 1102 was the target of derivative VIII. These results indicate that in both prokaryotic and eukaryotic RNA polymerases, domains H and I are in close proximity and participate to the active site. Images PMID:1408783

  20. Selective Gas-Phase Oxidation and Localization of Alkylated Cysteine Residues in Polypeptide Ions via Ion/Ion Chemistry.

    PubMed

    Pilo, Alice L; Zhao, Feifei; McLuckey, Scott A

    2016-09-01

    The thiol group in cysteine residues is susceptible to several post-translational modifications (PTMs), including prenylation, nitrosylation, palmitoylation, and the formation of disulfide bonds. Additionally, cysteine residues involved in disulfide bonds are commonly reduced and alkylated prior to mass spectrometric analysis. Several of these cysteine modifications, specifically S-alkyl modifications, are susceptible to gas-phase oxidation via selective ion/ion reactions with periodate anions. Multiply protonated peptides containing modified cysteine residues undergo complex formation upon ion/ion reaction with periodate anions. Activation of the ion/ion complexes results in oxygen transfer from the reagent to the modified sulfur residue to create a sulfoxide functionality. Further activation of the sulfoxide derivative yields abundant losses of the modification with the oxidized sulfur as a sulfenic acid (namely, XSOH) to generate a dehydroalanine residue. This loss immediately indicates the presence of an S-alkyl cysteine residue, and the mass of the loss can be used to easily deduce the type of modification. An additional step of activation can be used to localize the modification to a specific residue within the peptide. Selective cleavage to create c- and z-ions N-terminal to the dehydroalanine residue is often noted. As these types of ions are not typically observed upon collision-induced dissociation (CID), they can be used to immediately indicate where in the peptide the PTM was originally located. PMID:27476698

  1. Selective Gas-Phase Oxidation and Localization of Alkylated Cysteine Residues in Polypeptide Ions via Ion/Ion Chemistry.

    PubMed

    Pilo, Alice L; Zhao, Feifei; McLuckey, Scott A

    2016-09-01

    The thiol group in cysteine residues is susceptible to several post-translational modifications (PTMs), including prenylation, nitrosylation, palmitoylation, and the formation of disulfide bonds. Additionally, cysteine residues involved in disulfide bonds are commonly reduced and alkylated prior to mass spectrometric analysis. Several of these cysteine modifications, specifically S-alkyl modifications, are susceptible to gas-phase oxidation via selective ion/ion reactions with periodate anions. Multiply protonated peptides containing modified cysteine residues undergo complex formation upon ion/ion reaction with periodate anions. Activation of the ion/ion complexes results in oxygen transfer from the reagent to the modified sulfur residue to create a sulfoxide functionality. Further activation of the sulfoxide derivative yields abundant losses of the modification with the oxidized sulfur as a sulfenic acid (namely, XSOH) to generate a dehydroalanine residue. This loss immediately indicates the presence of an S-alkyl cysteine residue, and the mass of the loss can be used to easily deduce the type of modification. An additional step of activation can be used to localize the modification to a specific residue within the peptide. Selective cleavage to create c- and z-ions N-terminal to the dehydroalanine residue is often noted. As these types of ions are not typically observed upon collision-induced dissociation (CID), they can be used to immediately indicate where in the peptide the PTM was originally located.

  2. Role of cysteine-58 and cysteine-95 residues in the thiol di-sulfide oxidoreductase activity of Macrophage Migration Inhibitory Factor-2 of Wuchereria bancrofti.

    PubMed

    Chauhan, Nikhil; Hoti, S L

    2016-01-01

    Macrophage Migration Inhibitory Factor (MIF) is the first human cytokine reported and was thought to have a central role in the regulation of inflammatory responses. Homologs of this molecule have been reported in bacteria, invertebrates and plants. Apart from cytokine activity, it also has two catalytic activities viz., tautomerase and di-sulfide oxidoreductase, which appear to be involved in immunological functions. The CXXC catalytic site is responsible for di-sulfide oxidoreductase activity of MIF. We have recently reported thiol-disulfide oxidoreductase activity of Macrophage Migration Inhibitory Factor-2 of Wuchereria bancrofti (Wba-MIF-2), although it lacks the CXXC motif. We hypothesized that three conserved cysteine residues might be involved in the formation of di-sulfide oxidoreductase catalytic site. Homology modeling of Wba-MIF-2 showed that among the three cysteine residues, Cys58 and Cys95 residues came in close proximity (3.23Å) in the tertiary structure with pKa value 9, indicating that these residues might play a role in the di-sulfide oxidoreductase catalytic activity. We carried out site directed mutagenesis of these residues (Cys58Ser & Cys95Ser) and expressed mutant proteins in Escherichia coli. The mutant proteins did not show any oxidoreductase activity in the insulin reduction assay, thus indicating that these two cysteine residues are vital for the catalytic activity of Wba-MIF-2. PMID:26432350

  3. Mutagenesis of Zinc Ligand Residue Cys221 Reveals Plasticity in the IMP-1 Metallo-β-Lactamase Active Site

    PubMed Central

    Horton, Lori B.; Shanker, Sreejesh; Mikulski, Rose; Brown, Nicholas G.; Phillips, Kevin J.; Lykissa, Ernest; Venkataram Prasad, B. V.

    2012-01-01

    Metallo-β-lactamases catalyze the hydrolysis of a broad range of β-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 β-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of β-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward β-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 μM. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable. PMID:22908171

  4. Redox Biology: Computational Approaches to the Investigation of Functional Cysteine Residues

    PubMed Central

    Marino, Stefano M.

    2011-01-01

    Abstract Cysteine (Cys) residues serve many functions, such as catalysis, stabilization of protein structure through disulfides, metal binding, and regulation of protein function. Cys residues are also subject to numerous post-translational modifications. In recent years, various computational tools aiming at classifying and predicting different functional categories of Cys have been developed, particularly for structural and catalytic Cys. On the other hand, given complexity of the subject, bioinformatics approaches have been less successful for the investigation of regulatory Cys sites. In this review, we introduce different functional categories of Cys residues. For each category, an overview of state-of-the-art bioinformatics methods and tools is provided, along with examples of successful applications and potential limitations associated with each approach. Finally, we discuss Cys-based redox switches, which modify the view of distinct functional categories of Cys in proteins. Antioxid. Redox Signal. 15, 135–146. PMID:20812876

  5. Analysis of active site residues of the antiviral protein from summer leaves from Phytolacca americana by site-directed mutagenesis.

    PubMed

    Poyet, J L; Hoeveler, A; Jongeneel, C V

    1998-12-30

    The summer leaf isoform of the pokeweed (Phytolacca americana) antiviral protein, PAP II, was produced in high yields from inclusion bodies in recombinant E. coli. On the basis of its sequence similarity with the spring leaf isoform (PAP I) and with the A chain of ricin, a three-dimensional model of the protein was constructed as an aid in the design of active site mutants. PAP II variants mutated in residues Asp 88 (D88N), Tyr 117 (Y117S), Glu 172 (E172Q), Arg 175 (R175H) and a combination of Asp 88 and Arg 175 (D88N/R175H) were produced in E. coli and assayed for their ability to inhibit protein synthesis in a rabbit reticulocyte lysate. All of these mutations had effects deleterious to the enzymatic activity of PAP II. The results were interpreted in the light of three reaction mechanisms proposed for ribosome-inactivating proteins (RIPs). We conclude that none of the proposed mechanisms is entirely consistent with the data presented here.

  6. A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers.

    PubMed

    Bienert, Gerd P; Cavez, Damien; Besserer, Arnaud; Berny, Marie C; Gilis, Dimitri; Rooman, Marianne; Chaumont, François

    2012-07-01

    AQPs (aquaporins) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Among the different plant AQPs, PIPs (plasma membrane intrinsic proteins), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. In the present study, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulfide bond between two monomers. Although this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of ZmPIP1s (Zm is Zea mays), ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.

  7. Characterization of the active site of chloroperoxidase using physical techniques

    SciTech Connect

    Hall, K.S.

    1986-01-01

    Chloroperoxidase (CPO) and Cytochrome P-450, two very different hemeproteins, have been shown to have similar active sites by several techniques. Recent work has demonstrated thiolate ligation from a cysteine residue to the iron in P-450. A major portion of this research has been devoted to obtaining direct evidence that CPO also has a thiolate 5th ligand from a cysteine residue. This information will provide the framework for a detailed analysis of the structure-function relationships between peroxidases, catalase and cytochrome P-450 hemeproteins. To determine whether the 5th ligand is a cysteine, methionine or a unique amino acid, specific isotope enrichment experiments were used. Preliminary /sup 1/H-NMR studies show that the carbon monoxide-CPO complex has a peak in the upfield region corresponding to alpha-protons of a thiolate amino acid. C. fumago was grown on 95% D/sub 2/O media with a small amount of /sup 1/H-cysteine added. Under these conditions C. fumago slows down the biosynthesis of cysteine by at least 50% and utilizes the exogenous cysteine in the media. GC-MS was able to show that the methylene protons next to the sulfur atom in cysteine are 80-90% protonated while these positions in methionine are approximately 73% deuterated. Comparison of the /sup 1/H-NMR spectra of CO-CPO and CO-CPO indicate the presence of a cysteine ligand in chloroperoxidase.

  8. Cysteine residues of the porcine reproductive and respiratory syndrome virus ORF5a protein are not essential for virus viability.

    PubMed

    Sun, Lichang; Zhou, Yan; Liu, Runxia; Li, Yanhua; Gao, Fei; Wang, Xiaomin; Fan, Hongjie; Yuan, Shishan; Wei, Zuzhang; Tong, Guangzhi

    2015-02-01

    ORF5a protein was recently identified as a novel structural protein in porcine reproductive and respiratory syndrome virus (PRRSV). The ORF5a protein possesses two cysteines at positions 29 and 30 that are highly conserved among type 2 PRRSV. In this study, the significance of the ORF5a protein cysteine residues on virus replication was determined based on a type 2 PRRSV cDNA clone (pAJXM). Each cysteine was substituted by serine or glycine and the mutations were introduced into pAJXM. We found that the replacement of cysteine to glycine at position 30 was lethal for virus viability, but all serine mutant clones produced infectious progeny viruses. This data indicated that cysteine residues in the ORF5a protein were not essential for replication of type 2 PRRSV. The bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) assay were used to study ORF5a protein interacted with other enveloped proteins. These results showed that ORF5a protein interacted non-covalently with itself and interacted with GP4 and 2b protein. The replacement of cysteine to glycine at position 30 affected the ORF5a protein interacted non-covalently with itself, which may account for the lethal phenotype of mutants carrying substitution of cysteine to glycine at position 30.

  9. A conserved active site tyrosine residue of proline dehydrogenase helps enforce the preference for proline over hydroxyproline as the substrate.

    PubMed

    Ostrander, Elizabeth L; Larson, John D; Schuermann, Jonathan P; Tanner, John J

    2009-02-10

    Proline dehydrogenase (PRODH) catalyzes the oxidation of l-proline to Delta-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-l-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analogue l-tetrahydro-2-furoic acid were determined at resolutions of 1.75, 1.90, and 1.85 A, respectively. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline 3-fold and decreases the specificity for proline by factors of 20 (Y540S) and 50 (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding the substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate.

  10. A Conserved Active Site Tyrosine Residue of Proline Dehydrogenase Helps Enforce the Preference for Proline over Hydroxyproline as the Substrate

    SciTech Connect

    Ostrander, E.L.; Larson, J.D.; Schuermann, J.P.; Tanner, J.J.

    2009-03-02

    Proline dehydrogenase (PRODH) catalyzes the oxidation of L-proline to {Delta}-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-L-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analogue L-tetrahydro-2-furoic acid were determined at resolutions of 1.75, 1.90, and 1.85 {angstrom}, respectively. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline 3-fold and decreases the specificity for proline by factors of 20 (Y540S) and 50 (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding the substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate.

  11. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 1: Cysteine Residues and Glycans.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-02-01

    Due to their remarkable selectivity and specificity for cancer biomarkers, immunoconjugates have emerged as extremely promising vectors for the delivery of diagnostic radioisotopes and fluorophores to malignant tissues. Paradoxically, however, these tools for precision medicine are synthesized in a remarkably imprecise way. Indeed, the vast majority of immunoconjugates are created via the random conjugation of bifunctional probes (e.g., DOTA-NCS) to amino acids within the antibody (e.g., lysines). Yet antibodies have multiple copies of these residues throughout their macromolecular structure, making control over the location of the conjugation reaction impossible. This lack of site specificity can lead to the formation of poorly defined, heterogeneous immunoconjugates with suboptimal in vivo behavior. Over the past decade, interest in the synthesis and development of site-specifically labeled immunoconjugates--both antibody-drug conjugates as well as constructs for in vivo imaging--has increased dramatically, and a number of reports have suggested that these better defined, more homogeneous constructs exhibit improved performance in vivo compared to their randomly modified cousins. In this two-part review, we seek to provide an overview of the various methods that have been developed to create site-specifically modified immunoconjugates for positron emission tomography, single photon emission computed tomography, and fluorescence imaging. We will begin with an introduction to the structure of antibodies and antibody fragments. This is followed by the core of the work: sections detailing the four different approaches to site-specific modification strategies based on cysteine residues, glycans, peptide tags, and unnatural amino acids. These discussions will be divided into two installments: cysteine residues and glycans will be detailed in Part 1 of the review, while peptide tags and unnatural amino acids will be addressed in Part 2. Ultimately, we sincerely hope

  12. Posttranslational heterocyclization of cysteine and serine residues in the antibiotic microcin B17: distributivity and directionality.

    PubMed

    Kelleher, N L; Hendrickson, C L; Walsh, C T

    1999-11-23

    To produce the antibiotic Microcin B17, four Cys and four Ser residues are converted into four thiazoles and four oxazoles by the three subunit Microcin B17 synthetase. High-resolution mass spectrometry (MS) was used to monitor the kinetics of posttranslational heterocyclic ring formation (-20 Da per ring) and demonstrated the accumulation of all intermediates, from one to seven rings, indicating distributive processing. All of the intermediates could be converted by the enzyme to the eight ring product. Enzymatic chemoselectivity (Cys vs Ser cyclization rates) was assessed using iodoacetamido-salicylate to alkylate unreacted cysteines (+193 Da) in the 8 kDa biosynthetic intermediates; three of the first four rings formed were thiazoles, and by the five ring stage, all four of the cysteines had been heterocyclized while three of the original four serines remained uncyclized. Finally, tandem MS using a 9.4 T Fourier transform instrument with electrospray ionization was used to elaborate the major processing pathway: the first two rings formed are at the most amino proximal sites (Cys(41) then Ser(40)) followed by the remaining three cysteines at positions 48, 51, and 55. The cyclization of serines at positions 56, 62, and 65 then follows, with Ser(62) and Ser(65) the last to heterocyclize and the first of these at a slower rate. Thus, despite free dissociation of intermediates after each of seven ring-forming catalytic cycles, there is an overall directionality of ring formation from N-terminal to C-terminal sites. This remarkable regioselectivity is determined more by the substrate than the enzyme, due to a combination of (1) initial high-affinity binding of the posttranslational catalyst to the N-terminal propeptide of substrate 88mer, and (2) a chemoselectivity for thiazole over oxazole formation. This mechanism is consistent with antibiotic biosynthesis in vivo, yielding microcin with six, seven, and eight rings, all with bioactivity.

  13. INTERACTIONS OF DIFFERENT INHIBITORS WITH ACTIVE-SITE ASPARTYL RESIDUES OF HIV-1 PROTEASE AND POSSIBLE RELEVANCE TO PEPSINS

    PubMed Central

    Sayer, Jane M.; Louis, John M.

    2008-01-01

    The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors (symmetric cyclic urea inhibitor DMP323, non-hydrolysable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)) was assessed by differential scanning calorimetry. ΔTm values, defined as the difference in Tm for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 °C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by ΔTm ratios [ΔTm(PR)/ΔTm(PRD25N)] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (ΔTm ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (ΔTm(PR)/ΔTm(PRD29N) ≥ 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (ΔTm ratios of 1.4-2.2). Of the 9 FDA approved clinical HIV-1 protease inhibitors screened, APV, RTV and DRV competitively inhibit porcine pepsin with Ki values of 0.3, 0.6 and 2.14 μM, respectively. DSC results were consistent with this relatively weak binding of APV (ΔTm 2.7 °C) compared with the tight binding of AcPEP (ΔTm ≥17 °C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac-PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32 and Ser219 in pepsin, equivalent to Asp25, Asp25′ and Asp29 in PR, in the binding and stabilization of the pepsin/APV complex. PMID:18951411

  14. Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry

    PubMed Central

    Rombouts, Ine; Lagrain, Bert; Brunnbauer, Markus; Delcour, Jan A.; Koehler, Peter

    2013-01-01

    The concentration and composition of wheat gluten proteins and the presence, concentration and location of cysteine residues therein are important for wheat flour quality. However, it is difficult to identify gluten proteins, as they are an extremely polymorphic mixture of prolamins. We here present methods for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM) which, as compared to label-free analysis, substantially improve identification of cysteine-containing peptides in enzymic prolamin digests by electrospray ionization - tandem mass spectrometry. Both chymotrypsin and thermolysin yielded cysteine-containing peptides from different gluten proteins, but more proteins could be identified after chymotryptic digestion. In addition, to the best of our knowledge, we were the first to label prolamins with isotope coded affinity tags (ICAT), which are commonly used for quantitative proteomics. However, more peptides were detected after labeling gluten proteins with 4-VP and IDAM than with ICAT. PMID:23880742

  15. Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry.

    PubMed

    Rombouts, Ine; Lagrain, Bert; Brunnbauer, Markus; Delcour, Jan A; Koehler, Peter

    2013-01-01

    The concentration and composition of wheat gluten proteins and the presence, concentration and location of cysteine residues therein are important for wheat flour quality. However, it is difficult to identify gluten proteins, as they are an extremely polymorphic mixture of prolamins. We here present methods for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM) which, as compared to label-free analysis, substantially improve identification of cysteine-containing peptides in enzymic prolamin digests by electrospray ionization--tandem mass spectrometry. Both chymotrypsin and thermolysin yielded cysteine-containing peptides from different gluten proteins, but more proteins could be identified after chymotryptic digestion. In addition, to the best of our knowledge, we were the first to label prolamins with isotope coded affinity tags (ICAT), which are commonly used for quantitative proteomics. However, more peptides were detected after labeling gluten proteins with 4-VP and IDAM than with ICAT.

  16. On-line procedures for alkylation of cysteine residues with 3-bromopropylamine prior to protein sequence analysis.

    PubMed

    Jue, R A; Hale, J E

    1994-09-01

    We have previously shown that 3-bromopropylamine offers several advantages over other alkylating reagents in the modification and subsequent identification of cysteine residues by protein sequencing. We describe here simple on-sequencer procedures for alkylating cysteines in proteins which employ the reduction of cystines in proteins with tri-n-butylphosphine and concomitant alkylation of the resulting cysteines with 3-bromopropylamine. Addition of an aqueous acetone wash to a modified reaction cycle on the Applied Biosystems 477A sequencer removes excess 3-bromopropylamine. As a result, very little background in the first step of the sequence analysis is seen. Under these conditions, cysteines are readily modified and identified during sequencing. Moreover, very little preview of the next amino acid is observed, which indicates that the N-terminal amino acid is not appreciably alkylated by 3-bromopropylamine. On-sequencer methods have been developed for proteins spotted onto glass fiber filters and proteins electroblotted onto polyvinylidene difluoride membranes.

  17. Alanine substitutions of noncysteine residues in the cysteine-stabilized αβ motif

    PubMed Central

    Yang, Ying-Fang; Cheng, Kuo-Chang; Tsai, Ping-Hsing; Liu, Chung-Cheng; Lee, Tian-Ren; Ping-Chiang Lyu

    2009-01-01

    The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine-stabilized αβ motif (CSαβ) consists of an α-helix and an antiparallel triple-stranded β-sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty-two of 46 residue positions of VrD1 are altered by site-directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering. PMID:19533758

  18. (Function of active-site residues of ribulosebisphosphate carboxylase/oxygenase, Stockholm, Sweden, and visit to Uppsala, Sweden, August 6--12, 1989): Foreign trip report

    SciTech Connect

    Hartman, F.C.

    1989-08-22

    The traveler participated in the 8th International Congress on Photosynthesis by presenting a paper entitled ''Function of Active-Site Residues of Ribulosebisphosphate Carboxylase/Oxygenase'' and by chairing a discussion session on the same enzyme. Presentation concerning biological CO/sub 2/ fixation, chemical modifications of proteins, 3D structure of proteins, and site-directed mutagenesis were relevant to ongoing investigations of the Protein Engineering Program at ORNL's Biology Division.

  19. Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation.

    PubMed

    Shiraishi, Yasuhisa; Muramoto, Takashige; Nagatomo, Kazutaka; Shinmi, Daisuke; Honma, Emiko; Masuda, Kazuhiro; Yamasaki, Motoo

    2015-06-17

    Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

  20. Oxidative post-translational modifications of cysteine residues in plant signal transduction.

    PubMed

    Waszczak, Cezary; Akter, Salma; Jacques, Silke; Huang, Jingjing; Messens, Joris; Van Breusegem, Frank

    2015-05-01

    In plants, fluctuation of the redox balance by altered levels of reactive oxygen species (ROS) can affect many aspects of cellular physiology. ROS homeostasis is governed by a diversified set of antioxidant systems. Perturbation of this homeostasis leads to transient or permanent changes in the redox status and is exploited by plants in different stress signalling mechanisms. Understanding how plants sense ROS and transduce these stimuli into downstream biological responses is still a major challenge. ROS can provoke reversible and irreversible modifications to proteins that act in diverse signalling pathways. These oxidative post-translational modifications (Ox-PTMs) lead to oxidative damage and/or trigger structural alterations in these target proteins. Characterization of the effect of individual Ox-PTMs on individual proteins is the key to a better understanding of how cells interpret the oxidative signals that arise from developmental cues and stress conditions. This review focuses on ROS-mediated Ox-PTMs on cysteine (Cys) residues. The Cys side chain, with its high nucleophilic capacity, appears to be the principle target of ROS. Ox-PTMs on Cys residues participate in various signalling cascades initiated by plant stress hormones. We review the mechanistic aspects and functional consequences of Cys Ox-PTMs on specific target proteins in view of stress signalling events.

  1. Identification of zinc-ligated cysteine residues based on 13Calpha and 13Cbeta chemical shift data.

    PubMed

    Kornhaber, Gregory J; Snyder, David; Moseley, Hunter N B; Montelione, Gaetano T

    2006-04-01

    Although a significant number of proteins include bound metals as part of their structure, the identification of amino acid residues coordinated to non-paramagnetic metals by NMR remains a challenge. Metal ligands can stabilize the native structure and/or play critical catalytic roles in the underlying biochemistry. An atom's chemical shift is exquisitely sensitive to its electronic environment. Chemical shift data can provide valuable insights into structural features, including metal ligation. In this study, we demonstrate that overlapped 13Cbeta chemical shift distributions of Zn-ligated and non-metal-ligated cysteine residues are largely resolved by the inclusion of the corresponding 13Calpha chemical shift information, together with secondary structural information. We demonstrate this with a bivariate distribution plot, and statistically with a multivariate analysis of variance (MANOVA) and hierarchical logistic regression analysis. Using 287 13Calpha/13Cbeta shift pairs from 79 proteins with known three-dimensional structures, including 86 13Calpha and 13Cbeta shifts for 43 Zn-ligated cysteine residues, along with corresponding oxidation state and secondary structure information, we have built a logistic regression model that distinguishes between oxidized cystines, reduced (non-metal ligated) cysteines, and Zn-ligated cysteines. Classifying cysteines/cystines with a statistical model incorporating all three phenomena resulted in a predictor of Zn ligation with a recall, precision and F-measure of 83.7%, and an accuracy of 95.1%. This model was applied in the analysis of Bacillus subtilis IscU, a protein involved in iron-sulfur cluster assembly. The model predicts that all three cysteines of IscU are metal ligands. We confirmed these results by (i) examining the effect of metal chelation on the NMR spectrum of IscU, and (ii) inductively coupled plasma mass spectrometry analysis. To gain further insight into the frequency of occurrence of non-cysteine Zn

  2. Characterization of a novel y-type HMW-GS with eight cysteine residues from Triticum monococcum ssp. monococcum.

    PubMed

    Li, Zenglin; Li, Hongyu; Chen, Gang; Kou, Chunlan; Ning, Shunzong; Yuan, Zhongwei; Jiang, Qi; Zheng, Youliang; Liu, Dengcai; Zhang, Lianquan

    2015-11-15

    The composition and number of high-molecular-weight glutenin subunits (HMW-GSs) play important roles in determining the grain-processing quality of common wheat. The Glu-1Ay allele is silent in common wheat. In this study, an active y-type HMW-GS allele termed 1Ay8.2 (GenBank No. KP137569) was identified from Triticum monococcum L. ssp. monococcum (AmAm, 2n=2x=14), a species with a genome related to the A-genome of common wheat. Compared with previously reported active 1Ay subunits, this novel subunit contained an extra cysteine residue at position 103 of the amino acid sequence in the N-terminal region, in addition to the six cysteines in the N- and C-terminal regions found in most active 1Ay subunits and the one in the repetitive region that appears in only a few 1Ay alleles. This subunit was expressed in an amphiploid (AAAmAmBB, 2n=6x=42) between Triticum turgidum L. ssp. dicoccon and T. monococcum ssp. monococcum. This amphiploid could be used as a bridge to transfer 1Ay8.2 into common wheat cultivars. Replacing the silenced 1Ay in common wheat with the active 1Ay8.2 allele harboring an extra cysteine residue is expected to improve the quality by increasing the number of HMW-GSs and promoting the formation of covalent interactions through disulfide bonds with the extra cysteine residue.

  3. Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

    SciTech Connect

    Elliott, J.I.; Ljungdahl, L.G.

    1982-04-01

    The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4'-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K/sub m/ values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4'-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20 to 30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20 to 30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.

  4. Direct determination of the redox status of cysteine residues in proteins in vivo

    SciTech Connect

    Hara, Satoshi; Tatenaka, Yuki; Ohuchi, Yuya; Hisabori, Toru

    2015-01-02

    Highlights: • A new DNA-maleimide which is cleaved by UV irradiation, DNA-PCMal, was developed. • DNA-PCMal can be used like DNA-Mal to analyze the redox state of cysteine residues. • It is useful for detecting the thiol redox status of a protein in vivo by Western blotting method. • Thus, DNA-PCMal can be a powerful tool for redox proteomics analysis. - Abstract: The redox states of proteins in cells are key factors in many cellular processes. To determine the redox status of cysteinyl thiol groups in proteins in vivo, we developed a new maleimide reagent, a photocleavable maleimide-conjugated single stranded DNA (DNA-PCMal). The DNA moiety of DNA-PCMal is easily removed by UV-irradiation, allowing DNA-PCMal to be used in Western blotting applications. Thereby the state of thiol groups in intracellular proteins can be directly evaluated. This new maleimide compound can provide information concerning redox proteins in vivo, which is important for our understanding of redox networks in the cell.

  5. Predicting the redox state and secondary structure of cysteine residues using multi-dimensional classification analysis of NMR chemical shifts.

    PubMed

    Wang, Ching-Cheng; Lai, Wen-Chung; Chuang, Woei-Jer

    2016-09-01

    A tool for predicting the redox state and secondary structure of cysteine residues using multi-dimensional analyses of different combinations of nuclear magnetic resonance (NMR) chemical shifts has been developed. A data set of cysteine [Formula: see text], (13)C(α), (13)C(β), (1)H(α), (1)H(N), and (15)N(H) chemical shifts was created, classified according to redox state and secondary structure, using a library of 540 re-referenced BioMagResBank (BMRB) entries. Multi-dimensional analyses of three, four, five, and six chemical shifts were used to derive rules for predicting the structural states of cysteine residues. The results from 60 BMRB entries containing 122 cysteines showed that four-dimensional analysis of the C(α), C(β), H(α), and N(H) chemical shifts had the highest prediction accuracy of 100 and 95.9 % for the redox state and secondary structure, respectively. The prediction of secondary structure using 3D, 5D, and 6D analyses had the accuracy of ~90 %, suggesting that H(N) and [Formula: see text] chemical shifts may be noisy and made the discrimination worse. A web server (6DCSi) was established to enable users to submit NMR chemical shifts, either in BMRB or key-in formats, for prediction. 6DCSi displays predictions using sets of 3, 4, 5, and 6 chemical shifts, which shows their consistency and allows users to draw their own conclusions. This web-based tool can be used to rapidly obtain structural information regarding cysteine residues directly from experimental NMR data.

  6. Role of Non-Active-Site Residue Trp-93 in the Function and Stability of New Delhi Metallo-β-Lactamase 1

    PubMed Central

    Rehman, M. Tabish

    2015-01-01

    New Delhi metallo-β-lactamase-1 (NDM-1) is expressed by various members of Enterobacteriaceae as a defense mechanism to hydrolyze β-lactam antibiotics. Despite various studies showing the significance of active-site residues in the catalytic mechanism, there is a paucity of reports addressing the role of non-active-site residues in the structure and function of NDM-1. In this study, we investigated the significance of non-active-site residue Trp-93 in the structure and function of NDM-1. We cloned blaNDM-1 from an Enterobacter cloacae clinical strain (EC-15) and introduced the mutation of Trp-93 to Ala (yielding the Trp93Ala mutant) by PCR-based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of the Trp93Ala mutant were reduced 4- to 8-fold for ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem, and meropenem. The poor hydrolytic activity of the Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of the Trp93Ala mutant was reduced by 40 to 55% (the Km was reduced, while the kcat was similar to that of wild-type NDM-1 [wtNDM-1]). Heat-induced denaturation showed that the ΔGDo and Tm of Trp93Ala mutant were reduced by 1.8 kcal/mol and 4.8°C, respectively. Far-UV circular dichroism (CD) analysis showed that the α-helical content of the Trp93Ala mutant was reduced by 2.9%. The decrease in stability and catalytic efficiency of the Trp93Ala mutant was due to the loss of two hydrogen bonds with Ser-63 and Val-73 and hydrophobic interactions with Leu-65, Val-73, Gln-123, and Asp-124. The study provided insight into the role of non-active-site amino acid residues in the hydrolytic mechanism of NDM-1. PMID:26525789

  7. Role of cysteine residues in ribonuclease H from Escherichia coli. Site-directed mutagenesis and chemical modification.

    PubMed Central

    Kanaya, S; Kimura, S; Katsuda, C; Ikehara, M

    1990-01-01

    The role of the three cysteine residues at positions 13, 63 and 133 in Escherichia coli RNAase H, an enzyme that is sensitive to N-ethylmaleimide [Berkower, Leis & Hurwitz (1973) J. Biol. Chem. 248, 5914-5921], was examined by using both site-directed mutagenesis and chemical modification. Novel aspects that were found are as follows. First, none of the cysteine residues is required for activity. Secondly, chemical modification of either Cys-13 or Cys-133 with thiol-blocking reagents inactivates the enzyme, but that of Cys-63 does not. Thus the sensitivity of E. coli RNAase H to N-ethylmaleimide arises not from blocking of the thiol group but from steric hindrance by the modifying group incorporated at either Cys-13 or Cys-133. Images Fig. 2. PMID:2171503

  8. Application of iTRAQ Reagents to Relatively Quantify the Reversible Redox State of Cysteine Residues

    PubMed Central

    McDonagh, Brian; Martínez-Acedo, Pablo; Vázquez, Jesús; Padilla, C. Alicia; Sheehan, David; Bárcena, José Antonio

    2012-01-01

    Cysteines are one of the most rarely used amino acids, but when conserved in proteins they often play critical roles in structure, function, or regulation. Reversible cysteine modifications allow for potential redox regulation of proteins. Traditional measurement of the relative absolute quantity of a protein between two samples is not always necessarily proportional to the activity of the protein. We propose application of iTRAQ reagents in combination with a previous thiol selection method to relatively quantify the redox state of cysteines both within and between samples in a single analysis. Our method allows for the identification of the proteins, identification of redox-sensitive cysteines within proteins, and quantification of the redox status of individual cysteine-containing peptides. As a proof of principle, we applied this technique to yeast alcohol dehydrogenase-1 exposed in vitro to H2O2 and also in vivo to the complex proteome of the Gram-negative bacterium Bacillus subtilis. PMID:22844595

  9. Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Scotcher, Jenna; Clarke, David J.; Weidt, Stefan K.; Mackay, C. Logan; Hupp, Ted R.; Sadler, Peter J.; Langridge-Smith, Pat R. R.

    2011-05-01

    The tumor suppressor p53 is a redox-regulated transcription factor involved in cell cycle arrest, apoptosis and senescence in response to multiple forms of stress, as well as many other cellular processes such as DNA repair, glycolysis, autophagy, oxidative stress and differentiation. The discovery of cysteine-targeting compounds that cause re-activation of mutant p53 and the death of tumor cells in vivo has emphasized the functional importance of p53 thiols. Using a combination of top-down and middle-down FTICR mass spectrometry, we show that of the 10 Cys residues in the core domain of wild-type p53, Cys182 and Cys277 exhibit a remarkable preference for modification by the alkylating reagent N-ethylmaleimide. The assignment of Cys182 and Cys277 as the two reactive Cys residues was confirmed by site-directed mutagenesis. Further alkylation of p53 beyond Cys182 and Cys277 was found to trigger co-operative modification of the remaining seven Cys residues and protein unfolding. This study highlights the power of top-down FTICR mass spectrometry for analysis of the cysteine reactivity and redox chemistry in multiple cysteine-containing proteins.

  10. Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2' and P3' variations in extended substrates.

    PubMed Central

    Portaro, F C; Santos, A B; Cezari, M H; Juliano, M A; Juliano, L; Carmona, E

    2000-01-01

    We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S(4) and S(3) subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S(2)' and S(3)'. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P(4), P(3), P(2)' and P(3)' were made. The S(4) to S(2)' subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S(3)', indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S(4), S(3), S(2)' and S(3)' of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P(2)' respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P(3)'. Basic residues at P(3) and P(4) were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P(2)' (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His(111) (H111A) and His(110) (H110A) of cathepsin B led to an increase in k(cat) values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L. PMID:10727410

  11. Identification of key functional residues in the active site of human {beta}1,4-galactosyltransferase 7: a major enzyme in the glycosaminoglycan synthesis pathway.

    PubMed

    Talhaoui, Ibtissam; Bui, Catherine; Oriol, Rafael; Mulliert, Guillermo; Gulberti, Sandrine; Netter, Patrick; Coughtrie, Michael W H; Ouzzine, Mohamed; Fournel-Gigleux, Sylvie

    2010-11-26

    Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of β1,4-galactosyltransferase 7 (β4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human β4GalT7, we conducted a phylogenetic analysis of the β1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila β4GalT7-UDP as template. Two evolutionary conserved motifs, (163)DVD(165) and (221)FWGWGREDDE(230), are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type β4GalT7 and selected mutants identified Trp(224) as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp(228) acting as general base in the reaction catalyzed by human β4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human β4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human β4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics.

  12. Important role for phylogenetically invariant PP2Acalpha active site and C-terminal residues revealed by mutational analysis in Saccharomyces cerevisiae.

    PubMed Central

    Evans, D R; Hemmings, B A

    2000-01-01

    PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acalpha functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acalpha Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acalpha catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acalpha C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acalpha catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo. PMID:10978272

  13. Positioning of cysteine residues within the N-terminal portion of the BST-2/tetherin ectodomain is important for functional dimerization of BST-2.

    PubMed

    Welbourn, Sarah; Kao, Sandra; Du Pont, Kelly E; Andrew, Amy J; Berndsen, Christopher E; Strebel, Klaus

    2015-02-01

    BST-2/tetherin is a cellular host factor capable of restricting the release of a variety of enveloped viruses, including HIV-1. Structurally, BST-2 consists of an N-terminal cytoplasmic domain, a transmembrane domain, an ectodomain, and a C-terminal membrane anchor. The BST-2 ectodomain encodes three cysteine residues in its N-terminal half, each of which can contribute to the formation of cysteine-linked dimers. We previously reported that any one of the three cysteine residues is sufficient to produce functional BST-2 dimers. Here we investigated the importance of cysteine positioning on the ectodomain for functional dimerization of BST-2. Starting with a cysteine-free monomeric form of BST-2, individual cysteine residues were reintroduced at various locations throughout the ectodomain. The resulting BST-2 variants were tested for expression, dimerization, surface presentation, and inhibition of HIV-1 virus release. We found significant flexibility in the positioning of cysteine residues, although the propensity to form cysteine-linked dimers generally decreased with increasing distance from the N terminus. Interestingly, all BST-2 variants, including the one lacking all three ectodomain cysteines, retained the ability to form non-covalent dimers, and all of the BST-2 variants were efficiently expressed at the cell surface. Importantly, not all BST-2 variants capable of forming cysteine-linked dimers were functional, suggesting that cysteine-linked dimerization of BST-2 is necessary but not sufficient for inhibiting virus release. Our results expose new structural constraints governing the functional dimerization of BST-2, a property essential to its role as a restriction factor tethering viruses to the host cell.

  14. Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family†

    PubMed Central

    Sobrado, Pablo; Fitzpatrick, Paul F.

    2006-01-01

    The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide. We have previously identified tryptophan 2-monooxygenase as a homologue of l-amino acid oxidase [Sobrado, P., and Fitzpatrick, P. F. (2002) Arch. Biochem. Biophys. 402, 24–30]. On the basis of the sequence comparisons of the different LAAO family members, Arg98 of tryptophan 2-monooxygenase can be identified as an active site residue which interacts with the carboxylate of the amino acid substrate. The catalytic properties of R98K and R98A tryptophan 2-monooxygenase have been characterized to evaluate the role of this residue. Mutation of Arg98 to lysine decreases the first-order rate constant for flavin reduction by 180-fold and the second-order rate constant for flavin oxidation by 26-fold, has no significant effect on the Kd value for tryptophan or the Ki value for the competitive inhibitor indoleacetamide, and increases the Ki value for indolepyruvate less than 2-fold. Mutation of this residue to alanine decreases the rate constants for reduction and oxidation an additional 5- and 2-fold, respectively, and increases the Kd value for tryptophan and the Ki value for indolepyruvate by 31- and 17-fold, respectively, while having an only 2-fold effect on the Ki value for indoleacetamide. Both mutations increase the value of the primary deuterium isotope effect with tryptophan as a substrate, consistent with a later transition state. Both mutant enzymes catalyze a simple oxidase reaction, producing indolepyruvate and hydrogen peroxide. The pH dependences of the V/Ktrp values for the mutant enzymes show that the anionic form of the substrate is preferred but that the zwitterionic form is a substrate. The results are consistent with the interaction between Arg98 and the carboxylate of the amino acid substrate being critical for correct positioning of the substrate in the active site for efficient catalysis. PMID:14636049

  15. Structural Dissection of the Active Site of Thermotoga maritima β-Galactosidase Identifies Key Residues for Transglycosylating Activity.

    PubMed

    Talens-Perales, David; Polaina, Julio; Marín-Navarro, Julia

    2016-04-13

    Glycoside hydrolases, specifically β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from Thermotoga maritima has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis. Rational design of mutations was based on homologous sequence analysis and structural modeling. Analysis of mutant enzymes indicated that residue W959, or an alternative aromatic residue at this position, is critical for the synthesis of β-3'-galactosyl-lactose, the major GOS obtained with the wild-type enzyme. Mutants W959A and W959C, but not W959F, showed an 80% reduced synthesis of this GOS. Other substitutions, N574S, N574A, and F571L, increased the synthesis of β-3'-galactosyl-lactose about 40%. Double mutants F571L/N574S and F571L/N574A showed an increase of about 2-fold.

  16. Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

    SciTech Connect

    Eun Jin Kim; Jian Feng; Matthew R. Bramlett; Paul A. Lindahl

    2004-05-18

    OAK-B135 Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidation of CO to CO2 at a nickel-iron-sulfur active-site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semi-conserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was ''rescued'' by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also ''rescued'' by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no EPR signals originating from the C-cluster. Electronic absorption and metal analysis suggests that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity.

  17. Modification of residue 42 of the active site loop with a lysine-mimetic side chain rescues isochorismate-pyruvate lyase activity in Pseudomonas aeruginosa PchB.

    PubMed

    Olucha, José; Meneely, Kathleen M; Lamb, Audrey L

    2012-09-25

    PchB is an isochorismate-pyruvate lyase from Pseudomonas aeruginosa. A positively charged lysine residue is located in a flexible loop that behaves as a lid to the active site, and the lysine residue is required for efficient production of salicylate. A variant of PchB that lacks the lysine at residue 42 has a reduced catalytic free energy of activation of up to 4.4 kcal/mol. Construction of a lysine isosteric residue bearing a positive charge at the appropriate position leads to the recovery of 2.5-2.7 kcal/mol (about 60%) of the 4.4 kcal/mol by chemical rescue. Exogenous addition of ethylamine to the K42A variant leads to a neglible recovery of activity (0.180 kcal/mol, roughly 7% rescue), whereas addition of propylamine caused an additional modest loss in catalytic power (0.056 kcal/mol, or 2% loss). This is consistent with the view that (a) the lysine-42 residue is required in a specific conformation to stabilize the transition state and (b) the correct conformation is achieved for a lysine-mimetic side chain at site 42 in the course of loop closure, as expected for transition-state stabilization by the side chain ammonio function. That the positive charge is the main effector of transition state stabilization is shown by the construction of a lysine-isosteric residue capable of exerting steric effects and hydrogen bonding but not electrostatic effects, leading to a modest increase of catalytic power (0.267-0.505 kcal/mol of catalytic free energy, or roughly 6-11% rescue). PMID:22970849

  18. Thioredoxin A Active-Site Mutants Form Mixed Disulfide Dimers That Resemble Enzyme–Substrate Reaction Intermediates

    PubMed Central

    Kouwen, Thijs R.H.M.; Andréll, Juni; Schrijver, Rianne; Dubois, Jean-Yves F.; Maher, Megan J.; Iwata, So; Carpenter, Elisabeth P.; van Dijl, Jan Maarten

    2008-01-01

    Thioredoxin functions in nearly all organisms as the major thiol–disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as “mixed disulfide fishing” in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme–substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5Å) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes. PMID:18455736

  19. The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues

    SciTech Connect

    Kurooka, Hisanori; Sugai, Manabu; Mori, Kentaro; Yokota, Yoshifumi

    2013-04-19

    Highlights: •Sodium arsenite induces cytoplasmic accumulation of Id3. •Arsenite binds to closely spaced N-terminal cysteine residues of Id3. •N-terminal cysteines are essential for arsenite-induced nuclear export of Id3. •Nuclear export of Id3 counteracts its transcriptional repression activity. -- Abstract: Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite.

  20. Probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor N-bromoacetylethanolamine phosphate.

    PubMed Central

    Meng, M.; Chane, T. L.; Sun, Y. J.; Hsiao, C. D.

    1999-01-01

    Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the interconversion of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate by promoting an intrahydrogen transfer between C1 and C2. A conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. In the present study, this conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was subjected to mutational analysis, and the mutational effect on the inactivation kinetics by N-bromoacetylethanolamine phosphate was investigated. The substitution of His311 with alanine, asparagine, or glutamine resulted in the decrease of activity, in k(cat)/K(M), by a factor of 10(3), indicating the importance of this residue. N-bromoacetylethanolamine phosphate inactivated irreversibly the activity of wild-type phosphoglucose isomerase; however, His311 --> Ala became resistant to this inhibitor, indicating that His311 is located in the active site and is responsible for the inactivation of the enzyme by this active site-directed inhibitor. The pKa of His311 was estimated to be 6.31 according to the pH dependence of the inactivation. The proximity of this value with the pKa value of 6.35, determined from the pH dependence of k(cat)/K(M), supports a role of His311 as a general base in the catalysis. PMID:10595547

  1. The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants☆

    PubMed Central

    Tsoumpra, Maria K.; Muniz, Joao R.; Barnett, Bobby L.; Kwaasi, Aaron A.; Pilka, Ewa S.; Kavanagh, Kathryn L.; Evdokimov, Artem; Walter, Richard L.; Von Delft, Frank; Ebetino, Frank H.; Oppermann, Udo; Russell, R. Graham G.; Dunford, James E.

    2015-01-01

    Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding. PMID:26318908

  2. The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants.

    PubMed

    Tsoumpra, Maria K; Muniz, Joao R; Barnett, Bobby L; Kwaasi, Aaron A; Pilka, Ewa S; Kavanagh, Kathryn L; Evdokimov, Artem; Walter, Richard L; Von Delft, Frank; Ebetino, Frank H; Oppermann, Udo; Russell, R Graham G; Dunford, James E

    2015-12-01

    Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding. PMID:26318908

  3. Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability

    PubMed Central

    Yanagisawa, Tatsuo; Takahashi, Hideyuki; Suzuki, Takehiro; Masuda, Akiko; Dohmae, Naoshi; Yokoyama, Shigeyuki

    2016-01-01

    Translation elongation factor P (EF-P), a ubiquitous protein over the entire range of bacterial species, rescues ribosomal stalling at consecutive prolines in proteins. In Escherichia coli and Salmonella enterica, the post-translational β-lysyl modification of Lys34 of EF-P is important for the EF-P activity. The β-lysyl EF-P modification pathway is conserved among only 26–28% of bacteria. Recently, it was found that the Shewanella oneidensis and Pseudomonas aeruginosa EF-P proteins, containing an Arg residue at position 32, are modified with rhamnose, which is a novel post-translational modification. In these bacteria, EF-P and its Arg modification are both dispensable for cell viability, similar to the E. coli and S. enterica EF-P proteins and their Lys34 modification. However, in the present study, we found that EF-P and Arg32 are essential for the viability of the human pathogen, Neisseria meningitidis. We therefore analyzed the modification of Arg32 in the N. meningitidis EF-P protein, and identified the same rhamnosyl modification as in the S. oneidensis and P. aeruginosa EF-P proteins. N. meningitidis also has the orthologue of the rhamnosyl modification enzyme (EarP) from S. oneidensis and P. aeruginosa. Therefore, EarP should be a promising target for antibacterial drug development specifically against N. meningitidis. The pair of genes encoding N. meningitidis EF-P and EarP suppressed the slow-growth phenotype of the EF-P-deficient mutant of E. coli, indicating that the activity of N. meningitidis rhamnosyl–EF-P for rescuing the stalled ribosomes at proline stretches is similar to that of E. coli β-lysyl–EF-P. The possible reasons for the unique requirement of rhamnosyl–EF-P for N. meningitidis cells are that more proline stretch-containing proteins are essential and/or the basal ribosomal activity to synthesize proline stretch-containing proteins in the absence of EF-P is lower in this bacterium than in others. PMID:26840407

  4. The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1

    PubMed Central

    Skryhan, Katsiaryna; Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Marri, Lucia; Mellor, Silas B.; Glaring, Mikkel A.; Jensen, Poul E.; Palcic, Monica M.; Blennow, Andreas

    2015-01-01

    Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98–99%). In addition of being part of a redox directed activity “light switch”, reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings

  5. Chemical modification of xylanase from Trichosporon cutaneum shows the presence of carboxyl groups and cysteine residues essential for enzyme activity.

    PubMed

    Wen, L; Miao, Z W; Qing, W D

    1999-08-01

    The endo-beta-1,4-xylanase (EC 3.2.1.8) from Trichosporon cutaneum was chemically modified using amino acid-specific reagents. The enzyme does not bear arginines essential for activity, since 1,2-cyclohexanedione and 2,3-butanedione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Reaction of the enzyme with tetranitromethane and N-acetylimidazole did not result in a significant activity loss as a result of modification of tyrosine residues. The water-soluble carbodiimide 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide inactivated the xylanase rapidly and completely in a pseudo-first-order process, and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. A mixture of neutral xylooligomers provided significant protection of the enzyme against this carbodiimide inactivation. Reaction of the xylanase with 2,4,6-trinitrobenzene sulfonic acid did not result in a significant activity loss as a result of modification of lysine residues. Titration of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetamide and p-chloromercuribenzoate indicated the presence of a free/active thiol group. Xylan completely protected the enzyme from inactivation by p-hydroxymercuribenzoate, suggesting the presence of cysteine at the substrate-binding site. Inactivation of xylanase by p-hydroxymercuribenzoate could be restored by cysteine. PMID:10609644

  6. Contribution of cysteine residues to the structure and function of herpes simplex virus gH/gL

    SciTech Connect

    Cairns, Tina M. . E-mail: tmcairns@biochem.dental.upenn.edu; Landsburg, Daniel J. . E-mail: dlandsbu@temple.edu; Charles Whitbeck, J. . E-mail: whitbeck@biochem.dental.upenn.edu; Eisenberg, Roselyn J. . E-mail: roselyn@biochem.dental.upenn.edu; Cohen, Gary H. . E-mail: cohen@biochem.dental.upenn.edu

    2005-02-20

    In HSV types 1 and 2, gH forms a noncovalent heterodimer with gL. Previous studies demonstrated that the first 323 amino acids of gH1 and the first 161 amino acids of gL1 are sufficient for gH/gL binding. For gL1, substitution of any of its four cysteine (C) residues (all located within the gH/gL binding region) destroyed gH binding and function. Although gH1 contains 8 cysteines in its ectodomain, gH 2 contains 7 (C3 of gH1 is replaced by arginine in gH2). We found that mutation of any of the four C-terminal cysteines led to a reduction or loss of gH/gL function. Mutation of C5 or C6 in gH1 or gH2 rendered the proteins non-functional. However, substitution of C7 and/or C8 in gH1 has a definite negative impact on cell-cell fusion, although these mutations had less effect on complementation. Remarkably, all four gH1 N-terminal cysteines could be mutated simultaneously with little effect on fusion or complementation. As gH2 already lacks C3, we constructed a triple mutant (gH2-C1/2/4) which exhibited a similar phenotype. Since gH1 is known to bind gL2 and vice versa, we wondered whether binding of gH2 to the heterologous gL1 would enhance the fusion defect seen with the gH2-C2 mutant. The combination of mutant gH2-C2 with wild-type gL1 was nonfunctional in a cell-cell fusion assay. Interestingly, the reciprocal was not true, as gH1-C2 could utilize both gL1 and gL2. These findings suggest that there is a structural difference in the gH2 N-terminus as compared to gH1. We also present genetic evidence for at least one disulfide bond within gH2, between cysteines 2 and 4.

  7. Human single-chain urokinase is activated by the omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues.

    PubMed

    Järvinen, Hanna M; Laakkonen, Liisa; Haiko, Johanna; Johansson, Tiira; Juuti, Katri; Suomalainen, Marjo; Buchrieser, Carmen; Kalkkinen, Nisse; Korhonen, Timo K

    2013-08-01

    Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in omptin proteolysis.

  8. Mechanism of the Flavoprotein L-Hydroxynicotine Oxidase: Kinetic Mechanism, Substrate Specificity, Reaction Product, and Roles of Active-Site Residues.

    PubMed

    Fitzpatrick, Paul F; Chadegani, Fatemeh; Zhang, Shengnan; Roberts, Kenneth M; Hinck, Cynthia S

    2016-02-01

    The flavoprotein L-hydroxynicotine oxidase (LHNO) catalyzes an early step in the bacterial catabolism of nicotine. Although the structure of the enzyme establishes that it is a member of the monoamine oxidase family, LHNO is generally accepted to oxidize a carbon-carbon bond in the pyrrolidine ring of the substrate and has been proposed to catalyze the subsequent tautomerization and hydrolysis of the initial oxidation product to yield 6-hydroxypseudooxynicotine [Kachalova, G., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 4800-4805]. Analysis of the product of the enzyme from Arthrobacter nicotinovorans by nuclear magnetic resonance and continuous-flow mass spectrometry establishes that the enzyme catalyzes the oxidation of the pyrrolidine carbon-nitrogen bond, the expected reaction for a monoamine oxidase, and that hydrolysis of the amine to form 6-hydroxypseudooxynicotine is nonenzymatic. On the basis of the kcat/Km and kred values for (S)-hydroxynicotine and several analogues, the methyl group contributes only marginally (∼ 0.5 kcal/mol) to transition-state stabilization, while the hydroxyl oxygen and pyridyl nitrogen each contribute ∼ 4 kcal/mol. The small effects on activity of mutagenesis of His187, Glu300, or Tyr407 rule out catalytic roles for all three of these active-site residues.

  9. Functional constituents of the active site of human neutrophil collagenase.

    PubMed

    Mookhtiar, K A; Wang, F; Van Wart, H E

    1986-05-01

    A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10-phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue.

  10. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    SciTech Connect

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

  11. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    DOE PAGES

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog ofmore » uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.« less

  12. Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics.

    PubMed

    Toledo, José Carlos; Audi, Renata; Ogusucu, Renata; Monteiro, Gisele; Netto, Luis Eduardo Soares; Augusto, Ohara

    2011-05-01

    Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 ± 0.2) × 10⁷M⁻¹ s⁻¹) and peroxynitrite ((3.7 ± 0.4) × 10⁵ M⁻¹ s⁻¹) at pH 7.4 and 25°C.

  13. A single gamma-carboxyglutamic acid residue in a novel cysteine-rich secretory protein without propeptide.

    PubMed

    Hansson, Karin; Thämlitz, Ann-Marie; Furie, Bruce; Furie, Barbara C; Stenflo, Johan

    2006-10-24

    Gamma-glutamyl carboxylase catalyzes the modification of specific glutamyl residues to gamma-carboxyglutamyl (Gla) residues in precursor proteins that possess the appropriate gamma-carboxylation recognition signal within the propeptide region. We describe the immunopurification and first biochemical characterization of an invertebrate high molecular weight Gla-containing protein with homologues in mammals. The protein, named GlaCrisp, was isolated from the venom of the marine cone snail Conus marmoreus. GlaCrisp gave intense signals in Western blot experiments employing the Gla-specific antibody M3B, and the presence of Gla was chemically confirmed by amino acid analysis after alkaline hydrolysis. Characterization of a full-length cDNA clone encoding GlaCrisp deduced a precursor containing an N-terminal signal peptide but, unlike other Gla-containing proteins, no apparent propeptide. The predicted mature protein of 265 amino acid residues showed considerable sequence similarity to the widely distributed cysteine-rich secretory protein family and closest similarity (65% identity) to the recently described substrate-specific protease Tex31. In addition, two cDNA clones encoding the precursors of two isoforms of GlaCrisp were identified. The predicted precursor isoforms differed at three amino acid positions (-6, 9, and 25). Analysis by Edman degradation and nanoelectrospray ionization mass spectrometry, before and after methyl esterfication, identified a Gla residue at amino acid position 9 in GlaCrisp. This is the first example of a Gla-containing protein without an obvious gamma-carboxylation recognition site. The results define a new class of Gla proteins and support the notion that gamma-carboxylation of glutamyl residues is phylogenetically older than blood coagulation and the vertebrate lineage.

  14. Ab Initio MD Simulations of the Brønsted Acidity of Glutathione in Aqueous Solutions: Predicting pKa Shifts of the Cysteine Residue.

    PubMed

    Tummanapelli, Anil Kumar; Vasudevan, Sukumaran

    2015-12-10

    The tripeptide glutathione (GSH) is one of the most abundant peptides and the major repository for nonprotein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thiol-disulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influence the redox couple, and hence the pKa value of the cysteine residue of GSH is critical to its functioning. Here we report ab initio Car-Parrinello molecular dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. We show that the free-energy landscape for the protonation-deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared with the isolated cysteine amino acid.

  15. Complete replacement of basic amino acid residues with cysteines in Rickettsia prowazekii ATP/ADP translocase.

    PubMed

    Alexeyev, Mikhail F; Winkler, Herbert H

    2002-09-20

    The ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein are involved in electrostatic interactions with negatively charged substrates. We tested this hypothesis by individually mutating all basic residues in Tlc to Cys. Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than 80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited higher than wild-type (WT) activity. At least in one case this up-effect was additive and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT protein. Since in these two single mutants and in the corresponding double mutant K(m)'s were similar to that of WT protein, we conclude that Tlc may have evolved a mechanism that limits the transporter's exchange rate and that at least these two basic residues play a key role in that mechanism. Based on the alignment of 16 Tlc homologs, the loss of activity in the mutants poorly correlates with charge conservation within the Tlc family. Also, despite the presence of three positively charged and one negatively charged intramembrane residues, we have failed to identify potential charge pairs (salt bridges) by either charge reversal or charge neutralization approaches. PMID:12225862

  16. Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis.

    PubMed Central

    Keresztessy, Z; Brown, K; Dunn, M A; Hughes, M A

    2001-01-01

    The coding sequence of the mature cyanogenic beta-glucosidase (beta-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (K(m)=1.06 mM) and the synthetic p-nitrophenyl beta-D-glucopyranoside (PNP-Glc; K(m)=0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substrate complexes (pK(E)(1)=4.4-4.8, pK(E)(2)=6.7-7.2, pK(ES)(1)=3.9-4.4, pK(ES)(2)=8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased K(m) for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta-glucosidase. PMID:11139381

  17. Dissemination of lipid A deacylases (pagL) among gram-negative bacteria: identification of active-site histidine and serine residues.

    PubMed

    Geurtsen, Jeroen; Steeghs, Liana; Hove, Jan Ten; van der Ley, Peter; Tommassen, Jan

    2005-03-01

    Lipopolysaccharide (LPS) is one of the main constituents of the Gram-negative bacterial outer membrane. It usually consists of a highly variable O-antigen, a less variable core oligosaccharide, and a highly conserved lipid moiety, designated lipid A. Several bacteria are capable of modifying their lipid A architecture in response to external stimuli. The outer membrane-localized lipid A 3-O-deacylase, encoded by the pagL gene of Salmonella enterica serovar Typhimurium, removes the fatty acyl chain from the 3 position of lipid A. Although a similar activity was reported in some other Gram-negative bacteria, the corresponding genes could not be identified. Here, we describe the presence of pagL homologs in a variety of Gram-negative bacteria. Although the overall sequence similarity is rather low, a conserved domain could be distinguished in the C-terminal region. The activity of the Pseudomonas aeruginosa and Bordetella bronchiseptica pagL homologs was confirmed upon expression in Escherichia coli, which resulted in the removal of an R-3-hydroxymyristoyl group from lipid A. Upon deacylation by PagL, E. coli lipid A underwent another modification, which was the result of the activity of the endogenous palmitoyl transferase PagP. Furthermore, we identified a conserved histidine-serine couple as active site residues, suggesting a catalytic mechanism similar to serine hydrolases. The biological function of PagL remains unclear. However, because PagL homologs were found in both pathogenic and nonpathogenic species, PagL-mediated deacylation of lipid A probably does not have a dedicated role in pathogenicity.

  18. A Conserved Active Site Tyrosine Residue of Proline Dehydrogenase Helps Enforce the Preference for Proline over Hydroxyproline as the Substrate†,‡

    PubMed Central

    Ostrander, Elizabeth L.; Larson, John D.; Schuermann, Jonathan P.; Tanner, John J.

    2009-01-01

    Proline dehydrogenase (PRODH) catalyzes the oxidation of L-proline to Delta-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-L-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insights into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analog L-tetrahydro-2-furoic acid were determined at resolutions of 1.75 Å, 1.90 Å and 1.85 Å. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline three-fold and decreases the specificity for proline by factors of twenty (Y540S) and fifty (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate. PMID:19140736

  19. Threonine 79 is a hinge residue that governs the fidelity of DNA polymerase beta by helping to position the DNA within the active site.

    PubMed

    Maitra, Mausumi; Gudzelak, Andrew; Li, Shu-Xia; Matsumoto, Yoshihiro; Eckert, Kristin A; Jager, Joachim; Sweasy, Joann B

    2002-09-20

    DNA polymerase beta (pol beta) is an ideal system for studying the role of its different amino acid residues in the fidelity of DNA synthesis. In this study, the T79S variant of pol beta was identified using an in vivo genetic screen. T79S is located in the N-terminal 8-kDa domain of pol beta and has no contact with either the DNA template or the incoming dNTP substrate. The T79S protein produced 8-fold more multiple mutations in the herpes simplex virus type 1-thymidine kinase assay than wild-type pol beta. Surprisingly, T79S is a misincorporation mutator only when using a 3'-recessed primer-template. In the presence of a single nucleotide-gapped DNA substrate, T79S displays an antimutator phenotype when catalyzing DNA synthesis opposite template C and has similar fidelity as wild type opposite templates A, G, or T. Threonine 79 is located directly between two helix-hairpin-helix motifs located within the 8-kDa and thumb domains of pol beta. As the pol beta enzyme closes into its active form, the helix-hairpin-helix motifs appear to assist in the production and stabilization of a 90 degrees bend of the DNA. The function of the bent DNA is to present the templating base to the incoming nucleotide substrate. We propose that Thr-79 is part of a hydrogen bonding network within the helix-hairpin-helix motifs that is important for positioning the DNA within the active site. We suggest that alteration of Thr-79 to Ser disrupts this hydrogen bonding network and results in an enzyme that is unable to bend the DNA into the proper geometry for accurate DNA synthesis.

  20. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors.

    PubMed

    Zhang, Tinghu; Kwiatkowski, Nicholas; Olson, Calla M; Dixon-Clarke, Sarah E; Abraham, Brian J; Greifenberg, Ann K; Ficarro, Scott B; Elkins, Jonathan M; Liang, Yanke; Hannett, Nancy M; Manz, Theresa; Hao, Mingfeng; Bartkowiak, Bartlomiej; Greenleaf, Arno L; Marto, Jarrod A; Geyer, Matthias; Bullock, Alex N; Young, Richard A; Gray, Nathanael S

    2016-10-01

    Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities. PMID:27571479

  1. Mutation of active site serine residue with cysteine displays change in acyl-acceptor preference of β-peptidyl aminopeptidase from Pseudomonas aeruginosa PAO1.

    PubMed

    Arima, Jiro; Tanaka, Ayumi; Morimoto, Masazumi; Mori, Nobuhiro

    2014-02-01

    A β-peptidyl aminopeptidase, a peptidase belonging to the P1 family, catalyzes aminolysis in accordance with its hydrolytic activity. We specifically examined β-peptidyl aminopeptidase of Pseudomonas aeruginosa PAO1 (BapF) to assess the effects of mutation of catalytic Ser with Cys or Thr on its catalytic ability. Recombinant BapF and its S237C mutant exhibited p-nitroaniline release activity toward β-homo-Gly-p-nitroanilide (βhGly-pNA), but the products of the enzyme reaction differed completely from one another. Wild-type BapF showed βhGly-βhGly-pNA synthetic activity, but the product vanished in a few minutes and converted to free βhGly. In contrast, the product βhGly-βhGly-pNA was synthesized by S237C BapF efficiently without degradation, indicating that because of the mutation, the enzyme came to recognize only the amine group as an acyl acceptor instead of water. Furthermore, a difference in acyl acceptor preference between that of wild type and S237C BapF was observed. When using cysteamine as an acyl acceptor, βhGly-cysteamine was synthesized only in the reaction using S237C BapF. In contrast, S237C BapF was unable to synthesize βhGly-cystamine when using cystamine as an acyl acceptor, although it was synthesized by wild-type BapF. Such a dynamic change in the acyl acceptor by the mutation of catalytic Ser with Cys is regarded as a unique feature of family P1 peptidases.

  2. Amino-terminal cysteine residues differentially influence RGS4 protein plasma membrane targeting, intracellular trafficking, and function.

    PubMed

    Bastin, Guillaume; Singh, Kevin; Dissanayake, Kaveesh; Mighiu, Alexandra S; Nurmohamed, Aliya; Heximer, Scott P

    2012-08-17

    Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

  3. Phosphoribulokinase from Chlamydomonas reinhardtii: a Benson-Calvin cycle enzyme enslaved to its cysteine residues.

    PubMed

    Thieulin-Pardo, Gabriel; Remy, Thérèse; Lignon, Sabrina; Lebrun, Régine; Gontero, Brigitte

    2015-04-01

    Phosphoribulokinase (PRK) in the green alga Chlamydomonas reinhardtii is a finely regulated and well-studied enzyme of the Benson-Calvin cycle. PRK can form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small chloroplast protein CP12. This study aimed to determine the molecular determinants on PRK involved in the complex and the mechanism of action of a recently described novel regulation of PRK that involves glutathionylation. A combination of mass spectrometry, mutagenesis and activity analyses showed that Cys16, besides its role as the binding site of ATP, was also the site for S-glutathionylation. Previous kinetic analysis of the C55S mutant showed that in the oxidized inactive form of PRK, this residue formed a disulfide bridge with the Cys16 residue. This is the only bridge reported for PRK in the literature. Our data show for the first time that a disulfide bridge between Cys243 and Cys249 on PRK is required to form the PRK-GAPDH-CP12 complex. These results uncover a new mechanism for the PRK-GAPDH-CP12 formation involving a thiol disulfide exchange reaction with CP12 and identify Cys16 of PRK as a target of glutathionylation acting against oxidative stress. Although Cys16 is the key residue involved in binding ATP and acting as a defense against oxidative damage, the formation of the algal ternary complex requires the formation of another disulfide bridge on PRK involving Cys243 and Cys249.

  4. A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

    PubMed Central

    Beveridge, A. J.

    1996-01-01

    The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis. PMID:8819168

  5. Influences of Proline and Cysteine Residues on Fragment Yield in Matrix-Assisted Laser Desorption/Ionization In-Source Decay Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Asakawa, Daiki; Smargiasso, Nicolas; Quinton, Loïc; De Pauw, Edwin

    2014-06-01

    Matrix-assisted laser desorption/ionization in-source decay produces highly informative fragments for the sequencing of peptides/proteins. Among amino acids, cysteine and proline residues were found to specifically influence the fragment yield. As they are both frequently found in small peptide structures for which de novo sequencing is mandatory, the understanding of their specific behaviors would allow useful fragmentation rules to be established. In the case of cysteine, a c•/ w fragment pair originating from Xxx-Cys is formed by side-chain loss from the cysteine residue. The presence of a proline residue contributes to an increased yield of ISD fragments originating from N-Cα bond cleavage at Xxx1-Xxx2Pro, which is attributable to the cyclic structure of the proline residue. Our results suggest that the aminoketyl radical formed by MALDI-ISD generally induces the homolytic N-Cα bond cleavage located on the C-terminal side of the radical site. In contrast, N-Cα bond cleavage at Xxx-Pro produces no fragments and the N-Cα bond at the Xxx1-Xxx2Pro bond is alternatively cleaved via a heterolytic cleavage pathway.

  6. Pyroacm Resin: An Acetamidomethyl Derived Resin for Solid Phase Synthesis of Peptides through Side Chain Anchoring of C-Terminal Cysteine Residues.

    PubMed

    Juvekar, Vinayak; Gong, Young Dae

    2016-02-19

    The design, synthesis and utilization of an efficient acetamidomethyl derived resin for the peptide synthesis is presented using established Fmoc and Boc protocols via side chain anchoring. Cleavage of the target peptide from the resin is performed using carboxymethylsulfenyl chloride under mild conditions which gave in situ thiol-sulfenyl protection of the cysteine residues. The utility of the resin is successfully demonstrated through applications to the syntheses of model peptides and natural products Riparin 1.1 and Riparin 1.2.

  7. Phage display-mediated discovery of novel tyrosinase-targeting tetrapeptide inhibitors reveals the significance of N-terminal preference of cysteine residues and their functional sulfur atom.

    PubMed

    Lee, Yu-Ching; Hsiao, Nai-Wan; Tseng, Tien-Sheng; Chen, Wang-Chuan; Lin, Hui-Hsiung; Leu, Sy-Jye; Yang, Ei-Wen; Tsai, Keng-Chang

    2015-02-01

    Tyrosinase, a key copper-containing enzyme involved in melanin biosynthesis, is closely associated with hyperpigmentation disorders, cancer, and neurodegenerative diseases, and as such, it is an essential target in medicine and cosmetics. Known tyrosinase inhibitors possess adverse side effects, and there are no safety regulations; therefore, it is necessary to develop new inhibitors with fewer side effects and less toxicity. Peptides are exquisitely specific to their in vivo targets, with high potencies and relatively few off-target side effects. Thus, we systematically and comprehensively investigated the tyrosinase-inhibitory abilities of N- and C-terminal cysteine/tyrosine-containing tetrapeptides by constructing a phage-display random tetrapeptide library and conducting computational molecular docking studies on novel tyrosinase tetrapeptide inhibitors. We found that N-terminal cysteine-containing tetrapeptides exhibited the most potent tyrosinase-inhibitory abilities. The positional preference of cysteine residues at the N terminus in the tetrapeptides significantly contributed to their tyrosinase-inhibitory function. The sulfur atom in cysteine moieties of N- and C-terminal cysteine-containing tetrapeptides coordinated with copper ions, which then tightly blocked substrate-binding sites. N- and C-terminal tyrosine-containing tetrapeptides functioned as competitive inhibitors against mushroom tyrosinase by using the phenol ring of tyrosine to stack with the imidazole ring of His263, thus competing for the substrate-binding site. The N-terminal cysteine-containing tetrapeptide CRVI exhibited the strongest tyrosinase-inhibitory potency (with an IC50 of 2.7 ± 0.5 μM), which was superior to those of the known tyrosinase inhibitors (arbutin and kojic acid) and outperformed kojic acid-tripeptides, mimosine-FFY, and short-sequence oligopeptides at inhibiting mushroom tyrosinase.

  8. Redox-sensitive DNA binding by homodimeric Methanosarcina acetivorans MsvR is modulated by cysteine residues

    PubMed Central

    2013-01-01

    Background Methanoarchaea are among the strictest known anaerobes, yet they can survive exposure to oxygen. The mechanisms by which they sense and respond to oxidizing conditions are unknown. MsvR is a transcription regulatory protein unique to the methanoarchaea. Initially identified and characterized in the methanogen Methanothermobacter thermautotrophicus (Mth), MthMsvR displays differential DNA binding under either oxidizing or reducing conditions. Since MthMsvR regulates a potential oxidative stress operon in M. thermautotrophicus, it was hypothesized that the MsvR family of proteins were redox-sensitive transcription regulators. Results An MsvR homologue from the methanogen Methanosarcina acetivorans, MaMsvR, was overexpressed and purified. The two MsvR proteins bound the same DNA sequence motif found upstream of all known MsvR encoding genes, but unlike MthMsvR, MaMsvR did not bind the promoters of select genes involved in the oxidative stress response. Unlike MthMsvR that bound DNA under both non-reducing and reducing conditions, MaMsvR bound DNA only under reducing conditions. MaMsvR appeared as a dimer in gel filtration chromatography analysis and site-directed mutagenesis suggested that conserved cysteine residues within the V4R domain were involved in conformational rearrangements that impact DNA binding. Conclusions Results presented herein suggest that homodimeric MaMsvR acts as a transcriptional repressor by binding Ma PmsvR under non-reducing conditions. Changing redox conditions promote conformational changes that abrogate binding to Ma PmsvR which likely leads to de-repression. PMID:23865844

  9. Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus

    PubMed Central

    Zhang, Yuanwei; Zheng, Qingqing; Sun, Congcong; Song, Jinxing; Gao, Lina; Zhang, Shizhu; Muñoz, Alberto; Read, Nick D.; Lu, Ling

    2016-01-01

    Finely tuned changes in cytosolic free calcium ([Ca2+]c) mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation is a reversible post-translational modification involved in membrane protein trafficking between membranes and in their functional modulation. However, studies on the relationship between palmitoylation and calcium signaling have been limited. Here, we demonstrate that the yeast palmitoyl transferase ScAkr1p homolog, AkrA in Aspergillus nidulans, regulates [Ca2+]c homeostasis. Deletion of akrA showed marked defects in hyphal growth and conidiation under low calcium conditions which were similar to the effects of deleting components of the high-affinity calcium uptake system (HACS). The [Ca2+]c dynamics in living cells expressing the calcium reporter aequorin in different akrA mutant backgrounds were defective in their [Ca2+]c responses to high extracellular Ca2+ stress or drugs that cause ER or plasma membrane stress. All of these effects on the [Ca2+]c responses mediated by AkrA were closely associated with the cysteine residue of the AkrA DHHC motif, which is required for palmitoylation by AkrA. Using the acyl-biotin exchange chemistry assay combined with proteomic mass spectrometry, we identified protein substrates palmitoylated by AkrA including two new putative P-type ATPases (Pmc1 and Spf1 homologs), a putative proton V-type proton ATPase (Vma5 homolog) and three putative proteins in A. nidulans, the transcripts of which have previously been shown to be induced by extracellular calcium stress in a CrzA-dependent manner. Thus, our findings provide strong evidence that the AkrA protein regulates [Ca2+]c homeostasis by palmitoylating these protein candidates and give new insights the role of palmitoylation in the regulation of calcium-mediated responses to extracellular, ER or plasma membrane stress. PMID:27058039

  10. Conserved Cysteine Residue in the DNA-Binding Domain of the Bovine Papillomavirus Type 1 E2 Protein Confers Redox Regulation of the DNA- Binding Activity in Vitro

    NASA Astrophysics Data System (ADS)

    McBride, Alison A.; Klausner, Richard D.; Howley, Peter M.

    1992-08-01

    The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfydryl group(s) may be protected by DNA binding. Sensitivity to sulfydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.

  11. Amino acid sequence of the 203-residue fragment of the heavy chain of chicken gizzard myosin containing the SH1-type cysteine residue.

    PubMed

    Onishi, H; Maita, T; Miyanishi, T; Watanabe, S; Matsuda, G

    1986-12-01

    A fluorescent fragment of Mr = 23,800 was obtained by the papain digestion of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene diamine (abbreviated as IAEDANS)-modified chicken gizzard myosin. The fragment was isolated by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine-HCl followed by anion exchange chromatography on a QAE Sephadex A-50 column. This fragment contained 203 amino acid residues which could be assigned as a COOH-terminal part of the S-1 heavy chain based on the homology with the known sequence of rabbit skeletal myosin fragment. The amino acid sequence was K-G-M-F-R-T-V- G-Q-L-Y-K-E-Q-L-T-K-L-M-T-T-L-R-N-T-N-P-N-F-V-R-C-I-I-P-N-H-E-K-R-A- G-K-L-D-A-H-L-V-L-E-Q-L-R-C-N-G-V-L-E-G-I-R-I-C-R-Q-G-F-P-N-R-I-V-F-Q- E-F-R-Q-R-Y-E-I-L-A-A-N-A-I-P-K-G-F-M-D-G-K-Q-A-C-I-L-M -I-K-A-L-E-L- D-P-N-L-Y-R-I-G-Q-S-K-I-F-F-R-T-G-V-L-A-H-L-E-E-E-R-D-L-K- I-T-D-V-I-I-A- F-Q-A-Q-C-R-G-Y-L-A-R-K-A-F-A-K-R-Q-Q-Q-L-T-A-M-K-V-I-Q-R-N-C-A -A-Y-L-K-L-R-N-W-Q-W-W-R-L-F-T-K-V-K-P-L-L-Q-V-T-R. The cysteine residue which was modified with IAEDANS was of the SH1 type (Cys-65). Pro-197 was suggested to be the NH2-terminal boundary of the alpha-helical coiled-coil rod sequence of gizzard myosin, based on the homology with the nematode sequence reported by MacLachlan and Karn (Proc. Natl. Acad. Sci. U.S. 80, 4253-4257 (1983)). Three different COOH-terminal peptides (Val-Lys-Pro-Leu-Leu-Gln-Val-Thr-Arg, Val-Lys-Pro-Leu-Leu-Gln, and Val-Lys-Pro-Leu-Leu) were isolated from the tryptic digest of this fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

  12. Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation.

    PubMed Central

    Jensen, P E; Reid, J D; Hunter, C N

    2000-01-01

    The enzyme magnesium protoporphyrin chelatase catalyses the insertion of magnesium into protoporphyrin, the first committed step in chlorophyll biosynthesis. Magnesium chelatase from the cyanobacterium Synechocystis PCC6803 has been reconstituted in a highly active state as a result of purifying the constituent proteins from strains of Escherichia coli that overproduce the ChlH, ChlI and ChlD subunits. These individual subunits were analysed for their sensitivity to N-ethylmaleimide (NEM), in order to assess the roles that cysteine residues play in the partial reactions that comprise the catalytic cycle of Mg(2+) chelatase, such as the ATPase activity of ChlI, and the formation of ChlI-ChlD-MgATP and ChlH-protoporphyrin complexes. It was shown that NEM binds to ChlI and inhibits the ATPase activity of this subunit, and that prior incubation with MgATP affords protection against inhibition. Quantitative analysis of the effects of NEM binding on ChlI-catalysed ATPase activity showed that three out of four thiols per ChlI molecule are available to react with NEM, but only one cysteine residue per ChlI subunit is essential for ATPase activity. In contrast, the cysteines in ChlD are not essential for Mg(2+) chelatase activity, and the formation of the ChlI-ChlD-ATP complex can proceed with NEM-treated ChlI. Neither the ATPase activity of ChlI nor NEM-modifiable cysteines are therefore required to form the ChlI-ChlD-MgATP complex. However, this complex cannot catalyse magnesium chelation in the presence of the ChlH subunit, protoporphyrin and Mg(2+) ions. The simplest explanation for this is that in an intact Mg(2+) chelatase complex the ATPase activity of ChlI drives the chelation process. NEM binds to ChlH and inhibits the chelation reaction, and this effect can be partially alleviated by pre-incubating ChlH with magnesium and ATP. We conclude that cysteine residues play an important role in the chelation reaction, in respect of the ChlI-MgATP association, ATP

  13. SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion.

    PubMed

    Madu, Ikenna G; Belouzard, Sandrine; Whittaker, Gary R

    2009-10-25

    The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

  14. SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

    PubMed Central

    Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

    2013-01-01

    The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1–S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell–cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion. PMID:19717178

  15. SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

    SciTech Connect

    Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

    2009-10-25

    The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

  16. Formation of intersubunit disulfide bonds and properties of the single histidine and cysteine residues in each subunit relative to the decameric structure of cyanase.

    PubMed

    Anderson, P M; Korte, J J; Holcomb, T A; Cho, Y G; Son, C M; Sung, Y C

    1994-05-27

    Reaction of the single cysteine residue in each subunit of cyanase with certain SH reagents gives an active decameric derivative that dissociates reversibly to an inactive dimer derivative (Anderson, P. M., Johnson, W. V., Korte, J. J., Xiong, X., Sung, Y.-c., and Fuchs, J. A. (1988) J. Biol. Chem. 263, 5674-5680). Reaction of mixed disulfide dimer derivatives of cyanase with dithiothreitol at 0 degree C results in formation of a disulfide bond between the subunits in the dimer. The disulfide dimer was inactive and did not associate to a decamer; the intersubunit disulfide bond could not be formed when the dimers were associated as a decamer. The two SH groups apparently are in close proximity to each other in the dissociated dimer but not when the dimer is associated to a decamer. Substitution of glycine for the cysteine residue or of tyrosine, asparagine, glycine, valine, or leucine for the single histidine residue in each subunit gave mutant enzymes that were active. However, H113N, H113Y, and C83G were unstable at low temperature and/or ionic strength, dissociating reversibly to an inactive dimer. Efficient reassociation required the presence of bicarbonate or cyanate analog. The results are consistent with a proposed single site per subunit model explaining apparent half-site binding of substrates and the requirement of decameric structure for activity.

  17. Substitution of the cysteine 438 residue in the cytoplasmic tail of the glucagon-like peptide-1 receptor alters signal transduction activity.

    PubMed

    Vázquez, Patricia; Roncero, Isabel; Blázquez, Enrique; Alvarez, Elvira

    2005-04-01

    Several G-protein-coupled receptors contain cysteine residues in the C-terminal tail that may modulate receptor function. In this work we analysed the substitution of Cys438 by alanine in the glucagon-like peptide-1 (GLP-1) receptor (GLPR), which led to a threefold decrease in cAMP production, although endocytosis and cellular redistribution of GLP-1 receptor agonist-induced processes were unaffected. Additionally, cysteine residues in the C-terminal tail of several G-protein-coupled receptors were found to act as substrates for palmitoylation, which might modify the access of protein kinases to this region. His-tagged GLP-1 receptors incorporated 3H-palmitate. Nevertheless, substitution of Cys438 prevented the incorporation of palmitate. Accordingly, we also investigated the effect of substitution of the consensus sequence by protein kinase C (PKC) Ser431/432 in both wild-type and Ala438 GLP-1 receptors. Substitution of Ser431/432 by alanine did not modify the ability of wild-type receptors to stimulate adenylate cyclase or endocytosis and recycling processes. By contrast, the substitution of Ser431/432 by alanine in the receptor containing Ala438 increased the ability to stimulate adenylate cyclase. All types of receptors were mainly internalised through coated pits. Thus, cysteine 438 in the cytoplasmic tail of the GLP-1 receptor would regulate its interaction with G-proteins and the stimulation of adenylyl cyclase. Palmitoylation of this residue might control the access of PKC to Ser431/432.

  18. Comparative Structural Modeling of Six Old Yellow Enzymes (OYEs) from the Necrotrophic Fungus Ascochyta rabiei : Insight into Novel OYE Classes with Differences in Cofactor Binding, Organization of Active Site Residues and Stereopreferences

    PubMed Central

    Nizam, Shadab; Gazara, Rajesh Kumar; Verma, Sandhya; Singh, Kunal; Verma, Praveen Kumar

    2014-01-01

    Old Yellow Enzyme (OYE1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. Irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. In a recent study, we systematically identified OYE proteins from various fungi and classified them into three classes viz. Class I, II and III. However, there is no information about the structural organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Ascochyta rabiei, a filamentous phytopathogen which causes Ascochyta blight (AB) in chickpea possesses six OYEs (ArOYE1-6) belonging to the three OYE classes. Here we carried out comparative homology modeling of six ArOYEs representing all the three classes to get an in depth idea of structural and functional aspects of fungal OYEs. The predicted 3D structures of A. rabiei OYEs were refined and evaluated using various validation tools for their structural integrity. Analysis of FMN binding environment of Class III OYE revealed novel residues involved in interaction. The ligand para-hydroxybenzaldehyde (PHB) was docked into the active site of the enzymes and interacting residues were analyzed. We observed a unique active site organization of Class III OYE in comparison to Class I and II OYEs. Subsequently, analysis of stereopreference through structural features of ArOYEs was carried out, suggesting differences in R/S selectivity of these proteins. Therefore, our comparative modeling study provides insights into the FMN binding, active site organization and stereopreference of different classes of ArOYEs and indicates towards functional differences of these enzymes. This study provides the basis for future investigations towards the biochemical and functional characterization of these enigmatic enzymes. PMID:24776850

  19. Residues Distal to the Active Site Contribute to Enhanced Catalytic Activity of Variant and Hybrid β-Lactamases Derived from CTX-M-14 and CTX-M-15

    PubMed Central

    He, Dandan; Chiou, Jiachi; Zeng, Zhenling; Liu, Lanping; Chen, Xiaojie; Zeng, Li; Chan, Edward Wai Chi

    2015-01-01

    A variety of CTX-M-type extended-spectrum β-lactamases (ESBLs), including hybrid ones, have been reported in China that are uncommon elsewhere. To better characterize the substrate profiles and enzymatic mechanisms of these enzymes, we performed comparative kinetic analyses of both parental and hybrid CTX-M enzymes, including CTX-M-15, -132, -123, -64, -14 and -55, that are known to confer variable levels of β-lactam resistance in the host strains. All tested enzymes were susceptible to serine β-lactamase inhibitors, with sulbactam exhibiting the weakest inhibitory effects. CTX-M-55, which differs from CTX-M-15 by one substitution, A77V, displayed enhanced catalytic activity (kcat/Km) against expanded-spectrum cephalosporins (ESCs). CTX-M-55 exhibits higher structure stability, most likely by forming hydrophobic interactions between A77V and various key residues in different helices, thereby stabilizing the core architecture of the helix cluster, and indirectly contributes to a more stable active site conformation, which in turn shows higher catalytic efficiency and is more tolerant to temperature change. Analyses of the hybrids and their parental prototypes showed that evolution from CTX-M-15 to CTX-M-132, CTX-M-123, and CTX-M-64, characterized by gradual enhancement of catalytic activity to ESCs, was attributed to introduction of different substitutions to amino acids distal to the active site of CTX-M-15. Similarly, the increased hydrolytic activities against cephalosporins and sensitivity to β-lactamase inhibitors, clavulanic acid and sulbactam, of CTX-M-64 were partly due to the amino acids that were different from CTX-M-14 and located at both the C and N termini of CTX-M-64. These data indicate that residues distal to the active site of CTX-Ms contributed to their enhanced catalytic activities to ESCs. PMID:26169409

  20. Role of Cysteine Residues in the Carboxyl-Terminus of the Follicle-Stimulating Hormone Receptor in Intracellular Traffic and Postendocytic Processing.

    PubMed

    Melo-Nava, Brenda; Casas-González, Patricia; Pérez-Solís, Marco A; Castillo-Badillo, Jean; Maravillas-Montero, José L; Jardón-Valadez, Eduardo; Zariñán, Teresa; Aguilar-Rojas, Arturo; Gallay, Nathalie; Reiter, Eric; Ulloa-Aguirre, Alfredo

    2016-01-01

    Posttranslational modifications occurring during the biosynthesis of G protein-coupled receptors include glycosylation and palmitoylation at conserved cysteine residues located in the carboxyl-terminus of the receptor. In a number of these receptors, these modifications play an important role in receptor function and particularly, in intracellular trafficking. In the present study, the three cysteine residues present in the carboxyl-terminus of the human FSHR were replaced with glycine by site-directed mutagenesis. Wild-type and mutant (Cys627/629/655Gly) FSHRs were then transiently expressed in HEK-293 cells and analyzed for cell-surface plasma membrane expression, agonist-stimulated signaling and internalization, and postendocytic processing in the absence and presence of lysosome and/or proteasome inhibitors. Compared with the wild-type FSHR, the triple mutant FSHR exhibited ~70% reduction in plasma membrane expression as well as a profound attenuation in agonist-stimulated cAMP production and ERK1/2 phosphorylation. Incubation of HEK-293 cells expressing the wild-type FSHR with 2-bromopalmitate (palmitoylation inhibitor) for 6 h, decreased plasma membrane expression of the receptor by ~30%. The internalization kinetics and β-arrestin 1 and 2 recruitment were similar between the wild-type and triple mutant FSHR as disclosed by assays performed in non-equilibrium binding conditions and by confocal microscopy. Cells expressing the mutant FSHR recycled the internalized FSHR back to the plasma membrane less efficiently than those expressing the wild-type FSHR, an effect that was counteracted by proteasome but not by lysosome inhibition. These results indicate that replacement of the cysteine residues present in the carboxyl-terminus of the FSHR, impairs receptor trafficking from the endoplasmic reticulum/Golgi apparatus to the plasma membrane and its recycling from endosomes back to the cell surface following agonist-induced internalization. Since in the FSHR these

  1. Role of Cysteine Residues in the Carboxyl-Terminus of the Follicle-Stimulating Hormone Receptor in Intracellular Traffic and Postendocytic Processing

    PubMed Central

    Melo-Nava, Brenda; Casas-González, Patricia; Pérez-Solís, Marco A.; Castillo-Badillo, Jean; Maravillas-Montero, José L.; Jardón-Valadez, Eduardo; Zariñán, Teresa; Aguilar-Rojas, Arturo; Gallay, Nathalie; Reiter, Eric; Ulloa-Aguirre, Alfredo

    2016-01-01

    Posttranslational modifications occurring during the biosynthesis of G protein-coupled receptors include glycosylation and palmitoylation at conserved cysteine residues located in the carboxyl-terminus of the receptor. In a number of these receptors, these modifications play an important role in receptor function and particularly, in intracellular trafficking. In the present study, the three cysteine residues present in the carboxyl-terminus of the human FSHR were replaced with glycine by site-directed mutagenesis. Wild-type and mutant (Cys627/629/655Gly) FSHRs were then transiently expressed in HEK-293 cells and analyzed for cell-surface plasma membrane expression, agonist-stimulated signaling and internalization, and postendocytic processing in the absence and presence of lysosome and/or proteasome inhibitors. Compared with the wild-type FSHR, the triple mutant FSHR exhibited ~70% reduction in plasma membrane expression as well as a profound attenuation in agonist-stimulated cAMP production and ERK1/2 phosphorylation. Incubation of HEK-293 cells expressing the wild-type FSHR with 2-bromopalmitate (palmitoylation inhibitor) for 6 h, decreased plasma membrane expression of the receptor by ~30%. The internalization kinetics and β-arrestin 1 and 2 recruitment were similar between the wild-type and triple mutant FSHR as disclosed by assays performed in non-equilibrium binding conditions and by confocal microscopy. Cells expressing the mutant FSHR recycled the internalized FSHR back to the plasma membrane less efficiently than those expressing the wild-type FSHR, an effect that was counteracted by proteasome but not by lysosome inhibition. These results indicate that replacement of the cysteine residues present in the carboxyl-terminus of the FSHR, impairs receptor trafficking from the endoplasmic reticulum/Golgi apparatus to the plasma membrane and its recycling from endosomes back to the cell surface following agonist-induced internalization. Since in the FSHR these

  2. The dependence of Ag+ block of a potassium channel, murine kir2.1, on a cysteine residue in the selectivity filter.

    PubMed

    Dart, C; Leyland, M L; Barrett-Jolley, R; Shelton, P A; Spencer, P J; Conley, E C; Sutcliffe, M J; Stanfield, P R

    1998-08-15

    Externally applied Ag+ (100-200 nM) irreversibly blocked the strong inwardly rectifying K+ channel, Kir2.1. Mutation to serine of a cysteine residue at position 149 in the pore-forming H5 region of Kir2.1 abolished Ag+ blockage. To determine how many of the binding sites must be occupied by Ag+ before the channel is blocked, we measured the rate of channel block and found that our results were best fitted assuming that only one Ag+ ion need bind to eliminate channel current. We tested our hypothesis further by constructing covalently linked dimers and tetramers of Kir2.1 in which cysteine had been replaced by serine in one (dimer) or three (tetramer) of the linked subunits. When expressed, these constructs yielded functional channels with either two (dimer) or one (tetramer) cysteines per channel at position 149. Blockage in the tetramer was complete after sufficient exposure to 200 nM Ag+, a result that is also consistent with only one Ag+ being required to bind to Cys149 to block fully. The rate of development of blockage was 16 times slower than in wild-type channels; the rate was 4 times slower in channels formed from dimers.

  3. Intra-residue interactions in proteins: interplay between serine or cysteine side chains and backbone conformations, revealed by laser spectroscopy of isolated model peptides.

    PubMed

    Alauddin, Mohammad; Biswal, Himansu S; Gloaguen, Eric; Mons, Michel

    2015-01-21

    Intra-residue interactions play an important role in proteins by influencing local folding of the backbone. Taking advantage of the capability of gas phase experiments to provide relevant information on the intrinsic H-bonding pattern of isolated peptide chains, the intra-residue interactions of serine and cysteine residues, i.e., OH/SH···OC(i) C6 and NH(i···)O/S C5 interactions in Ser/Cys residues, are probed by laser spectroscopy of isolated peptides. The strength of these local side chain-main chain interactions, elegantly documented from their IR spectral features for well-defined conformations of the main chain, demonstrates that a subtle competition exists between the two types of intra-residue bond: the C6 H-bond is the major interaction with Ser, in contrast to Cys where C5 interaction takes over. The restricted number of conformers observed in the gas phase experiment with Ser compared to Cys (where both extended and folded forms are observed) also suggests a significant mediation role of these intra-residue interactions on the competition between the several main chain folding patterns. PMID:25482851

  4. Mechanism of Thiosulfate Oxidation in the SoxA Family of Cysteine-ligated Cytochromes

    PubMed Central

    Grabarczyk, Daniel B.; Chappell, Paul E.; Eisel, Bianca; Johnson, Steven; Lea, Susan M.; Berks, Ben C.

    2015-01-01

    Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism. PMID:25673696

  5. Mechanism of thiosulfate oxidation in the SoxA family of cysteine-ligated cytochromes.

    PubMed

    Grabarczyk, Daniel B; Chappell, Paul E; Eisel, Bianca; Johnson, Steven; Lea, Susan M; Berks, Ben C

    2015-04-01

    Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism.

  6. Reaction mechanism of the epsilon subunit of E. coli DNA polymerase III: insights into active site metal coordination and catalytically significant residues.

    PubMed

    Cisneros, G Andrés; Perera, Lalith; Schaaper, Roel M; Pedersen, Lars C; London, Robert E; Pedersen, Lee G; Darden, Thomas A

    2009-02-01

    The 28 kDa epsilon subunit of Escherichia coli DNA polymerase III is the exonucleotidic proofreader responsible for editing polymerase insertion errors. Here, we study the mechanism by which epsilon carries out the exonuclease activity. We performed quantum mechanics/molecular mechanics calculations on the N-terminal domain containing the exonuclease activity. Both the free-epsilon and a complex epsilon bound to a theta homologue (HOT) were studied. For the epsilon-HOT complex Mg(2+) or Mn(2+) were investigated as the essential divalent metal cofactors, while only Mg(2+) was used for free-epsilon. In all calculations a water molecule bound to the catalytic metal acts as the nucleophile for hydrolysis of the phosphate bond. Initially, a direct proton transfer to H162 is observed. Subsequently, the nucleophilic attack takes place followed by a second proton transfer to E14. Our results show that the reaction catalyzed with Mn(2+) is faster than that with Mg(2+), in agreement with experiment. In addition, the epsilon-HOT complex shows a slightly lower energy barrier compared to free-epsilon. In all cases the catalytic metal is observed to be pentacoordinated. Charge and frontier orbital analyses suggest that charge transfer may stabilize the pentacoordination. Energy decomposition analysis to study the contribution of each residue to catalysis suggests that there are several important residues. Among these, H98, D103, D129, and D146 have been implicated in catalysis by mutagenesis studies. Some of these residues were found to be structurally conserved on human TREX1, the exonuclease domains from E. coli DNA-Pol I, and the DNA polymerase of bacteriophage RB69.

  7. Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine.

    PubMed

    Pakavathkumar, Prateep; Sharma, Gyanesh; Kaushal, Vikas; Foveau, Bénédicte; LeBlanc, Andrea C

    2015-09-24

    Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases.

  8. Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine

    PubMed Central

    Pakavathkumar, Prateep; Sharma, Gyanesh; Kaushal, Vikas; Foveau, Bénédicte; LeBlanc, Andrea C.

    2015-01-01

    Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases. PMID:26400108

  9. TRPA1 is activated by direct addition of cysteine residues to the N-hydroxysuccinyl esters of acrylic and cinnamic acids.

    PubMed

    Sadofsky, Laura R; Boa, Andrew N; Maher, Sarah A; Birrell, Mark A; Belvisi, Maria G; Morice, Alyn H

    2011-01-01

    The nociceptor TRPA1 is thought to be activated through covalent modification of specific cysteine residues on the N terminal of the channel. The precise mechanism of covalent modification with unsaturated carbonyl-containing compounds is unclear, therefore by examining a range of compounds which can undergo both conjugate and/or direct addition reactions we sought to further elucidate the mechanism(s) whereby TRPA1 can be activated by covalent modification. Calcium signalling was used to determine the mechanism of activation of TRPA1 expressed in HEK293 cells with a series of related compounds which were capable of either direct and/or conjugate addition processes. These results were confirmed using physiological recordings with isolated vagus nerve preparations. We found negligible channel activation with chemicals which could only react with cysteine residues via conjugate addition such as acrylamide, acrylic acid, and cinnamic acid. Compounds able to react via either conjugate or direct addition, such as acrolein, methyl vinyl ketone, mesityl oxide, acrylic acid NHS ester, cinnamaldehyde and cinnamic acid NHS ester, activated TRPA1 in a concentration dependent manner as did compounds only capable of direct addition, namely propionic acid NHS ester and hydrocinnamic acid NHS ester. These compounds failed to activate TRPV1 expressed in HEK293 cells or mock transfected HEK293 cells. For molecules capable of direct or conjugate additions, the results suggest for the first time that TRPA1 may be activated preferentially by direct addition of the thiol group of TRPA1 cysteines to the agonist carbonyl carbon of α,β-unsaturated carbonyl-containing compounds.

  10. Effect of multiple cysteine substitutions on the functionality of human multidrug resistance protein 1 expressed in human embryonic kidney 293 cells: identification of residues essential for function.

    PubMed

    Qin, Lei; Tam, Shui-Pang; Deeley, Roger G

    2012-07-01

    Multidrug resistance protein 1 (MRP1) is a broad-specificity membrane transporter belonging to the C branch of the ATP binding cassette (ABC) superfamily. MRP1 confers resistance to various chemotherapeutic drugs and transports a wide range of conjugated organic anions. Several ABCC proteins, including MRP1, are unusual among ABC transporters in having a third membrane-spanning domain (MSD), MSD0, at their N termini. MRP1 lacking this additional MSD (ΔMRP1) is able to traffic to the plasma membrane of mammalian cells and to transport a number of well characterized substrates. A cysteineless (cysless) ΔMRP1 has been expressed in yeast and reported to be functional. However, we found that trafficking of such a construct in human cells was severely compromised, and, even when expressed in insect Sf21 cells, the protein had extremely low transport activity. Therefore, we have systematically examined the effects of substituting cysteines in the four domains of ΔMRP1, initially with alanine. These studies allowed us to identify five cysteines that cannot be replaced with alanine without inactivating the protein. Substitution of two of these residues with alternative amino acids has allowed us to produce an almost cysless form of ΔMRP1 that traffics to the plasma membrane and transports leukotriene C(4), 17β-estradiol 17-β-D-glucuronide, and estrone-3-sulfate with kinetic characteristics similar to those of the wild-type protein. The distribution of the remaining Cys residues is such that the protein will provide a useful template for a variety of cysteine based mutagenesis studies.

  11. Neutral sphingomyelinase-2 is a redox sensitive enzyme: role of catalytic cysteine residues in regulation of enzymatic activity through changes in oligomeric state.

    PubMed

    Dotson, P Patrick; Karakashian, Alexander A; Nikolova-Karakashian, Mariana N

    2015-02-01

    Neutral sphingomyelinase-2 (nSMase-2) is the major sphingomyelinase activated in response to pro-inflammatory cytokines and during oxidative stress. It is a membrane-bound 655 amino acid protein containing 22 cysteine residues. In this study, we expressed recombinant mouse nSMase-2 protein in Escherichia coli, and investigated whether nSMase-2 is a redox sensitive enzyme. Our results demonstrate that nSMase-2 exists as both monomers and multimers that are associated with high and low enzymatic activity respectively. Mutational analysis of nSMase-2 identified within its C-terminal catalytic domain several oxidant-sensitive cysteine residues that were shown to be involved in enzyme oligomerization. Changing Cys(617) to Ser for example is a gain-of-function mutation associated with a decreased propensity for oligomerization. Alternatively, nSMase-2 expression in a bacterial strain that lacks endogenous thioredoxin, Rosetta-gami2, results in increased oligomer formation and lower enzyme activity. Phenotypic rescue was accomplished by treating nSMase-2 lysates with recombinant human thioredoxin. This indicates that nSMase-2 may be a novel substrate for thioredoxin. FRET analysis confirmed the presence of nSMase-2 multimers in mammalian HEK cells and their localization to the plasma membrane. In conclusion, our results identify nSMase-2 as a redox-sensitive enzyme, whose basal activity is influenced by thioredoxin-mediated changes in its oligomeric state. PMID:25287744

  12. Cytoplasmic localization and redox cysteine residue of APE1/Ref-1 are associated with its anti-inflammatory activity in cultured endothelial cells.

    PubMed

    Park, Myoung Soo; Kim, Cuk-Seong; Joo, Hee Kyoung; Lee, Yu Ran; Kang, Gun; Kim, Soo Jin; Choi, Sunga; Lee, Sang Do; Park, Jin Bong; Jeon, Byeong Hwa

    2013-11-01

    Apurinic/apyrimidinic endonuclease1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in base excision DNA repair and transcriptional regulation of gene expression. APE1/Ref-1 is mainly localized in the nucleus, but cytoplasmic localization has also been reported. However, the functional role of cytoplasmic APE1/Ref-1 and its redox cysteine residue are still unknown. We investigated the role of cytoplasmic APE1/Ref-1 on tumor necrosis factor-α (TNF-α)-induced vascular cell adhesion molecule-1 (VCAM-1) expressions in endothelial cells. Endogenous APE1/Ref-1 was mainly observed in the nucleus, however, cytoplasmic APE1/Ref-1 was increased by TNF-α. Cytoplasmic APE1/Ref-1 expression was not blunted by cycloheximide, a protein synthesis inhibitor, suggesting cytoplasmic translocation of APE1/Ref-1. Transfection of an N-terminus deletion mutant APE1/Ref-1(29-318) inhibited TNF-α-induced VCAM-1 expression, indicating an anti-inflammatory role for APE1/Ref-1 in the cytoplasm. In contrast, redox mutant of APE1/Ref-1 (C65A/C93A) transfection led to increased TNF-α-induced VCAM-1 expression. Our findings suggest cytoplasmic APE1/Ref-1 localization and redox cysteine residues of APE1/Ref-1 are associated with its anti-inflammatory activity in endothelial cells.

  13. Identification of arginine 331 as an important active site residue in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli.

    PubMed Central

    Qamar, S.; Marsh, K.; Berry, A.

    1996-01-01

    Treatment of the Class II fructose-1,6-bisphosphate aldolase of Escherichia coli with the arginine-specific alpha-dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme. The enzyme is protected from inactivation by the substrate, fructose 1,6-bisphosphate, or by inorganic phosphate. Modification with [7-14C] phenylglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme. Sequence alignment of the eight known Class II FBP-aldolases shows that only one arginine residue is conserved in all the known sequences. This residue, Arg-331, was mutated to either alanine or glutamic acid. The mutant enzymes were much less susceptible to inactivation by phenylglyoxal. Measurement of the steady-state kinetic parameters revealed that mutation of Arg-331 dramatically increased the K(m) for fructose 1,6-bisphosphate. Comparatively small differences in the inhibitor constant Ki for dihydroxyacetone phosphate or its analogue, 2-phosphoglycolate, were found between the wild-type and mutant enzymes. In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3-phosphate was used as an inhibitor. Kinetic analysis of the oxidation of the carbanionic aldolase-substrate intermediate of the reaction by hexacyanoferrate (III) revealed that the K(m) for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6-bisphosphate was dramatically increased. Taken together, these results show that Arg-331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C-6 phosphate group of the substrate. PMID:8771208

  14. Chemical Synthesis of Proteins with Non-Strategically Placed Cysteines Using Selenazolidine and Selective Deselenization.

    PubMed

    Reddy, Post Sai; Dery, Shahar; Metanis, Norman

    2016-01-18

    Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125-residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C-terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N-terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild-type PHPT1 and an analogue in which the active-site histidine was substituted with the unnatural and isosteric amino acid β-thienyl-l-alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site. PMID:26636774

  15. Structure-Function Relationship of the Chloroplastic Glutaredoxin S12 with an Atypical WCSYS Active Site*S⃞

    PubMed Central

    Couturier, Jeremy; Koh, Cha San; Zaffagnini, Mirko; Winger, Alison M.; Gualberto, Jose Manuel; Corbier, Catherine; Decottignies, Paulette; Jacquot, Jean-Pierre; Lemaire, Stéphane D.; Didierjean, Claude; Rouhier, Nicolas

    2009-01-01

    Glutaredoxins (Grxs) are efficient catalysts for the reduction of mixed disulfides in glutathionylated proteins, using glutathione or thioredoxin reductases for their regeneration. Using GFP fusion, we have shown that poplar GrxS12, which possesses a monothiol 28WCSYS32 active site, is localized in chloroplasts. In the presence of reduced glutathione, the recombinant protein is able to reduce in vitro substrates, such as hydroxyethyldisulfide and dehydroascorbate, and to regenerate the glutathionylated glyceraldehyde-3-phosphate dehydrogenase. Although the protein possesses two conserved cysteines, it is functioning through a monothiol mechanism, the conserved C terminus cysteine (Cys87) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys87 exhibits a certain reactivity, since its pKa is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxS12 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cys29) and that the enzyme does not form inter- or intramolecular disulfides. Contrary to some plant Grxs, GrxS12 does not incorporate an iron-sulfur cluster in its wild-type form, but when the active site is mutated into YCSYS, it binds a [2Fe-2S] cluster, indicating that the single Trp residue prevents this incorporation. PMID:19158074

  16. Combined Use of Residual Dipolar Couplings and Solution X-ray Scattering To Rapidly Probe Rigid-Body Conformational Transitions in a Non-phosphorylatable Active-Site Mutant of the 128 kDa Enzyme I Dimer

    SciTech Connect

    Takayama, Yuki; Schwieters, Charles D.; Grishaev, Alexander; Ghirlando, Rodolfo; Clore, G. Marius

    2012-10-23

    The first component of the bacterial phosphotransferase system, enzyme I (EI), is a multidomain 128 kDa dimer that undergoes large rigid-body conformational transitions during the course of its catalytic cycle. Here we investigate the solution structure of a non-phosphorylatable active-site mutant in which the active-site histidine is substituted by glutamine. We show that perturbations in the relative orientations and positions of the domains and subdomains can be rapidly and reliably determined by conjoined rigid-body/torsion angle/Cartesian simulated annealing calculations driven by orientational restraints from residual dipolar couplings and shape and translation information afforded by small- and wide-angle X-ray scattering. Although histidine and glutamine are isosteric, the conformational space available to a Gln side chain is larger than that for the imidazole ring of His. An additional hydrogen bond between the side chain of Gln189 located on the EIN{sup {alpha}/{beta}} subdomain and an aspartate (Asp129) on the EIN{sup {alpha}} subdomain results in a small ({approx}9{sup o}) reorientation of the EIN{sup {alpha}} and EIN{sup {alpha}/{beta}} subdomains that is in turn propagated to a larger reorientation ({approx}26{sup o}) of the EIN domain relative to the EIC dimerization domain, illustrating the positional sensitivity of the EIN domain and its constituent subdomains to small structural perturbations.

  17. Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation

    SciTech Connect

    Boudreaux, David A.; Maiti, Tushar K.; Davies, Christopher W.; Das, Chittaranjan

    2010-07-06

    Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 {angstrom} from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarily at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal {beta}-hairpin at the distal site - a surface-exposed hydrophobic crevice 17 {angstrom} away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 {angstrom} of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.

  18. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  19. Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not.

    PubMed

    Singh, Pawan; Tripathi, Pankaj; Muniyappa, K

    2010-01-01

    Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site

  20. Cysteine pK(a) values for the bacterial peroxiredoxin AhpC.

    PubMed

    Nelson, Kimberly J; Parsonage, Derek; Hall, Andrea; Karplus, P Andrew; Poole, Leslie B

    2008-12-01

    Salmonella typhimurium AhpC is a founding member of the peroxiredoxin family, a ubiquitous group of cysteine-based peroxidases with high reactivity toward hydrogen peroxide, organic hydroperoxides, and peroxynitrite. For all of the peroxiredoxins, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), acts as a nucleophile in attacking the peroxide substrate, forming a cysteine sulfenic acid at the active site. Because thiolates are far stronger nucleophiles than thiol groups, it is generally accepted that cysteine-based peroxidases should exhibit pK(a) values lower than an unperturbed value of 8.3-8.5. In this investigation, several independent approaches were used to assess the pK(a) of the two cysteinyl residues of AhpC. Methods using two different iodoacetamide derivatives yielded unperturbed pK(a) values (7.9-8.7) for both cysteines, apparently due to reactivity with the wrong conformation of C(P) (i.e., locally unfolded and flipped out of the active site), as supported by X-ray crystallographic analyses. A functional pK(a) of 5.94 +/- 0.10 presumably reflecting the titration of C(P) within the fully folded active site was obtained by measuring AhpC competition with horseradish peroxidase for hydrogen peroxide; this value is quite similar to that obtained by analyzing the pH dependence of the epsilon(240) of wild-type AhpC (5.84 +/- 0.02) and similar to those obtained for two typical 2-cysteine peroxiredoxins from Saccharomyces cerevisiae (5.4 and 6.0). Thus, the pK(a) value of AhpC balances the need for a deprotonated thiol (at pH 7, approximately 90% of the C(P) would be deprotonated) with the fact that thiolates with higher pK(a) values are stronger nucleophiles. PMID:18986167

  1. The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions.

    PubMed

    Hartmann, Tobias; Schrapers, Peer; Utesch, Tillmann; Nimtz, Manfred; Rippers, Yvonne; Dau, Holger; Mroginski, Maria Andrea; Haumann, Michael; Leimkühler, Silke

    2016-04-26

    Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase. PMID:27054466

  2. Modification of the cysteine residues in IκBα kinase and NF-κB (p65) by xanthohumol leads to suppression of NF-κB–regulated gene products and potentiation of apoptosis in leukemia cells

    PubMed Central

    Harikumar, Kuzhuvelil B.; Kunnumakkara, Ajaikumar B.; Ahn, Kwang S.; Anand, Preetha; Krishnan, Sunil; Guha, Sushovan

    2009-01-01

    Xanthohumol (XN), a prenylated chalcone isolated from hop plant, exhibits anti-inflammatory, antiproliferative, and antiangiogenic properties through an undefined mechanism. Whether examined by intracellular esterase activity, phosphatidylserine externalization, DNA strand breaks, or caspase activation, we found that XN potentiated tumor necrosis factor–induced apoptosis in leukemia and myeloma cells. This enhancement of apoptosis correlated with down-regulation of nuclear factor-κB (NF-κB) survivin, bcl-xL, XIAP, cIAP1, cIAP2, cylin D1, and c-myc. XN down-regulated both constitutive and inducible NF-κB activation, inhibition of phosphorylation and degradation of IκBα, suppression of p65 nuclear translocation, and NF-κB–dependent reporter gene transcription. XN directly inhibited tumor necrosis factor-induced IκBα kinase (IKK) activation and a reducing agent abolished this inhibition, indicating the role of cysteine residue. XN had no effect on the IKK activity when cysteine residue 179 of IKK was mutated to alanine. XN also directly inhibited binding of p65 to DNA, a reducing agent reversed this effect, and mutation of cysteine residue 38 to serine of p65 abolished this effect. Thus, our results show that modification of cysteine residues of IKK and p65 by XN leads to inhibition of the NF-κB activation pathway, suppression of antiapoptotic gene products, and potentiation of apoptosis in leukemia cells. PMID:18952893

  3. Protein Topology Determines Cysteine Oxidation Fate: The Case of Sulfenyl Amide Formation among Protein Families

    PubMed Central

    Defelipe, Lucas A.; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A.; Turjanski, Adrián G.

    2015-01-01

    Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function. PMID:25741692

  4. Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells.

    PubMed

    Uprety, Rajendra; Luo, Ji; Liu, Jihe; Naro, Yuta; Samanta, Subhas; Deiters, Alexander

    2014-08-18

    We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light-activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase-amino acid interactions. As a proof-of-principle application, the genetic replacement of an active-site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150-fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.

  5. Modeling the Active Sites in Metalloenzymes 5. The Heterolytic Bond Cleavage of H2 in the [NiFe] Hydrogenase of DesulfoWibrio gigas by a Nucleophilic Addition Mechanism

    SciTech Connect

    Niu, Shuqiang; Hall, Michael B.

    2001-11-19

    The H2 activation catalyzed by an Fe(II)-Ni(III) model of the [NiFe] hydrogenase of DesulfoVibrio gigas has been investigated by density functional theory (DFT/B3LYP) calculations on the neutral and anionic active site complexes, [(CO)(CN)2Fe(Mu-SH)2Ni(SH)(SH2)]0 and [(CO)(CN)2Fe(Mu-SH)2Ni(SH)2]-. The results suggest that the reaction proceeds by a nucleophilic addition mechanism that cleaves the H-H bond heterolytically. The terminal cysteine residue Cys530 in the [NiFe] hydrogenase active site of the D. gigas enzyme plays a crucial role in the catalytic process by accepting the proton. The active site is constructed to provide access by this cysteine residue, and this role explains the change in activity observed when this cysteine is replaced by a selenocysteine. Furthermore, the optimized geometry of the transition state in the model bears a striking resemblance to the geometry of the active site as determined by X-ray crystallography.

  6. Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa-mediated synthesis of a new polyester: identification of catalytic residues.

    PubMed

    Amara, Amro A; Rehm, Bernd H A

    2003-09-01

    The class II PHA (polyhydroxyalkanoate) synthases [PHA(MCL) synthases (medium-chain-length PHA synthases)] are mainly found in pseudomonads and catalyse synthesis of PHA(MCL)s using CoA thioesters of medium-chain-length 3-hydroxy fatty acids (C6-C14) as a substrate. Only recently PHA(MCL) synthases from Pseudomonas oleovorans and Pseudomonas aeruginosa were purified and in vitro activity was achieved. A threading model of the P. aeruginosa PHA(MCL) synthase PhaC1 was developed based on the homology to the epoxide hydrolase (1ek1) from mouse which belongs to the alpha/beta-hydrolase superfamily. The putative catalytic residues Cys-296, Asp-452, His-453 and His-480 were replaced by site-specific mutagenesis. In contrast to class I and III PHA synthases, the replacement of His-480, which aligns with the conserved base catalyst of the alpha/beta-hydrolases, with Gln did not affect in vivo enzyme activity and only slightly in vitro enzyme activity. The second conserved histidine His-453 was then replaced by Gln, and the modified enzyme showed only 24% of wild-type in vivo activity, which indicated that His-453 might functionally replace His-480 in class II PHA synthases. Replacement of the postulated catalytic nucleophile Cys-296 by Ser only reduced in vivo enzyme activity to 30% of wild-type enzyme activity and drastically changed substrate specificity. Moreover, the C296S mutation turned the enzyme sensitive towards PMSF inhibition. The replacement of Asp-452 by Asn, which is supposed to be required as general base catalyst for elongation reaction, did abolish enzyme activity as was found for the respective amino acid residue of class I and III enzymes. In the threading model residues Cys-296, Asp-452, His-453 and His-480 reside in the core structure with the putative catalytic nucleophile Cys-296 localized at the highly conserved gamma-turns of the alpha/beta-hydrolases. Inhibitor studies indicated that catalytic histidines reside in the active site. The conserved

  7. Function of the active site lysine autoacetylation in Tip60 catalysis.

    PubMed

    Yang, Chao; Wu, Jiang; Zheng, Y George

    2012-01-01

    The 60-kDa HIV-Tat interactive protein (Tip60) is a key member of the MYST family of histone acetyltransferases (HATs) that plays critical roles in multiple cellular processes. We report here that Tip60 undergoes autoacetylation at several lysine residues, including a key lysine residue (i.e. Lys-327) in the active site of the MYST domain. The mutation of K327 to arginine led to loss of both the autoacetylation activity and the cognate HAT activity. Interestingly, deacetylated Tip60 still kept a substantial degree of HAT activity. We also investigated the effect of cysteine 369 and glutamate 403 in Tip60 autoacetylation in order to understand the molecular pathway of the autoacetylation at K327. Together, we conclude that the acetylation of K327 which is located in the active site of Tip60 regulates but is not obligatory for the catalytic activity of Tip60. Since acetylation at this key residue appears to be evolutionarily conserved amongst all MYST proteins, our findings provide an interesting insight into the regulatory mechanism of MYST activities. PMID:22470428

  8. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  9. Role of Cysteine Residues in the Structure, Stability, and Alkane Producing Activity of Cyanobacterial Aldehyde Deformylating Oxygenase

    PubMed Central

    Hayashi, Yuuki; Yasugi, Fumitaka; Arai, Munehito

    2015-01-01

    Aldehyde deformylating oxygenase (AD) is a key enzyme for alkane biosynthesis in cyanobacteria, and it can be used as a catalyst for alkane production in vitro and in vivo. However, three free Cys residues in AD may impair its catalytic activity by undesired disulfide bond formation and oxidation. To develop Cys-deficient mutants of AD, we examined the roles of the Cys residues in the structure, stability, and alkane producing activity of AD from Nostoc punctiforme PCC 73102 by systematic Cys-to-Ala/Ser mutagenesis. The C71A/S mutations reduced the hydrocarbon producing activity of AD and facilitated the formation of a dimer, indicating that the conserved Cys71, which is located in close proximity to the substrate-binding site, plays crucial roles in maintaining the activity, structure, and stability of AD. On the other hand, mutations at Cys107 and Cys117 did not affect the hydrocarbon producing activity of AD. Therefore, we propose that the C107A/C117A double mutant is preferable to wild type AD for alkane production and that the double mutant may be used as a pseudo-wild type protein for further improvement of the alkane producing activity of AD. PMID:25837679

  10. Role of cysteine residues in the structure, stability, and alkane producing activity of cyanobacterial aldehyde deformylating oxygenase.

    PubMed

    Hayashi, Yuuki; Yasugi, Fumitaka; Arai, Munehito

    2015-01-01

    Aldehyde deformylating oxygenase (AD) is a key enzyme for alkane biosynthesis in cyanobacteria, and it can be used as a catalyst for alkane production in vitro and in vivo. However, three free Cys residues in AD may impair its catalytic activity by undesired disulfide bond formation and oxidation. To develop Cys-deficient mutants of AD, we examined the roles of the Cys residues in the structure, stability, and alkane producing activity of AD from Nostoc punctiforme PCC 73102 by systematic Cys-to-Ala/Ser mutagenesis. The C71A/S mutations reduced the hydrocarbon producing activity of AD and facilitated the formation of a dimer, indicating that the conserved Cys71, which is located in close proximity to the substrate-binding site, plays crucial roles in maintaining the activity, structure, and stability of AD. On the other hand, mutations at Cys107 and Cys117 did not affect the hydrocarbon producing activity of AD. Therefore, we propose that the C107A/C117A double mutant is preferable to wild type AD for alkane production and that the double mutant may be used as a pseudo-wild type protein for further improvement of the alkane producing activity of AD.

  11. Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from bufo melanostictus.

    PubMed

    Liu, Wa; Ji, Senlin; Zhang, A-Mei; Han, Qinqin; Feng, Yue; Song, Yuzhu

    2013-01-01

    Cystatins are efficient inhibitors of papain-like cysteine proteinases, and they serve various important physiological functions. In this study, a novel cystatin, Cystatin-X, was cloned from a cDNA library of the skin of Bufo melanostictus. The single nonglycosylated polypeptide chain of Cystatin-X consisted of 102 amino acid residues, including seven cysteines. Evolutionary analysis indicated that Cystatin-X can be grouped with family 1 cystatins. It contains cystatin-conserved motifs known to interact with the active site of cysteine proteinases. Recombinant Cystatin-X expressed and purified from Escherichia coli exhibited obvious inhibitory activity against cathepsin B. rCystatin-X at a concentration of 8 µM inhibited nearly 80% of cathepsin B activity within 15 s, and about 90% of cathepsin B activity within 15 min. The Cystatin-X identified in this study can play an important role in host immunity and in the medical effect of B. melanostictus.

  12. Intra-membrane Signaling Between the Voltage-Gated Ca2+-Channel and Cysteine Residues of Syntaxin 1A Coordinates Synchronous Release

    PubMed Central

    Bachnoff, Niv; Cohen-Kutner, Moshe; Trus, Michael; Atlas, Daphne

    2013-01-01

    The interaction of syntaxin 1A (Sx1A) with voltage-gated calcium channels (VGCC) is required for depolarization-evoked release. However, it is unclear how the signal is transferred from the channel to the exocytotic machinery and whether assembly of Sx1A and the calcium channel is conformationally linked to triggering synchronous release. Here we demonstrate that depolarization-evoked catecholamine release was decreased in chromaffin cells infected with semliki forest viral vectors encoding Sx1A mutants, Sx1AC271V, or Sx1AC272V, or by direct oxidation of these Sx1A transmembrane (TM) cysteine residues. Mutating or oxidizing these highly conserved Sx1A Cys271 and Cys272 equally disrupted the Sx1A interaction with the channel. The results highlight the functional link between the VGCC and the exocytotic machinery, and attribute the redox sensitivity of the release process to the Sx1A TM C271 and C272. This unique intra-membrane signal-transduction pathway enables fast signaling, and triggers synchronous release by conformational-coupling of the channel with Sx1A. PMID:23567899

  13. Active site studies of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

    SciTech Connect

    Vlahos, C.J.

    1987-01-01

    The data presented delineate the complete amino acid sequence of E. coli KHG aldolase and also identify Lys-133, Glu-45, and Arg-49 as aminoacyl residues required for catalytic activity. Incubation of E. coli KHG aldolase with (/sup 14/C)pyruvate in the presence of NaCNBH/sub 3/ results in the incorporation of one mol of /sup 14/C per mol of enzyme subunit. Digestion of this enzyme-adduct with trypsin, followed by purification of the peptides, allowed for the isolation of a unique radioactive peptide. Its amino acid sequence showed that the pyruvate-binding (i.e., Schiff-base forming) lysine residue is located at position 133 in the intact enzyme. E. coli KHG aldolase activity is lost when the enzyme is reacted with bromopyruvate; saturation kinetics are observed. The substrates, pyruvate and KHG, protect the enzyme from inactivation. Both facts suggest that the reagent is active-site specific. Incubation of the aldolase with (3-/sup 14/C)bromopyruvate is associated with a concomitant loss of enzymatic activity and esterification of Glu-45; if the enzyme is denatured in the presence of excess bromopyruvate, Cys-159 and Cys-180 are also alkylated. Blocking the active-site lysine residue with pyruvate prevents Glu-45 from being esterified but does not eliminate alkylation of these two cysteine residues. Woodward's Reagent K was also found to inactivate the aldolase under conditions that are usually specific for carboxyl group modification. This aldolase is also inactivated by 1,2-cyclohexanedione. Loss of enzymatic activity occurs concomitantly with modification of one arginine residue per enzyme subunit. Treatment of the aldolase with the arginine-specific reagent, 4-(oxyacetyl)phenoxyacetic acid, followed by digestion with trypsin allowed for the isolation of a unique peptide and the identification of Arg-49 as the specific residue involved.

  14. Atypical thioredoxins in poplar: the glutathione-dependent thioredoxin-like 2.1 supports the activity of target enzymes possessing a single redox active cysteine.

    PubMed

    Chibani, Kamel; Tarrago, Lionel; Gualberto, José Manuel; Wingsle, Gunnar; Rey, Pascal; Jacquot, Jean-Pierre; Rouhier, Nicolas

    2012-06-01

    Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways.

  15. Functional consequences of sulfhydryl modification of the γ-aminobutyric acid transporter 1 at a single solvent-exposed cysteine residue.

    PubMed

    Omoto, Jaison J; Maestas, Matthew J; Rahnama-Vaghef, Ali; Choi, Ye E; Salto, Gerardo; Sanchez, Rachel V; Anderson, Cynthia M; Eskandari, Sepehr

    2012-12-01

    The aims of this study were to optimize the experimental conditions for labeling extracellularly oriented, solvent-exposed cysteine residues of γ-aminobutyric acid transporter 1 (GAT1) with the membrane-impermeant sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) and to characterize the functional and pharmacological consequences of labeling on transporter steady-state and presteady-state kinetic properties. We expressed human GAT1 in Xenopus laevis oocytes and used radiotracer and electrophysiological methods to assay transporter function before and after sulfhydryl modification with MTSET. In the presence of NaCl, transporter exposure to MTSET (1-2.5 mM for 5-20 min) led to partial inhibition of GAT1-mediated transport, and this loss of function was completely reversed by the reducing reagent dithiothreitol. MTSET treatment had no functional effect on the mutant GAT1 C74A, whereas the membrane-permeant reagents N-ethylmaleimide and tetramethylrhodamine-6-maleimide inhibited GABA transport mediated by GAT1 C74A. Ion replacement experiments indicated that MTSET labeling of GAT1 could be driven to completion when valproate replaced chloride in the labeling buffer, suggesting that valproate induces a GAT1 conformation that significantly increases C74 accessibility to the extracellular fluid. Following partial inhibition by MTSET, there was a proportional reduction in both the presteady-state and steady-state macroscopic signals, and the functional and pharmacological properties of the remaining signals were indistinguishable from those of unlabeled GAT1. Therefore, covalent modification of GAT1 at C74 results in completely nonfunctional as well as electrically silent transporters.

  16. VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications.

    PubMed

    Reina, Simona; Checchetto, Vanessa; Saletti, Rosaria; Gupta, Ankit; Chaturvedi, Deepti; Guardiani, Carlo; Guarino, Francesca; Scorciapino, Mariano Andrea; Magrì, Andrea; Foti, Salvatore; Ceccarelli, Matteo; Messina, Angela Anna; Mahalakshmi, Radhakrishnan; Szabo, Ildiko; De Pinto, Vito

    2016-01-19

    Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes. PMID:26760765

  17. VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications

    PubMed Central

    Saletti, Rosaria; Guardiani, Carlo; Guarino, Francesca; Scorciapino, Mariano Andrea; Magrì, Andrea; Foti, Salvatore; Ceccarelli, Matteo; Messina, Angela Anna; Mahalakshmi, Radhakrishnan; Szabo, Ildiko; De Pinto, Vito

    2016-01-01

    Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes. PMID:26760765

  18. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases.

    PubMed

    Liu, Pingyang; Torrens-Spence, Michael P; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2013-02-01

    Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development. PMID:22718265

  19. Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases.

    PubMed

    Liu, Pingyang; Torrens-Spence, Michael P; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2013-02-01

    Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development.

  20. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

    PubMed Central

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W.; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. PMID:27276217

  1. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae.

    PubMed

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. PMID:27276217

  2. Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli.

    PubMed Central

    Darimont, B.; Stehlin, C.; Szadkowski, H.; Kirschner, K.

    1998-01-01

    Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-1-deoxyribulose 5'-phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones. A strain of E. coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady-state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184. PMID:9605328

  3. Cysteine residues in the large extracellular loop (EC2) are essential for the function of the stress-regulated glycoprotein M6a.

    PubMed

    Fuchsova, Beata; Fernández, María E; Alfonso, Julieta; Frasch, Alberto C

    2009-11-13

    Gpm6a was identified as a stress-responsive gene in the hippocampal formation. This gene is down-regulated in the hippocampus of both socially and physically stressed animals, and this effect can be reversed by antidepressant treatment. Previously we showed that the stress-regulated protein M6a is a key modulator for neurite outgrowth and filopodium/spine formation. In the present work, mutational analysis was used to characterize the action of M6a at the molecular level. We show that four cysteines 162, 174, 192, and 202 within EC2 are functionally crucial sites. The presence of cysteines 162 and 202 is essential for the efficient cell surface expression of the M6a protein. In contrast, cysteines 174 and 192, which form a disulfide bridge as shown by biochemical analysis, are not required for the efficient surface expression of M6a. Their mutation to alanine does not interfere with the localization of M6a to filopodial protrusions in primary hippocampal neurons. The neurons expressing C174A and/or C192A mutants display decreased filopodia number. In non-permeabilized cells, these mutant proteins are not recognized by a function-blocking monoclonal antibody directed to M6a. Moreover, neurons in contact with axons expressing C174A/C192A mutant display significantly lower density of presynaptic clusters over their dendrites. Taken together, this study demonstrates that cysteines in the EC2 domain are critical for the role of M6a in filopodium outgrowth and synaptogenesis. PMID:19737934

  4. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  5. Protein cysteine modifications: (2) reactivity specificity and topics of medicinal chemistry and protein engineering.

    PubMed

    Nagahara, Noriyuki; Matsumura, Tomohiro; Okamoto, Ryo; Kajihara, Yasuhiro

    2009-01-01

    Cysteine (cysteinyl residue) modifications in proteins result in diversity in protein functions. The reaction specificity of a protein with a modified cysteine residue is determined by the overall conditions of the protein, including the spatial position of the cysteine residue, electrostatic interactions between cysteine residue and other charged residues, spatial interactions between the cysteine residue and a chemical compound, electrophilicity of the chemical compound, and the pH of the solution. In cysteine-dependant enzymes, each specific type of cysteine modification characterizes the catalytic mechanism of the enzyme. Recently, the catalytic mechanisms of peroxiredoxins and cysteine proteases, which contain a cysteine residue(s) in their catalytic sites, have been elucidated. In the catalytic process of peroxiredoxins, a sulfenyl intermediate is formed by oxidation of the catalytic cysteine residue. On the other hand, in cysteine proteases, the catalytic cysteine residue reacts with the carboxyl carbon of a peptide substrate to form an intermediate complex via S-alkylation. In this review, we introduce the most current information on the applications of cysteine thiol chemistry for in vitro glycoprotein synthesis. Recently, a glycoprotein (monocyte chemotactic protein-3), containing an intact human complex-type sialyloligosaccharide has been chemically synthesized. The procedure used for this could have applications in the development of new protein-based drugs, including antineoplastic drugs and antibiotics. It can also potentially be applied for improving the half-life and reducing the toxicity of these drugs, and for preventing the development of multidrug resistance.

  6. Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

    PubMed

    Wahba, Haytham M; Lecoq, Lauriane; Stevenson, Michael; Mansour, Ahmed; Cappadocia, Laurent; Lafrance-Vanasse, Julien; Wilkinson, Kevin J; Sygusch, Jurgen; Wilcox, Dean E; Omichinski, James G

    2016-02-23

    In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL. PMID:26820485

  7. Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

    PubMed

    Wahba, Haytham M; Lecoq, Lauriane; Stevenson, Michael; Mansour, Ahmed; Cappadocia, Laurent; Lafrance-Vanasse, Julien; Wilkinson, Kevin J; Sygusch, Jurgen; Wilcox, Dean E; Omichinski, James G

    2016-02-23

    In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

  8. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  9. Peptide-formation on cysteine-containing peptide scaffolds

    NASA Technical Reports Server (NTRS)

    Chu, B. C.; Orgel, L. E.

    1999-01-01

    Monomeric cysteine residues attached to cysteine-containing peptides by disulfide bonds can be activated by carbonyldiimidazole. If two monomeric cysteine residues, attached to a 'scaffold' peptide Gly-Cys-Glyn-Cys-Glu10, (n = 0, 1, 2, 3) are activated, they react to form the dipeptide Cys-Cys. in 25-65% yield. Similarly, the activation of a cysteine residue attached to the 'scaffold' peptide Gly-Cys-Gly-Glu10 in the presence of Arg5 leads to the formation of Cys-Arg5 in 50% yield. The significance of these results for prebiotic chemistry is discussed.

  10. Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

    NASA Technical Reports Server (NTRS)

    Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

    2016-01-01

    Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

  11. Crystal Structure of Mammalian Cysteine dioxygenase: A Novel Mononuclear Iron Center for Cysteine Thiol Oxidation

    SciTech Connect

    Simmons,C.; Liu, Q.; Huang, Q.; Hao, Q.; Begley, T.; Karplus, P.; Stipanuk, M.

    2006-01-01

    Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteinesulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or to the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5 Angstroms resolution, and these results confirm the canonical cupin {beta}-sandwich fold and the rare cysteinyl-tyrosine intramolecular crosslink (between Cys93 and Tyr157) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His86, His88, and His140) and a water molecule. Attempts to acquire a structure with bound ligand using either co-crystallization or soaks with cysteine revealed the formation of a mixed disulfide involving Cys164 near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploring the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine linkage.

  12. Factor D of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site.

    PubMed Central

    Johnson, D M; Gagnon, J; Reid, K B

    1980-01-01

    The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat 'group-specific protease' [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined. Images Fig. 1. Fig. 2. PMID:6821372

  13. Natural cysteine protease inhibitors in protozoa: Fifteen years of the chagasin family.

    PubMed

    Costa, Tatiana F R; Lima, Ana Paula C A

    2016-03-01

    Chagasin-type inhibitors comprise natural inhibitors of papain-like cysteine proteases that are distributed among Protist, Bacteria and Archaea. Chagasin was identified in the pathogenic protozoa Trypanosoma cruzi as an approximately 11 kDa protein that is a tight-binding and highly thermostable inhibitor of papain, cysteine cathepsins and endogenous parasite cysteine proteases. It displays an Imunoglobulin-like fold with three exposed loops to one side of the molecule, where amino acid residues present in conserved motifs at the tips of each loop contact target proteases. Differently from cystatins, the loop 2 of chagasin enters the active-site cleft, making direct contact with the catalytic residues, while loops 4 and 6 embrace the enzyme from the sides. Orthologues of chagasin are named Inhibitors of Cysteine Peptidases (ICP), and share conserved overall tri-dimensional structure and mode of binding to proteases. ICPs are tentatively distributed in three families: in family I42 are grouped chagasin-type inhibitors that share conserved residues at the exposed loops; family I71 contains Plasmodium ICPs, which are large proteins having a chagasin-like domain at the C-terminus, with lower similarity to chagasin in the conserved motif at loop 2; family I81 contains Toxoplasma ICP. Recombinant ICPs tested so far can inactivate protozoa cathepsin-like proteases and their mammalian counterparts. Studies on their biological roles were carried out in a few species, mainly using transgenic protozoa, and the conclusions vary. However, in all cases, alterations in the levels of expression of chagasin/ICPs led to substantial changes in one or more steps of parasite biology, with higher incidence in influencing their interaction with the hosts. We will cover most of the findings on chagasin/ICP structural and functional properties and overview the current knowledge on their roles in protozoa.

  14. Zinc-binding cysteines: diverse functions and structural motifs.

    PubMed

    Pace, Nicholas J; Weerapana, Eranthie

    2014-01-01

    Cysteine residues are known to perform essential functions within proteins, including binding to various metal ions. In particular, cysteine residues can display high affinity toward zinc ions (Zn2+), and these resulting Zn2+-cysteine complexes are critical mediators of protein structure, catalysis and regulation. Recent advances in both experimental and theoretical platforms have accelerated the identification and functional characterization of Zn2+-bound cysteines. Zn2+-cysteine complexes have been observed across diverse protein classes and are known to facilitate a variety of cellular processes. Here, we highlight the structural characteristics and diverse functional roles of Zn2+-cysteine complexes in proteins and describe structural, computational and chemical proteomic technologies that have enabled the global discovery of novel Zn2+-binding cysteines.

  15. Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

    NASA Astrophysics Data System (ADS)

    Lórenz-Fonfría, Víctor A.; Muders, Vera; Schlesinger, Ramona; Heberle, Joachim

    2014-12-01

    Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the (conductive) P2380 intermediate decays with τ ≈ 40 ms and 200 ms after pulsed excitation. The vibrational changes between the closed and the conductive states were analyzed in the X-H stretching region (X = O, S, N), comprising vibrational changes of water molecules, sulfhydryl groups of cysteine side chains and changes of the amide A of the protein backbone. The O-H stretching vibrations of "dangling" water molecules were detected in two different states of the protein using H218O exchange. Uncoupling experiments with a 1:1 mixture of H2O:D2O provided the natural uncoupled frequencies of the four O-H (and O-D) stretches of these water molecules, each with a very weakly hydrogen-bonded O-H group (3639 and 3628 cm-1) and with the other O-H group medium (3440 cm-1) to moderately strongly (3300 cm-1) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the "dangling" water molecules within the protein, providing a better understanding of their functional relevance in CaChR1.

  16. Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1.

    PubMed

    Lórenz-Fonfría, Víctor A; Muders, Vera; Schlesinger, Ramona; Heberle, Joachim

    2014-12-14

    Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the (conductive) P2(380) intermediate decays with τ ≈ 40 ms and 200 ms after pulsed excitation. The vibrational changes between the closed and the conductive states were analyzed in the X-H stretching region (X = O, S, N), comprising vibrational changes of water molecules, sulfhydryl groups of cysteine side chains and changes of the amide A of the protein backbone. The O-H stretching vibrations of "dangling" water molecules were detected in two different states of the protein using H2 (18)O exchange. Uncoupling experiments with a 1:1 mixture of H2O:D2O provided the natural uncoupled frequencies of the four O-H (and O-D) stretches of these water molecules, each with a very weakly hydrogen-bonded O-H group (3639 and 3628 cm(-1)) and with the other O-H group medium (3440 cm(-1)) to moderately strongly (3300 cm(-1)) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the "dangling" water molecules within the protein, providing a better understanding of their functional relevance in CaChR1. PMID:25494778

  17. Mutagenesis of varicella-zoster virus glycoprotein I (gI) identifies a cysteine residue critical for gE/gI heterodimer formation, gI structure, and virulence in skin cells.

    PubMed

    Oliver, Stefan L; Sommer, Marvin H; Reichelt, Mike; Rajamani, Jaya; Vlaycheva-Beisheim, Leonssia; Stamatis, Shaye; Cheng, Jason; Jones, Carol; Zehnder, James; Arvin, Ann M

    2011-05-01

    Varicella-zoster virus (VZV) is the alphaherpesvirus that causes chicken pox (varicella) and shingles (zoster). The two VZV glycoproteins gE and gI form a heterodimer that mediates efficient cell-to-cell spread. Deletion of gI yields a small-plaque-phenotype virus, ΔgI virus, which is avirulent in human skin using the xenograft model of VZV pathogenesis. In the present study, 10 mutant viruses were generated to determine which residues were required for the typical function of gI. Three phosphorylation sites in the cytoplasmic domain of gI were not required for VZV virulence in vivo. Two deletion mutants mapped a gE binding region in gI to residues 105 to 125. A glycosylation site, N116, in this region did not affect virulence. Substitution of four cysteine residues highly conserved in the Alphaherpesvirinae established that C95 is required for gE/gI heterodimer formation. The C95A and Δ105-125 (with residues 105 to 125 deleted) viruses had small-plaque phenotypes with reduced replication kinetics in vitro similar to those of the ΔgI virus. The Δ105-125 virus was avirulent for human skin in vivo. In contrast, the C95A mutant replicated in vivo but with significantly reduced kinetics compared to those of the wild-type virus. In addition to abolished gE/gI heterodimer formation, gI from the C95A or the Δ105-125 mutant was not recognized by monoclonal antibodies that detect the canonical conformation of gI, demonstrating structural disruption of gI in these viruses. This alteration prevented gI incorporation into virus particles. Thus, residues C95 and 105 to 125 are critical for gI structure required for gE/gI heterodimer formation, virion incorporation, and ultimately, effective viral spread in human skin.

  18. Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.

    PubMed Central

    Bazan, J F; Fletterick, R J

    1988-01-01

    Proteases that are encoded by animal picornaviruses and plant como- and potyviruses form a related group of cysteine-active-center enzymes that are essential for virus maturation. We show that these proteins are homologous to the family of trypsin-like serine proteases. In our model, the active-site nucleophile of the trypsin catalytic triad, Ser-195, is changed to a Cys residue in these viral proteases. The other two residues of the triad, His-57 and Asp-102, are otherwise absolutely conserved in all the viral protease sequences. Secondary structure analysis of aligned sequences suggests the location of the component strands of the twin beta-barrel trypsin fold in the viral proteases. Unexpectedly, the 2a and 3c subclasses of viral cysteine proteases are, respectively, homologous to the small and large structural subclasses of trypsin-like serine proteases. This classification allows the molecular mapping of residues from viral sequences onto related tertiary structures; we precisely identify amino acids that are strong determinants of specificity for both small and large viral cysteine proteases. Images PMID:3186696

  19. ROSICS: CHEMISTRY AND PROTEOMICS OF CYSTEINE MODIFICATIONS IN REDOX BIOLOGY

    PubMed Central

    Kim, Hee-Jung; Ha, Sura; Lee, Hee Yoon; Lee, Kong-Joo

    2015-01-01

    Post-translational modifications (PTMs) occurring in proteins determine their functions and regulations. Proteomic tools are available to identify PTMs and have proved invaluable to expanding the inventory of these tools of nature that hold the keys to biological processes. Cysteine (Cys), the least abundant (1–2%) of amino acid residues, are unique in that they play key roles in maintaining stability of protein structure, participating in active sites of enzymes, regulating protein function and binding to metals, among others. Cys residues are major targets of reactive oxygen species (ROS), which are important mediators and modulators of various biological processes. It is therefore necessary to identify the Cys-containing ROS target proteins, as well as the sites and species of their PTMs. Cutting edge proteomic tools which have helped identify the PTMs at reactive Cys residues, have also revealed that Cys residues are modified in numerous ways. These modifications include formation of disulfide, thiosulfinate and thiosulfonate, oxidation to sulfenic, sulfinic, sulfonic acids and thiosulfonic acid, transformation to dehydroalanine (DHA) and serine, palmitoylation and farnesylation, formation of chemical adducts with glutathione, 4-hydroxynonenal and 15-deoxy PGJ2, and various other chemicals. We present here, a review of relevant ROS biology, possible chemical reactions of Cys residues and details of the proteomic strategies employed for rapid, efficient and sensitive identification of diverse and novel PTMs involving reactive Cys residues of redox-sensitive proteins. We propose a new name, “ROSics,” for the science which describes the principles of mode of action of ROS at molecular levels. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Rapid Commun. Mass Spec Rev 34:184–208, 2015. PMID:24916017

  20. ROSics: chemistry and proteomics of cysteine modifications in redox biology.

    PubMed

    Kim, Hee-Jung; Ha, Sura; Lee, Hee Yoon; Lee, Kong-Joo

    2015-01-01

    Post-translational modifications (PTMs) occurring in proteins determine their functions and regulations. Proteomic tools are available to identify PTMs and have proved invaluable to expanding the inventory of these tools of nature that hold the keys to biological processes. Cysteine (Cys), the least abundant (1-2%) of amino acid residues, are unique in that they play key roles in maintaining stability of protein structure, participating in active sites of enzymes, regulating protein function and binding to metals, among others. Cys residues are major targets of reactive oxygen species (ROS), which are important mediators and modulators of various biological processes. It is therefore necessary to identify the Cys-containing ROS target proteins, as well as the sites and species of their PTMs. Cutting edge proteomic tools which have helped identify the PTMs at reactive Cys residues, have also revealed that Cys residues are modified in numerous ways. These modifications include formation of disulfide, thiosulfinate and thiosulfonate, oxidation to sulfenic, sulfinic, sulfonic acids and thiosulfonic acid, transformation to dehydroalanine (DHA) and serine, palmitoylation and farnesylation, formation of chemical adducts with glutathione, 4-hydroxynonenal and 15-deoxy PGJ2, and various other chemicals. We present here, a review of relevant ROS biology, possible chemical reactions of Cys residues and details of the proteomic strategies employed for rapid, efficient and sensitive identification of diverse and novel PTMs involving reactive Cys residues of redox-sensitive proteins. We propose a new name, "ROSics," for the science which describes the principles of mode of action of ROS at molecular levels.

  1. The cysteine proteinases of the pineapple plant.

    PubMed Central

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-01-01

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct. Images Fig. 4. Fig. 5. PMID:2327970

  2. The cysteine proteinases of the pineapple plant.

    PubMed

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-03-15

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

  3. Correlation of loss of activity of human aldehyde dehydrogenase with reaction of bromoacetophenone with glutamic acid-268 and cysteine-302 residues. Partial-sites reactivity of aldehyde dehydrogenase.

    PubMed Central

    Abriola, D P; MacKerell, A D; Pietruszko, R

    1990-01-01

    Bromoacetophenone (2-bromo-1-phenylethanone) has been characterized as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) [MacKerell, MacWright & Pietruszko (1986) Biochemistry 25, 5182-5189], and has been shown to react specifically with the Glu-268 residue [Abriola, Fields, Stein, MacKerell & Pietruszko (1987) Biochemistry 26, 5679-5684] with an apparent inactivation stoichiometry of two molecules of bromoacetophenone per molecule of enzyme. The specificity of bromoacetophenone for reaction with Glu-268, however, is not absolute, owing to the extreme reactivity of this reagent. When bromo[14C]acetophenone was used to label the human cytoplasmic E1 isoenzyme radioactively and tryptic fragmentation was carried out, peptides besides that containing Glu-268 were found to have reacted with reagent. These peptides were purified by h.p.l.c. and analysed by sequencing and scintillation counting to quantify radioactive label in the material from each cycle of sequencing. Reaction of bromoacetophenone with the aldehyde dehydrogenase molecule during enzyme activity loss occurs with two residues, Glu-268 and Cys-302. The activity loss, however, appears to be proportional to incorporation of label at Glu-268. The large part of incorporation of label at Cys-302 occurs after the activity loss is essentially complete. With both Glu-268 and Cys-302, however, the incorporation of label stops after one molecule of bromoacetophenone has reacted with each residue. Reaction with other residues continues after activity loss is complete. PMID:1968743

  4. Kinetic evidence for surface residues influencing the active site of Coprinus cinereus peroxidase: analysis of the pH dependence of G154E, P90H and P90H-G154E substrate entrance mutants.

    PubMed

    Di Cerbo, P; Welinder, K G; Schiødt, C B

    2001-01-12

    Three mutants of Coprinus cinereus peroxidase (CIP) were made to mimic the substrate entrance histidine 82-glutamic acid 146 pair of the substrate channel in lignin peroxidase (LIP). Compound I formation of LIP has a low pH optimum around pH 3, while optimal formation of CIP compound I is obtained at pH 6-11. The mutants were glycine 154-->glutamic acid (G154E), proline 90-->histidine (P90H) and the double mutant P90H-G154E. All three showed kinetics of compound I formation similar to that of wt CIP between pH 3 and 9. However, the stability of compound I was strongly affected by these mutations. In wt CIP compound I is stable for approximately 30 min, while compound I of the mutants were stable for 5 s or less. The P90H and P90H-G154E mutants showed pK(a) values for the alkaline transition at least one pH unit lower than for wt CIP and the G154E mutant. We suggest that the changed electrostatic field results in destabilisation of the oxidised heme in compound I and II and that the P90H residue increases the electrostatic potential in the distal cavity thereby decreasing the pK(a) for the alkaline transition.

  5. Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis.

    PubMed Central

    Witte, M M; Dickson, R C

    1988-01-01

    LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae. Both proteins have a single "zinc finger" which plays a role in DNA binding. We previously hypothesized (L. V. Wray, M. M. Witte, R. C. Dickson, and M. I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger. In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein. Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding. Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity. These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix. Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought. Images PMID:3146691

  6. Modulation of Active Site Electronic Structure by the Protein Matrix to Control [NiFe] Hydrogenase Reactivity

    SciTech Connect

    Smith, Dayle MA; Raugei, Simone; Squier, Thomas C.

    2014-09-30

    Control of the reactivity of the nickel center of the [NiFe] hydrogenase and other metalloproteins commonly involves outer coordination sphere ligands that act to modify the geometry and physical properties of the active site metal centers. We carried out a combined set of classical molecular dynamics and quantum/classical mechanics calculations to provide quantitative estimates of how dynamic fluctuations of the active site within the protein matrix modulate the electronic structure at the catalytic center. Specifically we focused on the dynamics of the inner and outer coordination spheres of the cysteinate-bound Ni–Fe cluster in the catalytically active Ni-C state. There are correlated movements of the cysteinate ligands and the surrounding hydrogen-bonding network, which modulate the electron affinity at the active site and the proton affinity of a terminal cysteinate. On the basis of these findings, we hypothesize a coupling between protein dynamics and electron and proton transfer reactions critical to dihydrogen production.

  7. Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

    PubMed Central

    Macomber, Lee; Minkara, Mona S.; Hausinger, Robert P.; Merz, Kenneth M.

    2015-01-01

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  8. Defining critical residues for substrate binding to 1-deoxy-D-xylulose 5-phosphate synthase--active site substitutions stabilize the predecarboxylation intermediate C2α-lactylthiamin diphosphate.

    PubMed

    Brammer Basta, Leighanne A; Patel, Hetalben; Kakalis, Lazaros; Jordan, Frank; Freel Meyers, Caren L

    2014-06-01

    1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the formation of DXP from pyruvate and D-glyceraldehyde 3-phosphate (GraP) in a thiamin diphosphate-dependent manner, and is the first step in the essential pathway to isoprenoids in human pathogens. Understanding the mechanism of this unique enzyme is critical for developing new anti-infective agents that selectively target isoprenoid biosynthesis. The present study used mutagenesis and a combination of protein fluorescence, CD and kinetics experiments to investigate the roles of Arg420, Arg478 and Tyr392 in substrate binding and catalysis. The results support a random sequential, preferred order mechanism, and predict that Arg420 and Arg478 are involved in binding of the acceptor substrate, GraP. D-Glyceraldehyde, an alternative acceptor substrate lacking the phosphoryl group predicted to interact with Arg420 and Arg478, also accelerates decarboxylation of the predecarboxylation intermediate C2α-lactylthiamin diphosphate (LThDP) on DXP synthase, indicating that this binding interaction is not absolutely required, and that the hydroxyaldehyde sufficiently triggers decarboxylation. Unexpectedly, Tyr392 contributes to GraP affinity, and is not required for LThDP formation or its GraP-promoted decarboxylation. Time-resolved CD spectroscopy and NMR experiments indicate that LThDP is significantly stabilized on R420A and Y392F variants as compared with wild-type DXP synthase in the absence of acceptor substrate, but these substitutions do not appear to affect the rate of GraP-promoted LThDP decarboxylation in the presence of high levels of GraP, and LThDP formation remains the rate-limiting step. These results suggest a role of these residues in promoting GraP binding, which in turn facilitates decarboxylation, and also highlight interesting differences between DXP synthase and other thiamin diphosphate-dependent enzymes.

  9. Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1.

    PubMed

    Urzica, Eugen; Pierik, Antonio J; Mühlenhoff, Ulrich; Lill, Roland

    2009-06-01

    Iron-sulfur (Fe/S) protein maturation in the eukaryotic cytosol and nucleus requires conserved components of the essential CIA machinery. The CIA protein Nar1 performs a specific function in transferring an Fe/S cluster that is assembled de novo on the Cfd1-Nbp35 scaffold to apoproteins. Here, we used systematic site-directed mutagenesis and a combination of in vitro and in vivo studies to show that Nar1 holds two Fe/S clusters at conserved N- and C-terminal cysteine motifs. A wealth of biochemical studies suggests that the assembly of these Fe/S clusters on Nar1 cannot be studied in Escherichia coli, as the recombinant protein does not contain the native Fe/S clusters. We therefore followed Fe/S cluster incorporation directly in yeast by a (55)Fe radiolabeling method in vivo, and we measured the functional consequences of Nar1 mutations in the assembly of cytosolic Fe/S proteins. We find that both Fe/S clusters are essential for Nar1 function and cell viability. Molecular modeling using a structurally but not functionally related bacterial iron-only hydrogenase as a template provided compelling structural explanations for our mutational data. The C-terminal Fe/S cluster is stably buried within Nar1, whereas the N-terminal one is exposed at the protein surface and hence may be more easily lost. Insertion of an Fe/S cluster into the C-terminal location depends on the N-terminal motif, suggesting the participation of the latter motif in the assembly process of the C-terminal cluster. The vicinity of the two Fe/S centers suggests a close functional cooperation during cytosolic Fe/S protein maturation.

  10. Selenocysteine Positional Variants Reveal Contributions to Copper Binding From Cysteine Residues in Domains 2 And 3 of Human Copper Chaperone for Superoxide Dismutase

    SciTech Connect

    Barry, A.N.; Clark, K.M.; Otoikhian, A.; Donk, W.A.van der; Blackburn, N.J.

    2009-05-11

    The human copper chaperone for superoxide dismutase binds copper both in an Atx1-like MTCQSC motif in domain 1 and via a multinuclear cluster formed by two CXC motifs at the D3 dimer interface. The composition of the Cu(I) cluster has been investigated previously by mutagenesis of the CXC motif, and by construction of a CXU selenocysteine derivative, which has permitted XAS studies at both Cu and Se absorption edges. Here, we report the semisynthesis and spectroscopic characterization of a series of derivatives with the sequences 243-CACA, 243-CAUA, 243-UACA, and 243-UAUA in the D1 double mutant (C22AC25A) background, prepared by expressed protein ligation of Sec-containing tetrapeptides to an hCCS-243 truncation. By varying the position of the Se atom in the CXC motif, we have been able to show that Se is always bridging (2 Se-Cu) rather than terminal (1 Se-Cu). Substitution of both D3 Cys residues by Sec in the UAUA variant does not eliminate the Cu-S contribution, confirming our previous description of the cluster as most likely a Cu{sub 4}S{sub 6} species, and suggesting that D2 Cys residues contribute to the cluster. As predicted by this model, when Cys residues C141, C144, and C227 are mutated to alanine either individually or together as a triple mutant, the cluster nuclearity is dramatically attenuated. These data suggest that Cys residues in D2 of hCCS are involved in the formation, stability, and redox potential of the D3 cluster. The significance of these finding to the SOD1 thiol/disulfide oxidase activity are discussed in terms of a model in which a similar multinuclear cluster may form in the CCS-SOD heterodimer.

  11. Probes of the Catalytic Site of Cysteine Dioxygenase

    SciTech Connect

    Chai,S.; Bruyere, J.; Maroney, M.

    2006-01-01

    The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the a-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ a-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by {alpha}-ketoglutarate.

  12. Discovery of Potent Cysteine-Containing Dipeptide Inhibitors against Tyrosinase: A Comprehensive Investigation of 20 × 20 Dipeptides in Inhibiting Dopachrome Formation.

    PubMed

    Tseng, Tien-Sheng; Tsai, Keng-Chang; Chen, Wang-Chuan; Wang, Yeng-Tseng; Lee, Yu-Ching; Lu, Chung-Kuang; Don, Ming-Jaw; Chang, Chang-Yu; Lee, Ching-Hsiao; Lin, Hui-Hsiung; Hsu, Hung-Ju; Hsiao, Nai-Wan

    2015-07-15

    Tyrosinase is an essential copper-containing enzyme required for melanin synthesis. The overproduction and abnormal accumulation of melanin cause hyperpigmentation and neurodegenerative diseases. Thus, tyrosinase is promising for use in medicine and cosmetics. Our previous study identified a natural product, A5, resembling the structure of the dipeptide WY and apparently inhibiting tyrosinase. Here, we comprehensively estimated the inhibitory capability of 20 × 20 dipeptides against mushroom tyrosinase. We found that cysteine-containing dipeptides, directly blocking the active site of tyrosinase, are highly potent in inhibition; in particular, N-terminal cysteine-containing dipeptides markedly outperform the C-terminal-containing ones. The cysteine-containing dipeptides, CE, CS, CY, and CW, show comparative bioactivities, and tyrosine-containing dipeptides are substrate-like inhibitors. The dipeptide PD attenuates 16.5% melanin content without any significant cytotoxicity. This study reveals the functional role of cysteine residue positional preference and the selectivity of specific amino acids in cysteine-containing dipeptides against tyrosinase, aiding in developing skin-whitening products. PMID:26083974

  13. Activation Mechanism of the Bacteroides fragilis Cysteine Peptidase, Fragipain.

    PubMed

    Herrou, Julien; Choi, Vivian M; Bubeck Wardenburg, Juliane; Crosson, Sean

    2016-07-26

    Enterotoxigenic Bacteroides fragilis produces a secreted metalloprotease known as B. fragilis toxin (BFT), which contributes to anaerobic sepsis, colitis, and colonic malignancy in mouse models of disease. A C11 family cysteine protease, fragipain (Fpn), directly activates BFT in the B. fragilis cell by removing the BFT prodomain. Fpn is itself a proenzyme and is autoactivated upon cleavage at an arginine residue in its activation loop. We have defined the proteolytic active site of Fpn, demonstrated that Fpn autoactivation can occur by an in trans loop cleavage mechanism, and characterized structural features of the Fpn activation loop that control peptidase activity against several substrates, including BFT. An arginine residue at the autocleavage site determines the fast activation kinetics of Fpn relative to the homologous C11 protease, PmC11, which is cleaved at lysine. Arginine to alanine substitution at the cleavage site ablated peptidase activity, as did partial truncation of the Fpn activation loop. However, complete truncation of the activation loop yielded an uncleaved, pro form of Fpn that was active as a peptidase against both Fpn and BFT substrates. Thus, Fpn can be transformed into an active peptidase in the absence of activation loop cleavage. This study provides insight into the mechanism of fragipain activation and, more generally, defines the role of the C11 activation loop in the control of peptidase activity and substrate specificity.

  14. Protein modification by acrolein: Formation and stability of cysteine adducts

    PubMed Central

    Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

    2010-01-01

    The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intra-molecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein-acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues. PMID:19231900

  15. Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase*

    PubMed Central

    Lakshminarasimhan, Mahadevan; Madzelan, Peter; Nan, Ruth; Milkovic, Nicole M.; Wilson, Mark A.

    2010-01-01

    Isocyanide (formerly isonitrile) hydratase (EC 4.2.1.103) is an enzyme of the DJ-1 superfamily that hydrates isocyanides to yield the corresponding N-formamide. In order to understand the structural basis for isocyanide hydratase (ICH) catalysis, we determined the crystal structures of wild-type and several site-directed mutants of Pseudomonas fluorescens ICH at resolutions ranging from 1.0 to 1.9 Å. We also developed a simple UV-visible spectrophotometric assay for ICH activity using 2-naphthyl isocyanide as a substrate. ICH contains a highly conserved cysteine residue (Cys101) that is required for catalysis and interacts with Asp17, Thr102, and an ordered water molecule in the active site. Asp17 has carboxylic acid bond lengths that are consistent with protonation, and we propose that it activates the ordered water molecule to hydrate organic isocyanides. In contrast to Cys101 and Asp17, Thr102 is tolerant of mutagenesis, and the T102V mutation results in a substrate-inhibited enzyme. Although ICH is similar to human DJ-1 (1.6 Å C-α root mean square deviation), structural differences in the vicinity of Cys101 disfavor the facile oxidation of this residue that is functionally important in human DJ-1 but would be detrimental to ICH activity. The ICH active site region also exhibits surprising conformational plasticity and samples two distinct conformations in the crystal. ICH represents a previously uncharacterized clade of the DJ-1 superfamily that possesses a novel enzymatic activity, demonstrating that the DJ-1 core fold can evolve diverse functions by subtle modulation of the environment of a conserved, reactive cysteine residue. PMID:20630867

  16. Histidine at the active site of Neurospora tyrosinase.

    PubMed

    Pfiffner, E; Lerch, K

    1981-10-13

    The involvement of histidyl residues as potential ligands to the binuclear active-site copper of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was shown to be unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Copper measurements of photooxidized, reconstituted apoenzyme demonstrated the loss of binding of one copper atom per mole of enzyme as a consequence of photosensitized oxidation of three out of nine histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. The data obtained show the preferential modification of histidyl residues 188, 193, and 289 and suggest that they constitute metal ligands to one of the two active-site copper atoms. Substitution of copper by cobalt was found to afford complete protection of the histidyl residues from being modified by dye-sensitized photooxidation. PMID:6458322

  17. Using catalytic atom maps to predict the catalytic functions present in enzyme active sites.

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-09-18

    Catalytic atom maps (CAMs) are minimal models of enzyme active sites. The structures in the Protein Data Bank (PDB) were examined to determine if proteins with CAM-like geometries in their active sites all share the same catalytic function. We combined the CAM-based search protocol with a filter based on the weighted contact number (WCN) of the catalytic residues, a measure of the "crowdedness" of the microenvironment around a protein residue. Using this technique, a CAM based on the Ser-His-Asp catalytic triad of trypsin was able to correctly identify catalytic triads in other enzymes within 0.5 Å rmsd of the CAM with 96% accuracy. A CAM based on the Cys-Arg-(Asp/Glu) active site residues from the tyrosine phosphatase active site achieved 89% accuracy in identifying this type of catalytic functionality. Both of these CAMs were able to identify active sites across different fold types. Finally, the PDB was searched to locate proteins with catalytic functionality similar to that present in the active site of orotidine 5'-monophosphate decarboxylase (ODCase), whose mechanism is not known with certainty. A CAM, based on the conserved Lys-Asp-Lys-Asp tetrad in the ODCase active site, was used to search the PDB for enzymes with similar active sites. The ODCase active site has a geometry similar to that of Schiff base-forming Class I aldolases, with lowest aldolase rmsd to the ODCase CAM at 0.48 Å. The similarity between this CAM and the aldolase active site suggests that ODCase has the correct catalytic functionality present in its active site for the generation of a nucleophilic lysine. PMID:22909276

  18. Using Catalytic Atom Maps to Predict the Catalytic Functions Present in Enzyme Active Sites

    PubMed Central

    Nosrati, Geoffrey R.; Houk, K. N.

    2012-01-01

    Catalytic Atom Maps (CAMs) are minimal models of enzyme active sites. The structures in the Protein Data Bank (PDB) were examined to determine if proteins with CAM-like geometries in their active sites all share the same catalytic function. We combined the CAM-based search protocol with a filter based on the weighted contact number (WCN) of the catalytic residues, a measure of the “crowdedness” of the microenvironment around a protein residue. Using this technique, a CAM based on the Ser-His-Asp catalytic triad of trypsin was able to correctly identify catalytic triads in other enzymes within 0.5 Å RMSD of the Catalytic Atom Map with 96% accuracy. A CAM based on the Cys-Arg-(Asp/Glu) active site residues from the tyrosine phosphatase active site achieved 89% accuracy in identifying this type of catalytic functionality. Both of these Catalytic Atom Maps were able to identify active sites across different fold types. Finally, the PDB was searched to locate proteins with catalytic functionality similar to that present in the active site of orotidine 5′-monophosphate decarboxylase (ODCase), whose mechanism is not known with certainty. A CAM, based on the conserved Lys-Asp-Lys-Asp tetrad in the ODCase active site, was used to search the PDB for enzymes with similar active sites. The ODCase active site has a geometry similar to that of Schiff base-forming Class I aldolases, with lowest aldolase RMSD to the ODCase CAM at 0.48 Å. The similarity between this CAM and the aldolase active site suggests that ODCase has the correct catalytic functionality present in its active site for the generation of a nucleophilic lysine. PMID:22909276

  19. Computer simulation of the active site of human serum cholinesterase

    SciTech Connect

    Kefang Jiao; Song Li; Zhengzheng Lu

    1996-12-31

    The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positive electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.

  20. Key role of cysteine residues and sulfenic acids in thermal- and H2O2-mediated modification of β-lactoglobulin.

    PubMed

    Krämer, Anna C; Thulstrup, Peter W; Lund, Marianne N; Davies, Michael J

    2016-08-01

    Oxidation results in protein deterioration in mammals, plants, foodstuffs and pharmaceuticals, via changes in amino acid composition, fragmentation, aggregation, solubility, hydrophobicity, conformation, function and susceptibility to digestion. This study investigated whether and how individual or combined treatment with heat, a commonly encountered factor in industrial processing, and H2O2 alters the structure and composition of the major whey protein β-lactoglobulin. Thermal treatment induced reducible cross-links, with this being enhanced by low H2O2 concentrations, but decreased by high concentrations, where fragmentation was detected. Cross-linking was prevented when the single free Cys121 residue was blocked with iodoacetamide. Low concentrations of H2O2 added before heating depleted thiols, with H2O2 alone, or H2O2 added after heating, having lesser effects. A similar pattern was detected for methionine loss and methionine sulfoxide formation. Tryptophan loss was only detected with high levels of H2O2, and no other amino acid was affected, indicating that sulfur-centered amino acids are critical targets. No protection against aggregation was provided by high concentrations of the radical scavenger 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), consistent with molecular oxidation, rather than radical reactions, being the major process. Sulfenic acid formation was detected by Western blotting and LC-MS/MS peptide mass-mapping of dimedone-treated protein, consistent with these species being significant intermediates in heat-induced cross-linking, especially in the presence of H2O2. Studies using circular dichroism and intrinsic fluorescence indicate that H2O2 increases unfolding during heating. These mechanistic insights provide potential strategies for modulating the extent of modification of proteins exposed to thermal and oxidant treatment.

  1. Identification of the reactive cysteinyl residue and ATP binding site in Bacillus cereus glutamine synthetase by chemical modification.

    PubMed

    Nakano, Y; Itoh, M; Tanaka, E; Kimura, K

    1990-02-01

    Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2(+)-dependent activity and activated Mn2(+)-dependent activity. FSBA inactivated only Mn2(+)-dependent activity. Mg2+ plus Mn2(+)-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.

  2. A novel allosteric mechanism in the cysteine peptidase cathepsin K discovered by computational methods

    NASA Astrophysics Data System (ADS)

    Novinec, Marko; Korenč, Matevž; Caflisch, Amedeo; Ranganathan, Rama; Lenarčič, Brigita; Baici, Antonio

    2014-02-01

    Allosteric modifiers have the potential to fine-tune enzyme activity. Therefore, targeting allosteric sites is gaining increasing recognition as a strategy in drug design. Here we report the use of computational methods for the discovery of the first small-molecule allosteric inhibitor of the collagenolytic cysteine peptidase cathepsin K, a major target for the treatment of osteoporosis. The molecule NSC13345 is identified by high-throughput docking of compound libraries to surface sites on the peptidase that are connected to the active site by an evolutionarily conserved network of residues (protein sector). The crystal structure of the complex shows that NSC13345 binds to a novel allosteric site on cathepsin K. The compound acts as a hyperbolic mixed modifier in the presence of a synthetic substrate, it completely inhibits collagen degradation and has good selectivity for cathepsin K over related enzymes. Altogether, these properties qualify our methodology and NSC13345 as promising candidates for allosteric drug design.

  3. Redox regulation of epidermal growth factor receptor signaling through cysteine oxidation.

    PubMed

    Truong, Thu H; Carroll, Kate S

    2012-12-18

    Epidermal growth factor receptor (EGFR) exemplifies the family of receptor tyrosine kinases that mediate numerous cellular processes, including growth, proliferation, and differentiation. Moreover, gene amplification and EGFR mutations have been identified in a number of human malignancies, making this receptor an important target for the development of anticancer drugs. In addition to ligand-dependent activation and concomitant tyrosine phosphorylation, EGFR stimulation results in the localized generation of H(2)O(2) by NADPH-dependent oxidases. In turn, H(2)O(2) functions as a secondary messenger to regulate intracellular signaling cascades, largely through the modification of specific cysteine residues within redox-sensitive protein targets, including Cys797 in the EGFR active site. In this review, we highlight recent advances in our understanding of the mechanisms that underlie redox regulation of EGFR signaling and how these discoveries may form the basis for the development of new therapeutic strategies for targeting this and other H(2)O(2)-modulated pathways.

  4. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  5. Structure and mechanism of mouse cysteine dioxygenase

    PubMed Central

    McCoy, Jason G.; Bailey, Lucas J.; Bitto, Eduard; Bingman, Craig A.; Aceti, David J.; Fox, Brian G.; Phillips, George N.

    2006-01-01

    Cysteine dioxygenase (CDO) catalyzes the oxidation of l-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 Å. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical β-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal binding site. The structure further revealed a covalent linkage between the side chains of Cys-93 and Tyr-157, the cysteine of which is conserved only in eukaryotic proteins. Metal analysis showed that the recombinant enzyme contained a mixture of iron, nickel, and zinc, with increased iron content associated with increased catalytic activity. Details of the predicted active site are used to present and discuss a plausible mechanism of action for the enzyme. PMID:16492780

  6. Catalysis: Elusive active site in focus

    NASA Astrophysics Data System (ADS)

    Labinger, Jay A.

    2016-08-01

    The identification of the active site of an iron-containing catalyst raises hopes of designing practically useful catalysts for the room-temperature conversion of methane to methanol, a potential fuel for vehicles. See Letter p.317

  7. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  8. Amphibian DNases I are characterized by a C-terminal end with a unique, cysteine-rich stretch and by the insertion of a serine residue into the Ca2+-binding site.

    PubMed Central

    Takeshita, H; Yasuda, T; Iida, R; Nakajima, T; Mori, S; Mogi, K; Kaneko, Y; Kishi, K

    2001-01-01

    We purified four amphibian deoxyribonucleases I from the pancreases of one toad, two frog and one newt species, by using three different column chromatography methods in sequence. Each of the purified enzymes had a molecular mass of approx. 40 kDa and an optimal pH for activity of approx. 8.0. These values were significantly greater than those for other vertebrate DNases I. The full-length cDNA encoding each amphibian DNase I was constructed from the total RNA of the pancreas by using rapid amplification of cDNA ends. Nucleotide sequence analyses revealed two structural characteristics unique to amphibian DNases I: a stretch of approx. 70 amino acids with a high cysteine content (approx. 15%) in the C-terminal region, and the insertion of a serine residue at position 205 (in a domain containing an essential Ca2+-binding site). Expression analysis of a series of mutant constructs indicated that both of these structures are essential in generating the active form of the enzyme. 'DNase I signature sequences', which are well conserved in other vertebrate DNases I, could not be found in any of the amphibian DNases I tested, whereas a 'somatomedin B motif' was identified in the Cys-rich stretches of all four. Although DNase I has so far been considered to be a secretory glycoprotein, amphibian DNase I seems to be non-glycosylated. These structural findings indicate strongly that amphibian DNases I are situated in a unique position on the phylogenetic tree of the DNase I family. PMID:11439097

  9. Characterization of the Cysteine Content in Proteins Utilizing Cysteine Selenylation with 266 nm Ultraviolet Photodissociation (UVPD)

    NASA Astrophysics Data System (ADS)

    Parker, W. Ryan; Brodbelt, Jennifer S.

    2016-08-01

    Characterization of the cysteine content of proteins is a key aspect of proteomics. By defining both the total number of cysteines and their bound/unbound state, the number of candidate proteins considered in database searches is significantly constrained. Herein we present a methodology that utilizes 266 nm UVPD to count the number of free and bound cysteines in intact proteins. In order to attain this goal, proteins were derivatized with N-(phenylseleno)phthalimide (NPSP) to install a selectively cleavable Se-S bond upon 266 UVPD. The number of Se-S bonds cleaved upon UVPD, a process that releases SePh moieties, corresponds to the number of cysteine residues per protein.

  10. The Structures of the C185S and C185A Mutants of Sulfite Oxidase Reveal Rearrangement of the Active Site

    SciTech Connect

    Qiu, James A.; Wilson, Heather L.; Pushie, M. Jake; Kisker, Caroline; George, Graham N.; Rajagopalan, K.V.

    2010-11-03

    Sulfite oxidase (SO) catalyzes the physiologically critical conversion of sulfite to sulfate. Enzymatic activity is dependent on the presence of the metal molybdenum complexed with a pyranopterin-dithiolene cofactor termed molybdopterin. Comparison of the amino acid sequences of SOs from a variety of sources has identified a single conserved Cys residue essential for catalytic activity. The crystal structure of chicken liver sulfite oxidase indicated that this residue, Cys185 in chicken SO, coordinates the Mo atom in the active site. To improve our understanding of the role of this residue in the catalytic mechanism of sulfite oxidase, serine and alanine variants at position 185 of recombinant chicken SO were generated. Spectroscopic and kinetic studies indicate that neither variant is capable of sulfite oxidation. The crystal structure of the C185S variant was determined to 1.9 {angstrom} resolution and to 2.4 {angstrom} resolution in the presence of sulfite, and the C185A variant to 2.8 {angstrom} resolution. The structures of the C185S and C185A variants revealed that neither the Ser or Ala side chains appeared to closely interact with the Mo atom and that a third oxo group replaced the usual cysteine sulfur ligand at the Mo center, confirming earlier extended X-ray absorption fine structure spectroscopy (EXAFS) work on the human C207S mutant. An unexpected result was that in the C185S variant, in the absence of sulfite, the active site residue Tyr322 became disordered as did the loop region flanking it. In the C185S variant crystallized in the presence of sulfite, the Tyr322 residue relocalized to the active site. The C185A variant structure also indicated the presence of a third oxygen ligand; however, Tyr322 remained in the active site. EXAFS studies of the Mo coordination environment indicate the Mo atom is in the oxidized Mo{sup VI} state in both the C185S and C185A variants of chicken SO and show the expected trioxodithiolene active site. Density

  11. Structural and Immunological Characteristics of a 28-Kilodalton Cruzipain-Like Cysteine Protease of Paragonimus westermani Expressed in the Definitive Host Stage

    PubMed Central

    Yun, Doo-Hee; Chung, Joon-Yong; Chung, Young-Bae; Bahk, Young-Yil; Kang, Shin-Yong; Kong, Yoon; Cho, Seung-Yull

    2000-01-01

    A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adult Paragonimus westermani, termed Pw28CCP, was isolated from an adult cDNA library. The cDNA contained a single open reading frame of 975 bp encoding 325 amino acids, which exhibited the structural motif and domain organization characteristic of cysteine proteases of non-cathepsin Bs including a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine residues as well as active sites in the mature catalytic region. Analysis of its phylogenetic position revealed that this novel enzyme belonged to the cruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determined from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a location in the intestinal epithelium, suggesting that this enzyme could be secreted and involved in nutrient uptake and immune modulation. The recombinant protein expressed in Escherichia coli was used to assess antigenicity by immunoblotting with sera from patients with active paragonimiasis and from those with other parasitic infections. The resulting sensitivity of 86.2% (56 of 65 samples) and specificity of 98% (147 of 150 samples) suggest its potential as an antigen for use in immunodiagnosis. PMID:11063501

  12. Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site*

    PubMed Central

    Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

    2014-01-01

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser105 residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T5015, the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability. PMID:24448805

  13. Sequence comparison, molecular modeling, and network analysis predict structural diversity in cysteine proteases from the Cape sundew, Drosera capensis.

    PubMed

    Butts, Carter T; Zhang, Xuhong; Kelly, John E; Roskamp, Kyle W; Unhelkar, Megha H; Freites, J Alfredo; Tahir, Seemal; Martin, Rachel W

    2016-01-01

    Carnivorous plants represent a so far underexploited reservoir of novel proteases with potentially useful activities. Here we investigate 44 cysteine proteases from the Cape sundew, Drosera capensis, predicted from genomic DNA sequences. D. capensis has a large number of cysteine protease genes; analysis of their sequences reveals homologs of known plant proteases, some of which are predicted to have novel properties. Many functionally significant sequence and structural features are observed, including targeting signals and occluding loops. Several of the proteases contain a new type of granulin domain. Although active site residues are conserved, the sequence identity of these proteases to known proteins is moderate to low; therefore, comparative modeling with all-atom refinement and subsequent atomistic MD-simulation is used to predict their 3D structures. The structure prediction data, as well as analysis of protein structure networks, suggest multifarious variations on the papain-like cysteine protease structural theme. This in silico methodology provides a general framework for investigating a large pool of sequences that are potentially useful for biotechnology applications, enabling informed choices about which proteins to investigate in the laboratory. PMID:27471585

  14. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  15. Sulfur K-edge XAS and DFT calculations on nitrile hydratase: geometric and electronic structure of the non-heme iron active site.

    PubMed

    Dey, Abhishek; Chow, Marina; Taniguchi, Kayoko; Lugo-Mas, Priscilla; Davin, Steven; Maeda, Mizuo; Kovacs, Julie A; Odaka, Masafumi; Hodgson, Keith O; Hedman, Britt; Solomon, Edward I

    2006-01-18

    The geometric and electronic structure of the active site of the non-heme iron enzyme nitrile hydratase (NHase) is studied using sulfur K-edge XAS and DFT calculations. Using thiolate (RS(-))-, sulfenate (RSO(-))-, and sulfinate (RSO(2)(-))-ligated model complexes to provide benchmark spectral parameters, the results show that the S K-edge XAS is sensitive to the oxidation state of S-containing ligands and that the spectrum of the RSO(-) species changes upon protonation as the S-O bond is elongated (by approximately 0.1 A). These signature features are used to identify the three cysteine residues coordinated to the low-spin Fe(III) in the active site of NHase as CysS(-), CysSOH, and CysSO(2)(-) both in the NO-bound inactive form and in the photolyzed active form. These results are correlated to geometry-optimized DFT calculations. The pre-edge region of the X-ray absorption spectrum is sensitive to the Z(eff) of the Fe and reveals that the Fe in [FeNO](6) NHase species has a Z(eff) very similar to that of its photolyzed Fe(III) counterpart. DFT calculations reveal that this results from the strong pi back-bonding into the pi antibonding orbital of NO, which shifts significant charge from the formally t(2)(6) low-spin metal to the coordinated NO. PMID:16402841

  16. Histidine 407, a phantom residue in the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex, activates reductive acetylation of lipoamide on the E2 subunit. An explanation for conservation of active sites between the E1 subunit and transketolase.

    PubMed

    Nemeria, Natalia; Arjunan, Palaniappa; Brunskill, Andrew; Sheibani, Farzad; Wei, Wen; Yan, Yan; Zhang, Sheng; Jordan, Frank; Furey, William

    2002-12-31

    Least squares alignment of the E. coli pyruvate dehydrogenase multienzyme complex E1 subunit and yeast transketolase crystal structures indicates a general structural similarity between the two enzymes and provides a plausible location for a short-loop region in the E1 structure that was unobserved due to disorder. The residue H407, located in this region, is shown to be able to penetrate the active site. Suggested by this comparison, the H407A E1 variant was created, and H407 was shown to participate in the reductive acetylation of both an independently expressed lipoyl domain and the intact 1-lipoyl E2 subunit. While the H407A substitution only modestly affected the reaction through pyruvate decarboxylation (ca. 14% activity compared to parental E1), the overall complex has a much impaired activity, at most 0.15% compared to parental E1. Isothermal titration calorimetry measurements show that the binding of the lipoyl domain to the H407A E1 variant is much weaker than that to parental E1. At the same time, mass spectrometric measurements clearly demonstrate much impaired reductive acetylation of the independently expressed lipoyl domain and of the intact 1-lipoyl E2 by the H407A variant compared to the parental E1. A proposal is presented to explain the remarkable conservation of the three-dimensional structure at the active centers of the E. coli E1 subunit and transketolase on the basis of the parallels in the ligation-type reactions carried out and the need to protonate a very weak acid, a dithiolane sulfur atom in the former, and a carbonyl oxygen atom in the latter. PMID:12501174

  17. Π-Clamp-mediated cysteine conjugation.

    PubMed

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J; Santos, Michael S; Van Voorhis, Troy; Pentelute, Bradley L

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the 'π-clamp', that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

  18. π-Clamp-mediated cysteine conjugation

    NASA Astrophysics Data System (ADS)

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; van Voorhis, Troy; Pentelute, Bradley L.

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the ‘π-clamp’, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

  19. Π-Clamp-mediated cysteine conjugation.

    PubMed

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J; Santos, Michael S; Van Voorhis, Troy; Pentelute, Bradley L

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the 'π-clamp', that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics. PMID:26791894

  20. π-Clamp Mediated Cysteine Conjugation

    PubMed Central

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; Van Voorhis, Troy; Pentelute, Bradley L.

    2016-01-01

    Site-selective functionalization of complex molecules is a grand challenge in chemistry. Protecting groups or catalysts must be used to selectively modify one site among many that are similarly reactive. General strategies are rare such the local chemical environment around the target site is tuned for selective transformation. Here we show a four amino acid sequence (Phe-Cys-Pro-Phe), which we call the “π-clamp”, tunes the reactivity of its cysteine thiol for the site-selective conjugation with perfluoroaromatic reagents. We used the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues (e.g. antibodies and cysteine-based enzymes), which was impossible with prior cysteine modification methods. The modified π-clamp antibodies retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates (ADCs) for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach for site-selective chemistry and provides opportunities to modify biomolecules for research and therapeutics. PMID:26791894

  1. Arrested cell proliferation through cysteine protease activity of eukaryotic ribosomal protein S4.

    PubMed

    Yadaiah, Madasu; Sudhamalla, Babu; Rao, P Nageswara; Roy, Karnati R; Ramakrishna, Dasari; Hussain Syed, Gulam; Ramaiah, Kolluru V A; Bhuyan, Abani K

    2013-02-01

    S4 is an integral protein of the smaller subunit of cytosolic ribosome. In prokaryotes, it regulates the synthesis of ribosomal proteins by feedback inhibition of the α-operon gene expression, and it facilitates ribosomal RNA synthesis by direct binding to RNA polymerase. However, functional roles of S4 in eukaryotes are poorly understood, although its deficiency in humans is thought to produce Turner syndrome. We report here that wheat S4 is a cysteine protease capable of abrogating total protein synthesis in an actively translating cell-free system of rabbit reticulocytes. The translation-blocked medium, imaged by atomic force microscopy, scanning electron microscopy, and transmission electron microscopy, shows dispersed polysomes, and the disbanded polyribosome elements aggregate to form larger bodies. We also show that human embryonic kidney cells transfected with recombinant wheat S4 are unable to grow and proliferate. The mutant S4 protein, where the putative active site residue Cys 41 is replaced by a phenylalanine, can neither suppress protein synthesis nor arrest cell proliferation, suggesting that the observed phenomenon arises from the cysteine protease attribute of S4. The results also inspire many questions concerning in vivo significance of extraribosomal roles of eukaryotic S4 performed through its protease activity.

  2. Electrostatic fields in the active sites of lysozymes.

    PubMed

    Sun, D P; Liao, D I; Remington, S J

    1989-07-01

    Considerable experimental evidence is in support of several aspects of the mechanism that has been proposed for the catalytic activity of lysozyme. However, the enzymatically catalyzed hydrolysis of polysaccharides proceeds over 5 orders of magnitude faster than that of model compounds that mimic the configuration of the substrate in the active site of the enzyme. Although several possible explanations for this rate enhancement have been discussed elsewhere, a definitive mechanism has not emerged. Here we report striking results obtained by classical electrodynamics, which suggest that bond breakage and the consequent separation of charge in lysozyme is promoted by a large electrostatic field across the active site cleft, produced in part by a very asymmetric distribution of charged residues on the enzyme surface. Lysozymes unrelated in amino acid sequence have similar distributions of charged residues and electric fields. The results reported here suggest that the electrostatic component of the rate enhancement is greater than 9 kcal.mol-1. Thus, electrostatic interactions may play a more important role in the enzymatic mechanism than has generally been appreciated.

  3. Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation

    PubMed Central

    Kim, Soohyun; Kim, Hyori; Chung, Junho

    2016-01-01

    For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process. PMID:26764487

  4. Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation.

    PubMed

    Yoon, Aerin; Shin, Jung Won; Kim, Soohyun; Kim, Hyori; Chung, Junho

    2016-01-01

    For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process.

  5. A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

    SciTech Connect

    Somarowthu, Srinivas; Brodkin, Heather R.; D’Aquino, J. Alejandro; Ringe, Dagmar; Ondrechen, Mary Jo; Beuning, Penny J.

    2012-07-11

    Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

  6. Cysteine Modification: Probing Channel Structure, Function and Conformational Change.

    PubMed

    Akabas, Myles H

    2015-01-01

    Cysteine substitution has been a powerful tool to investigate the structure and function of proteins. It has been particularly useful for studies of membrane proteins in their native environment, embedded in phospholipid membranes. Among the 20 amino acids, cysteine is uniquely reactive. This reactivity has motivated the synthesis of a wide array of sulfhydryl reactive chemicals. The commercially available array of sulfhydryl reactive reagents has allowed investigators to probe the local steric and electrostatic environment around engineered cysteines and to position fluorescent, paramagnetic and mass probes at specific sites within proteins and for distance measurements between pairs of sites. Probing the reactivity and accessibility of engineered cysteines has been extensively used in Substituted Cysteine Accessibility Method (SCAM) investigations of ion channels, membrane transporters and receptors. These studies have successfully identified the residues lining ion channels, agonist/antagonist and allosteric modulator binding sites, and regions whose conformation changes as proteins transition between different functional states. The thousands of cysteine-substitution mutants reported in the literature demonstrate that, in general, mutation to cysteine is well tolerated. This has allowed systematic studies of residues in transmembrane segments and in other parts of membrane proteins. Finally, by inserting pairs of cysteines and assaying their ability to form disulfide bonds, changes in proximity and mobility relationships between specific positions within a protein can be inferred. Thus, cysteine mutagenesis has provided a wealth of data on the structure of membrane proteins in their functional environment. This data can complement the structural insights obtained from the burgeoning number of crystal structures of detergent solubilized membrane proteins whose functional state is often uncertain. This article will review the use of cysteine mutagenesis to probe

  7. Radiation inactivation study of aminopeptidase: probing the active site

    NASA Astrophysics Data System (ADS)

    Jamadar, V. K.; Jamdar, S. N.; Mohan, Hari; Dandekar, S. P.; Harikumar, P.

    2004-04-01

    Ionizing radiation inactivated purified chicken intestinal aminopeptidase in media saturated with gases in the order N 2O>N 2>air. The D 37 values in the above conditions were 281, 210 and 198 Gy, respectively. OH radical scavengers such as t-butanol and isopropanol effectively nullified the radiation-induced damage in N 2O. The radicals (SCN) 2•-, Br 2•- and I 2•- inactivated the enzyme, pointing to the involvement of aromatic amino acids and cysteine in its catalytic activity. The enzyme exhibited fluorescence emission at 340 nm which is characteristic of tryptophan. The radiation-induced loss of activity was accompanied by a decrease in the fluorescence of the enzyme suggesting a predominant influence on tryptophan residues. The enzyme inhibition was associated with a marked increase in the Km and a decrease in the Vmax and kcat values, suggesting an irreversible alteration in the catalytic site. The above observations were confirmed by pulse radiolysis studies.

  8. Active site mutants of Escherichia coli dethiobiotin synthetase: effects of mutations on enzyme catalytic and structural properties.

    PubMed

    Yang, G; Sandalova, T; Lohman, K; Lindqvist, Y; Rendina, A R

    1997-04-22

    Five active site residues, Thr11, Glu12, Lys15, Lys37, and Ser41, implicated by the protein crystal structure studies of Escherichia coli DTBS, were mutated to determine their function in catalysis and substrate binding. Nine mutant enzymes, T11V, E12A, E12D, K15Q, K37L, K37Q, K37R, S41A, and S41C, were overproduced in an E. coli strain lacking a functional endogenous DTBS gene and purified to homogeneity. Replacement of Thr11 with valine resulted in a 24,000-fold increase in the Km(ATP) with little or no change in the Kd(ATP), KM(DAPA) and DTBS k(cat), suggesting an essential role for this residue in the steady-state affinity for ATP. The two Glu12 mutants showed essentially wild-type DTBS activity (slightly elevated k(cat)'s). Unlike wild-type DTBS, E12A had the same apparent KM(DAPA) at subsaturating and saturating ATP concentrations, indicating a possible role for Glu12 in the binding synergy between DAPA and ATP. The mutations in Lys15 and Lys37 resulted in loss of catalytic activity (0.01% and <0.9% of wild-type DTBS k(cat) for K15Q and the Lys37 mutant enzymes, respectively) and higher KM's for both DAPA (40-fold and >100-fold higher than wild-type for the K15Q and Lys37 mutant enzymes, respectively) and ATP (1800-fold and >10-fold higher than wild-type for K15Q and the K37 mutant enzymes, respectively). These results strongly suggest that Lys15 and Lys37 are crucial to both catalysis and substrate binding. S41A and S41C had essentially the same k(cat) as wild-type and had moderate increases in the DAPA and ATP KM and Kd (ATP) values. Replacement of Ser41 with cysteine resulted in larger effects than replacement with alanine. These data suggest that the H-bond between N7 of DAPA and the Ser41 side chain is not very important for catalysis. The catalytic behavior of these mutant enzymes was also studied by pulse-chase experiments which produced results consistent with the steady-state kinetic analyses. X-ray crystallographic studies of four mutant enzymes, S

  9. Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site*S

    PubMed Central

    Maresso, Anthony W.; Wu, Ruiying; Kern, Justin W.; Zhang, Rongguang; Janik, Dorota; Missiakas, Dominique M.; Duban, Mark-Eugene; Joachimiak, Andrzej; Schneewind, Olaf

    2011-01-01

    Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (β-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via β-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery. PMID:17545669

  10. A novel cysteine desulfurase influencing organosulfur compounds in Lentinula edodes

    PubMed Central

    Liu, Ying; Lei, Xiao-Yu; Chen, Lian-Fu; Bian, Yin-Bing; Yang, Hong; Ibrahim, Salam A.; Huang, Wen

    2015-01-01

    Organosulfur compounds are the basis for the unique aroma of Lentinula edodes, and cysteine sulfoxide lyase (C-S lyase) is the key enzyme in this trait. The enzyme from Alliium sativum has been crystallized and well-characterized; however, there have been no reports of the characterization of fungi C-S lyase at the molecular level. We identified a L. edodes C-S lyase (Lecsl), cloned a gene of Csl encoded Lecsl and then combined modeling, simulations, and experiments to understand the molecular basis of the function of Lecsl. Our analysis revealed Lecsl to be a novel cysteine desulfurase and not a type of cysteine sulfoxide lyase. The pyridoxal-5-phosphate (PLP) molecule bonded tightly to Lecsl to form a Lecsl-PLP complex. Moreover, the Lecsl had one active center that served to bind two kinds of substrates, S-methyl-L-cysteine sulfoxide and L-cysteine, and had both cysteine sulfoxide lyase and cysteine desulfurase activity. We found that the amino acid residue Asn393 was essential for the catalytic activity of Lecsl and that the gene Csl encoded a novel cysteine desulfurase to influence organosulfur compounds in L. edodes. Our results provide a new insight into understanding the formation of the unique aroma of L. edodes. PMID:26054293

  11. A novel cysteine desulfurase influencing organosulfur compounds in Lentinula edodes.

    PubMed

    Liu, Ying; Lei, Xiao-Yu; Chen, Lian-Fu; Bian, Yin-Bing; Yang, Hong; Ibrahim, Salam A; Huang, Wen

    2015-01-01

    Organosulfur compounds are the basis for the unique aroma of Lentinula edodes, and cysteine sulfoxide lyase (C-S lyase) is the key enzyme in this trait. The enzyme from Alliium sativum has been crystallized and well-characterized; however, there have been no reports of the characterization of fungi C-S lyase at the molecular level. We identified a L. edodes C-S lyase (Lecsl), cloned a gene of Csl encoded Lecsl and then combined modeling, simulations, and experiments to understand the molecular basis of the function of Lecsl. Our analysis revealed Lecsl to be a novel cysteine desulfurase and not a type of cysteine sulfoxide lyase. The pyridoxal-5-phosphate (PLP) molecule bonded tightly to Lecsl to form a Lecsl-PLP complex. Moreover, the Lecsl had one active center that served to bind two kinds of substrates, S-methyl-L-cysteine sulfoxide and L-cysteine, and had both cysteine sulfoxide lyase and cysteine desulfurase activity. We found that the amino acid residue Asn393 was essential for the catalytic activity of Lecsl and that the gene Csl encoded a novel cysteine desulfurase to influence organosulfur compounds in L. edodes. Our results provide a new insight into understanding the formation of the unique aroma of L. edodes.

  12. Mutation at a Strictly Conserved, Active Site Tyrosine in the Copper Amine Oxidase Leads to Uncontrolled Oxygenase Activity

    SciTech Connect

    Chen, Zhi-wei; Datta, Saumen; DuBois, Jennifer L.; Klinman, Judith P.; Mathews, F. Scott

    2010-09-07

    The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

  13. Development of nitrile-based peptidic inhibitors of cysteine cathepsins.

    PubMed

    Frizler, Maxim; Stirnberg, Marit; Sisay, Mihiret Tekeste; Gütschow, Michael

    2010-01-01

    It is now becoming clear that several papain-like cysteine cathepsins are involved in the pathophysiology of diseases such as osteoporosis, autoimmune disorders, and cancer. Therefore, the development of potent and selective cathepsin inhibitors is an attractive subject for medicinal chemists. New advances have been made for nitrile-based inhibitors, leading to the identification of the cathepsin K inhibitor odanacatib and other candidates with potential for therapeutic use. This review summarizes the development of peptidic and peptidomimetic compounds with an electrophilic nitrile 'warhead' as inhibitors of the cysteine cathepsins B, S, L, C, and K. Peptide nitriles have been shown to reversibly react with the active site cysteine under formation of a covalent thioimidate adduct. The structural optimization with respect to the positions P3, P2, P1, P1', and P2' resulted in the identification of potent and selective inhibitors of the corresponding cathepsins. The underlying structure-activity relationships are discussed herein. PMID:20166952

  14. Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease.

    PubMed

    Hu, Xin; Compton, Jaimee R; Leary, Dagmar H; Olson, Mark A; Lee, Michael S; Cheung, Jonah; Ye, Wenjuan; Ferrer, Mark; Southall, Noel; Jadhav, Ajit; Morazzani, Elaine M; Glass, Pamela J; Marugan, Juan; Legler, Patricia M

    2016-05-31

    The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.-) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, (475)NVCWAK(480), which differs from papain's (CGS(25)CWAFS), and the enzyme lacks a transition state-stabilizing residue homologous to Gln-19 in papain. To understand the roles of conserved residues in catalysis, we determined the structure of the free enzyme and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a β-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme; one or both of the conformers may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 μM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in kcat/Km, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold decreases in kcat/Km. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle East Respiratory Syndrome (MERS) virus. Mutation of another motif residue, K480A, led to a 9

  15. Crystal Structure of a Hidden Protein, YcaC, a Putative Cysteine Hydrolase from Pseudomonas aeruginosa, with and without an Acrylamide Adduct

    PubMed Central

    Grøftehauge, Morten K.; Truan, Daphne; Vasil, Adriana; Denny, Paul W.; Vasil, Michael L.; Pohl, Ehmke

    2015-01-01

    As part of the ongoing effort to functionally and structurally characterize virulence factors in the opportunistic pathogen Pseudomonas aeruginosa, we determined the crystal structure of YcaC co-purified with the target protein at resolutions of 2.34 and 2.56 Å without a priori knowledge of the protein identity or experimental phases. The three-dimensional structure of YcaC adopts a well-known cysteine hydrolase fold with the putative active site residues conserved. The active site cysteine is covalently bound to propionamide in one crystal form, whereas the second form contains an S-mercaptocysteine. The precise biological function of YcaC is unknown; however, related prokaryotic proteins have functions in antibacterial resistance, siderophore production and NADH biosynthesis. Here, we show that YcaC is exceptionally well conserved across both bacterial and fungal species despite being non-ubiquitous. This suggests that whilst YcaC may not be part of an integral pathway, the function could confer a significant evolutionary advantage to microbial life. PMID:26184183

  16. Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge.

    PubMed

    Tara, S; Elcock, A H; Kirchhoff, P D; Briggs, J M; Radic, Z; Taylor, P; McCammon, J A

    1998-12-01

    It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant kon, for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction.

  17. Crystal Structure of a Sulfur Carrier Protein Complex Found in the Cysteine Biosynthetic Pathway of Mycobacterium tuberculosis

    SciTech Connect

    Jurgenson, Christopher T.; Burns, Kristin E.; Begley, Tadhg P.; Ealick, Steven E.

    2008-10-02

    The structure of the protein complex CysM-CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 {angstrom} resolution. CysM (Rv1336) is a PLP-containing {beta}-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 {angstrom} resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM-CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF-ThiS and MoeB-MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.

  18. Molecular characterization and expression analysis of Cathepsin B and L cysteine proteases from rock bream (Oplegnathus fasciatus).

    PubMed

    Whang, Ilson; De Zoysa, Mahanama; Nikapitiya, Chamilani; Lee, Youngdeuk; Kim, Yucheol; Lee, Sukkyoung; Oh, Chulhong; Jung, Sung-Ju; Oh, Myung-Joo; Choi, Cheol Young; Yeo, Sang-Yeob; Kim, Bong-Seok; Kim, Se-Jae; Lee, Jehee

    2011-03-01

    Cathepsins are lysosomal cysteine proteases of the papain family that play an important role in intracellular protein degradation and turn over within the lysosomal system. In the present study, full-length sequences of cathepsin B (RbCathepsin B) and L (RbCathepsin L) were identified after transcriptome sequencing of rock bream Oplegnathus fasciatus mixed tissue cDNA. Cathepsin B was composed of 330 amino acid residues with 36 kDa predicted molecular mass. RbCathepsin L contained 336 amino acid residues encoding for a 38 kDa predicted molecular mass protein. The sequencing analysis results showed that both cathepsin B and L contain the characteristic papain family cysteine protease signature and active sites for the eukaryotic thiol proteases of cysteine, asparagine and histidine. In addition, RbCathepsin L contained EF hand Ca(2+) binding and cathepsin propeptide inhibitor domains. The rock bream cathepsin B and L showed the highest amino acid identity of 90 and 95% to Lutjanus argentimaculatus cathepsin B and Lates calcarifer cathepsin L, respectively. By phylogenetic analysis, cathepsin B and L exhibited a high degree of evolutionary relationship to respective cathepsin family members of the papain superfamily. Quantitative real-time RT-PCR analysis results confirmed that the expression of cathepsin B and L genes was constitutive in all examined tissues isolated from un-induced rock bream. Moreover, activation of RbCathepsin B and L mRNA was observed in both lipopolysaccharide (LPS) and Edwardsiella tarda challenged liver and blood cells, indicating a role of immune response in rock bream. PMID:21195770

  19. Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

    PubMed Central

    Tomkinson, B; Wernstedt, C; Hellman, U; Zetterqvist, O

    1987-01-01

    The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases. PMID:3313395

  20. Active Sites Environmental Monitoring Program: Action levels

    SciTech Connect

    Ashwood, J.S.; Ashwood, T.L.

    1991-10-01

    The Active Sites Environmental Monitoring Program (ASEMP) was established at Oak Ridge National Laboratory to provide for early leak detection and to monitor performance of the active low-level waste disposal facilities in Solid Waste Storage Area (SWSA) 6 and the transuranic waste storage areas in SWSA 5 North. Early leak detection is accomplished by sampling runoff, groundwater, and perched water in burial trenches. Sample results are compared to action levels that represent background contamination by naturally occurring and fallout-derived radionuclides. 15 refs., 3 figs., 12 tabs.

  1. Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

    SciTech Connect

    Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.; Fairlie, W. Douglas; Colman, Peter M.; Crabb, Brendan S.; Smith, Brian J.

    2009-08-28

    The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putative nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.

  2. The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

    SciTech Connect

    Bradshaw, William J.; Kirby, Jonathan M.; Thiyagarajan, Nethaji; Chambers, Christopher J.; Davies, Abigail H.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi

    2014-07-01

    The crystal structure of Cwp84, an S-layer protein from Clostridium difficile is presented for the first time. The cathepsin L-like fold of cysteine protease domain, a newly observed ‘lectin-like’ domain and several other features are described. Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA) into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Å resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33–497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.

  3. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors.

    PubMed

    Siklos, Marton; BenAissa, Manel; Thatcher, Gregory R J

    2015-11-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy.

  4. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

    PubMed Central

    Siklos, Marton; BenAissa, Manel; Thatcher, Gregory R.J.

    2015-01-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy. PMID:26713267

  5. Cysteine-Based Redox Switches in Enzymes

    PubMed Central

    Klomsiri, Chananat; Karplus, P. Andrew

    2011-01-01

    Abstract The enzymes involved in metabolism and signaling are regulated by posttranslational modifications that influence their catalytic activity, rates of turnover, and targeting to subcellular locations. Most prominent among these has been phosphorylation/dephosphorylation, but now a distinct class of modification coming to the fore is a set of versatile redox modifications of key cysteine residues. Here we review the chemical, structural, and regulatory aspects of such redox regulation of enzymes and discuss examples of how these regulatory modifications often work in concert with phosphorylation/dephosphorylation events, making redox dependence an integral part of many cell signaling processes. Included are the emerging roles played by peroxiredoxins, a family of cysteine-based peroxidases that now appear to be major players in both antioxidant defense and cell signaling. Antioxid. Redox Signal. 14, 1065–1077. PMID:20799881

  6. Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

    SciTech Connect

    Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong

    2012-06-05

    A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

  7. Contribution of active-site glutamine to rate enhancement in ubiquitin carboxy terminal hydrolases

    PubMed Central

    Boudreaux, David; Chaney, Joseph; Maiti, Tushar K.; Das, Chittaranjan

    2012-01-01

    Ubiquitin carboxy terminal hydrolases (UCHs) are cysteine proteases featuring a classical cysteine-histidine-aspartate catalytic triad, also a highly conserved glutamine thought to be a part of the oxyanion hole. However, the contribution of this side chain to the catalysis by UCH enzymes is not known. Herein, we demonstrate that the glutamine side chain contributes to rate enhancement in UCHL1, UCHL3 and UCHL5. Mutation of the glutamine to alanine in these enzymes impairs the catalytic efficiency mainly due to a 16 to 30-fold reduction in kcat, which is consistent with a loss of approximately 2 kcal/mol in transition-state stabilization. However, the contribution to transition-state stabilization observed here is rather modest for the side chain’s role in oxyanion stabilization. Interestingly, we discovered that the carbonyl oxygen of this side chain is engaged in a C—H•••O hydrogen-bonding contact with the CεH group of the catalytic histidine. Upon further analysis, we found that this interaction is a common active-site structural feature in most cysteine proteases, including papain, belonging to families with the QCH(N/D) type of active-site configuration. It is possible that removal of the glutamine side chain might have abolished the C—H•••O interaction, which typically accounts for 2 kcal/mol of stabilization, leading to the effect on catalysis observed here. Additional studies performed on UCHL3 by mutating the glutamine to glutamate (strong C—H•••O acceptor but oxyanion destabilizer) and to lysine (strong oxyanion stabilizer but lacking C—H•••O hydrogen-bonding property) suggest that the C—H•••O hydrogen bond could contribute to catalysis. PMID:22284438

  8. Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the

  9. Roles of histidines 154 and 189 and aspartate 139 in the active site of serine acetyltransferase from Haemophilus influenzae.

    PubMed

    Guan, Rong; Roderick, Steven L; Huang, Bin; Cook, Paul F

    2008-06-17

    A crystal structure of serine acetyltransferase (SAT) with cysteine bound in the serine subsite of the active site shows that both H154 and H189 are within hydrogen-bonding distance to the cysteine thiol [Olsen, L. R., Huang, B., Vetting, M. W., and Roderick, S. L. (2004) Biochemistry 43, 6013 -6019]. In addition, H154 is in an apparent dyad linkage with D139. The structure suggests that H154 is the most likely catalytic general base and that H189 and D139 may also play important roles during the catalytic reaction. Site-directed mutagenesis was performed to mutate each of these three residues to Asn, one at a time. The V1/Et value of all of the single mutant enzymes decreased, with the largest decrease (approximately 1240-fold) exhibited by the H154N mutant enzyme. Mutation of both histidines, H154N/H189N, gave a V1/Et approximately 23700-fold lower than that of the wild-type enzyme. An increase in K Ser was observed for the H189N, D139N, and H154N/H189N mutant enzymes, while the H154N mutant enzyme gave an 8-fold decrease in K Ser. For all three single mutant enzymes, V1/Et and V1/K Ser Et decrease at low pH and give a pKa of about 7, while the V1/Et of the double mutant enzyme was pH independent. The solvent deuterium kinetic isotope effects on V 1 and V1/K Ser decreased compared to wild type for the H154N mutant enzyme and increased for the H189N mutant enzyme but was about the same as that of wild type for D139N and H154N/H189N. Data suggest that H154, H189, and D139 play different catalytic roles for SAT. H154 likely serves as a general base, accepting a proton from the beta-hydroxyl of serine as the tetrahedral intermediate is formed upon nucleophilic attack on the thioester carbonyl of acetyl-CoA. However, activity is not completely lost upon elimination of H154, and thus, H189 may be able to serve as a backup general base at a lower efficiency compared to H154; it also aids in binding and orienting the serine substrate. Aspartate 139, in dyad linkage with

  10. Expression, purification, crystallization and X-ray crystallographic studies of different redox states of the active site of thioredoxin 1 from the whiteleg shrimp Litopenaeus vannamei

    PubMed Central

    Campos-Acevedo, Adam A.; Garcia-Orozco, Karina D.; Sotelo-Mundo, Rogerio R.; Rudiño-Piñera, Enrique

    2013-01-01

    Thioredoxin (Trx) is a 12 kDa cellular redox protein that belongs to a family of small redox proteins which undergo reversible oxidation to produce a cystine disulfide bond through the transfer of reducing equivalents from the catalytic site cysteine residues (Cys32 and Cys35) to a disulfide substrate. In this study, crystals of thioredoxin 1 from the Pacific whiteleg shrimp Litopenaeus vannamei (LvTrx) were successfully obtained. One data set was collected from each of four crystals at 100 K and the three-dimensional structures of the catalytic cysteines in different redox states were determined: reduced and oxidized forms at 2.00 Å resolution using data collected at a synchrotron-radiation source and two partially reduced structures at 1.54 and 1.88 Å resolution using data collected using an in-house source. All of the crystals belonged to space group P3212, with unit-cell parameters a = 57.5 (4), b = 57.5 (4), c = 118.1 (8) Å. The asymmetric unit contains two subunits of LvTrx, with a Matthews coefficient (V M) of 2.31 Å3 Da−1 and a solvent content of 46%. Initial phases were determined by molecular replacement using the crystallographic model of Trx from Drosophila melanogaster as a template. In the present work, LvTrx was overexpressed in Escherichia coli, purified and crystallized. Structural analysis of the different redox states at the Trx active site highlights its reactivity and corroborates the existence of a dimer in the crystal. In the crystallographic structures the dimer is stabilized by several interactions, including a disulfide bridge between Cys73 of each LvTrx monomer, a hydrogen bond between the side chain of Asp60 of each monomer and several hydrophobic interactions, with a noncrystallographic twofold axis. PMID:23695560

  11. An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

    SciTech Connect

    Guidinger, P.F.; Nowak, T. )

    1991-09-10

    The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 {plus minus} 0.025 M{sup {minus}1} min{sup {minus}1}. Inactivation by pyridoxal 5{prime}-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7,700 {plus minus} 860 m{sup {minus}1} min{sup {minus}1}. Treatment of the enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of k{sub obs} vs pH suggests this active-site lysine has a pK{sub a} of 8.1 and a pH-independent rate constant of inactivation of 47,700 m{sup {minus}1} min{sup {minus}1}. Proton relaxation rate measurements suggest that pyridoxal 5{prime}-phosphate modification alters binding of the phosphate-containing substrates. {sup 31}P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme {center dot}Mn{sup 2+}{center dot}substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5{prime}-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.

  12. Topology of transmembrane proteins by scanning cysteine accessibility mutagenesis methodology.

    PubMed

    Zhu, Quansheng; Casey, Joseph R

    2007-04-01

    Integral membrane proteins of the plasma membrane span from the inside to the outside of the cell. The primary structural element of integral membrane proteins is their topology: the pattern in which the protein traverses the membrane. A full description of topology, defining which parts of the protein face outside versus inside, goes a long way toward understanding the folding of these proteins. Many approaches have been established to define membrane protein topology. Here, we present the technique of scanning cysteine accessibility mutagenesis (SCAM). This approach uses the unique chemical reactivity of the cysteine sulfhydryl to probe membrane protein structure. Individual cysteine residues are introduced into the target protein by mutagenesis. The ability to chemically react these residues using sulfhydryl-directed reagents (either membrane permeant or impermeant) defines each site as either extracellular or intracellular, thus establishing topology of a location. This analysis performed on many sites in the protein will define the protein's topology. PMID:17367716

  13. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; Udell, Hannah S.; Yik, Eric J.; Bergeman, Lai F.; Fox, Brian G.

    2015-01-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  14. Sulfur K-Edge XAS and DFT Calculations on NitrileHydratase: Geometric and Electronic Structure of the Non-heme Iron Active Site

    SciTech Connect

    Dey, Abhishek; Chow, Marina; Taniguchi, Kayoko; Lugo-Mas, Priscilla; Davin, Steven; Maeda, Mizuo; Kovacs, Julie A.; Odaka, Masafumi; Hodgson, Keith O.; Hedman, Britt; Solomon, Edward I.; /SLAC, SSRL

    2006-09-28

    The geometric and electronic structure of the active site of the non-heme iron enzyme nitrile hydratase (NHase) is studied using sulfur K-edge XAS and DFT calculations. Using thiolate (RS{sup -})-, sulfenate (RSO{sup -})-, and sulfinate (RSO{sub 2}{sup -})-ligated model complexes to provide benchmark spectral parameters, the results show that the S K-edge XAS is sensitive to the oxidation state of S-containing ligands and that the spectrum of the RSO- species changes upon protonation as the S-O bond is elongated (by {approx}0.1 {angstrom}). These signature features are used to identify the three cysteine residues coordinated to the low-spin Fe{sup III} in the active site of NHase as CysS{sup -}, CysSOH, and CysSO{sub 2}{sup -} both in the NO-bound inactive form and in the photolyzed active form. These results are correlated to geometry-optimized DFT calculations. The pre-edge region of the X-ray absorption spectrum is sensitive to the Z{sub eff} of the Fe and reveals that the Fe in [FeNO]{sup 6} NHase species has a Z{sub eff} very similar to that of its photolyzed Fe{sup III} counterpart. DFT calculations reveal that this results from the strong {pi} back-bonding into the {pi}* antibonding orbital of NO, which shifts significant charge from the formally t{sub 2}{sup 6} low-spin metal to the coordinated NO.

  15. Characterization of active sites in zeolite catalysts

    SciTech Connect

    Eckert, J.; Bug, A.; Nicol, J.M.

    1997-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Atomic-level details of the interaction of adsorbed molecules with active sites in catalysts are urgently needed to facilitate development of more effective and/or environmentally benign catalysts. To this end the authors have carried out neutron scattering studies combined with theoretical calculations of the dynamics of small molecules inside the cavities of zeolite catalysts. The authors have developed the use of H{sub 2} as a probe of adsorption sites by observing the hindered rotations of the adsorbed H{sub 2} molecule, and they were able to show that an area near the four-rings is the most likely adsorption site for H{sub 2} in zeolite A while adsorption of H{sub 2} near cations located on six-ring sites decreases in strength as Ni {approximately} Co > Ca > Zn {approximately} Na. Vibrational and rotational motions of ethylene and cyclopropane adsorption complexes were used as a measure for zeolite-adsorbate interactions. Preliminary studies of the binding of water, ammonia, and methylamines were carried out in a number of related guest-host materials.

  16. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  17. Analysis of structural changes in active site of luciferase adsorbed on nanofabricated hydrophilic Si surface by molecular-dynamics simulations

    SciTech Connect

    Nishiyama, Katsuhiko; Hoshino, Tadatsugu

    2007-05-21

    Interactions between luciferase and a nanofabricated hydrophilic Si surface were explored by molecular-dynamics simulations. The structural changes in the active-site residues, the residues affecting the luciferin binding, and the residues affecting the bioluminescence color were smaller on the nanofabricated hydrophilic Si surface than on both a hydrophobic Si surface and a hydrophilic Si surface. The nanofabrication and wet-treatment techniques are expected to prevent the decrease in activity of luciferase on the Si surface.

  18. Human liver alcohol dehydrogenase: amino acid substitution in the beta 2 beta 2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme.

    PubMed Central

    Jörnvall, H; Hempel, J; Vallee, B L; Bosron, W F; Li, T K

    1984-01-01

    The homodimeric Oriental beta 2 beta 2 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the beta 1 beta 1 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the beta 2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the beta 1 form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the beta 1 beta 1 and beta 2 beta 2 isozymes, including both a lower pH optimum and higher turnover number of beta 2 beta 2, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme. PMID:6374651

  19. Control of active sites in flocculation: Concept of equivalent active sites''

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Flocculation and dispersion of solids are strong functions of the amount and conformation of the adsorbed polymer. Regions of dispersion and flocculation of solids with particular polymer molecules may be deduced from saturation adsorption data. The concept of equivalent active sites'' is proposed to explain flocculation and dispersion behavior irrespective of the amount or conformation of the adsorbed polymer. The concept has been further extended to study the selective flocculation process.

  20. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  1. Structural characterization and expression analysis of a novel cysteine protease inhibitor from Haliotis discus hannai Ino.

    PubMed

    Ding, Jianfeng; Li, Li; Zhang, Guofan

    2015-02-01

    The sequence of the cysteine protease inhibitor gene of Haliotis discus hannai (designated HdCpi) was determined using the RACE method. The full-length HdCpi cDNA is 1049 bp long, and contains an open reading frame of 813 bp, encoding a 271-amino-acid protein with a calculated molecular mass of 29.83 kDa and an isoelectric point of 8.57. The deduced amino acid sequence of HdCpi contains two cystatin-like domains, and each has the structural features of the cystatin family, including three evolutionarily conserved motifs known to interact with the active sites of cysteine peptidases: the Gly residue at the N-terminus (Gly(65) and Gly(160)), the Gln-X-Val-X-Gly motif (Q(106)IVSG(110) and Q(202)VVAG(206)), and the less conserved motif at the C-terminus (S(136)W(137) and A(254)W(255)). Many putative transcription-factor-binding sites involved in the immune system and cancer occur in the promoter region of HdCpi. Quantitative real-time RT-PCR detected HdCpi expression in all the tissues examined and in the gills of abalone challenged with the bacterium Vibrio anguillarum. HdCpi transcripts were expressed in the mantle, gill, digestive tract, hemocytes, and muscle, and increased HdCpi expression was observed after bacterial stimulation. These results suggest that HdCpi is a biologically active protease inhibitor that is likely to be involved in the antibacterial response of the abalone. PMID:25463299

  2. Structural characterization and expression analysis of a novel cysteine protease inhibitor from Haliotis discus hannai Ino.

    PubMed

    Ding, Jianfeng; Li, Li; Zhang, Guofan

    2015-02-01

    The sequence of the cysteine protease inhibitor gene of Haliotis discus hannai (designated HdCpi) was determined using the RACE method. The full-length HdCpi cDNA is 1049 bp long, and contains an open reading frame of 813 bp, encoding a 271-amino-acid protein with a calculated molecular mass of 29.83 kDa and an isoelectric point of 8.57. The deduced amino acid sequence of HdCpi contains two cystatin-like domains, and each has the structural features of the cystatin family, including three evolutionarily conserved motifs known to interact with the active sites of cysteine peptidases: the Gly residue at the N-terminus (Gly(65) and Gly(160)), the Gln-X-Val-X-Gly motif (Q(106)IVSG(110) and Q(202)VVAG(206)), and the less conserved motif at the C-terminus (S(136)W(137) and A(254)W(255)). Many putative transcription-factor-binding sites involved in the immune system and cancer occur in the promoter region of HdCpi. Quantitative real-time RT-PCR detected HdCpi expression in all the tissues examined and in the gills of abalone challenged with the bacterium Vibrio anguillarum. HdCpi transcripts were expressed in the mantle, gill, digestive tract, hemocytes, and muscle, and increased HdCpi expression was observed after bacterial stimulation. These results suggest that HdCpi is a biologically active protease inhibitor that is likely to be involved in the antibacterial response of the abalone.

  3. Formylglycine, a post-translationally generated residue with unique catalytic capabilities and biotechnology applications.

    PubMed

    Appel, Mason J; Bertozzi, Carolyn R

    2015-01-16

    Formylglycine (fGly) is a catalytically essential residue found almost exclusively in the active sites of type I sulfatases. Formed by post-translational oxidation of cysteine or serine side chains, this aldehyde-functionalized residue participates in a unique and highly efficient catalytic mechanism for sulfate ester hydrolysis. The enzymes that produce fGly, formylglycine-generating enzyme (FGE) and anaerobic sulfatase-maturating enzyme (anSME), are as unique and specialized as fGly itself. FGE especially is structurally and mechanistically distinct, and serves the sole function of activating type I sulfatase targets. This review summarizes the current state of knowledge regarding the mechanism by which fGly contributes to sulfate ester hydrolysis, the molecular details of fGly biogenesis by FGE and anSME, and finally, recent biotechnology applications of fGly beyond its natural catalytic function.

  4. Formylglycine, a Post-Translationally Generated Residue with Unique Catalytic Capabilities and Biotechnology Applications

    PubMed Central

    Appel, Mason J.; Bertozzi, Carolyn R.

    2015-01-01

    Formylglycine (fGly) is a catalytically essential residue found almost exclusively in the active sites of type I sulfatases. Formed by post-translational oxidation of cysteine or serine side chains, this aldehyde-functionalized residue participates in a unique and highly efficient catalytic mechanism for sulfate ester hydrolysis. The enzymes that produce fGly, formylglycine-generating enzyme (FGE) and anaerobic sulfatase-maturating enzyme (anSME), are as unique and specialized as fGly itself. FGE especially is structurally and mechanistically distinct, and serves the sole function of activating type I sulfatase targets. This review summarizes the current state of knowledge regarding the mechanism by which fGly contributes to sulfate ester hydrolysis, the molecular details of fGly biogenesis by FGE and anSME, and finally, recent biotechnology applications of fGly beyond its natural catalytic function. PMID:25514000

  5. Dynamics and Mechanism of Efficient DNA Repair Reviewed by Active-Site Mutants

    NASA Astrophysics Data System (ADS)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

    2010-06-01

    Photolyases repair the UV-induced pyrimidine dimers in damage DNA via a photoreaction which includes a series of light-driven electron transfers between the two-electron-reduced flavin cofactor FADH^- and the dimer. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of several active-site residues. With femtosecond resolution, we observed the significant change in the forward electron transfer from the excited FADH^- to the dimer and the back electron transfer from the repaired thymines by mutation of E274A, R226A, R342A, N378S and N378C. We also found that the mutation of E274A accelerates the bond-breaking of the thymine dimer. The dynamics changes are consistent with the quantum yield study of these mutants. These results suggest that the active-site residues play a significant role, structurally and chemically, in the DNA repair photocycle.

  6. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    SciTech Connect

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  7. Structure and mechanism leading to formation of the cysteine sulfinate product complex of a biomimetic cysteine dioxygenase model.

    PubMed

    Sallmann, Madleen; Kumar, Suresh; Chernev, Petko; Nehrkorn, Joscha; Schnegg, Alexander; Kumar, Devesh; Dau, Holger; Limberg, Christian; de Visser, Sam P

    2015-05-11

    Cysteine dioxygenase is a unique nonheme iron enzyme that is involved in the metabolism of cysteine in the body. It contains an iron active site with an unusual 3-His ligation to the protein, which contrasts with the structural features of common nonheme iron dioxygenases. Recently, some of us reported a truly biomimetic model for this enzyme, namely a trispyrazolylborato iron(II) cysteinato complex, which not only has a structure very similar to the enzyme-substrate complex but also represents a functional model: Treatment of the model with dioxygen leads to cysteine dioxygenation, as shown by isolating the cysteine part of the product in the course of the work-up. However, little is known on the conversion mechanism and, so far, not even the structure of the actual product complex had been characterised, which is also unknown in case of the enzyme. In a multidisciplinary approach including density functional theory calculations and X-ray absorption spectroscopy, we have now determined the structure of the actual sulfinato complex for the first time. The Cys-SO2 (-) functional group was found to be bound in an η(2) -O,O-coordination mode, which, based on the excellent resemblance between model and enzyme, also provides the first support for a corresponding binding mode within the enzymatic product complex. Indeed, this is again confirmed by theory, which had predicted a η(2) -O,O-binding mode for synthetic as well as the natural enzyme.

  8. Noncovalent intermolecular interactions between dehydroepiandrosterone and the active site of human dehydroepiandrosterone sulphotransferase: A density functional theory based treatment

    NASA Astrophysics Data System (ADS)

    Astani, Elahe; Heshmati, Emran; Chen, Chun-Jung; Hadipour, Nasser L.; Shekarsaraei, Setareh

    2016-04-01

    A theoretical study was performed to characterize noncovalent intermolecular interactions, especially hydrogen bond (HB), in the active site of enzyme human dehydroepiandrosterone sulphotransferase (SULT2A1/DHEA) using the local (M06-L) and hybrid (M06, M06-2X) meta-GGA functionals of density functional theory (DFT). Results revealed that DHEA is able to form HBs with residues His99, Tyr231, Met137 and Met16 in the active site of the SULT2A1/DHEA. It was found that DHEA interacts with the other residues through electrostatic and Van der Waals interactions.

  9. The cysteine-cluster motif of c-Yes, Lyn and FAK as a suppressive module for the kinases.

    PubMed

    Rahman, Mohammad Aminur; Senga, Takeshi; Oo, Myat Lin; Hasegawa, Hitoki; Biswas, Md Helal Uddin; Mon, Naing Naing; Huang, Pengyu; Ito, Satoko; Yamamoto, Tadashi; Hamaguchi, Michinari

    2008-04-01

    The Src family of non-receptor protein tyrosine kinases plays a critical role in the progression of human cancers so that the development of its specific inhibitors is important as a therapeutic tool. We previously reported that cysteine residues in the cysteine-cluster (CC) motif of v-Src were critical for the kinase inactivation by the SH-alkylating agents such as N-(9-acridinyl) maleimide (NAM), whereas other cysteine residues were dispensable. We found similar CC-motifs in other Src-family kinases and a non-Src-family kinase, FAK. In this study, we explored the function of the CC-motif in Yes, Lyn and FAK. While Src has four cysteines in the CC-motif, c-Yes and Lyn have three and two of the four cysteines, respectively. Two conserved cysteines of the Src family kinases, corresponding to Cys487 and Cys498 of Src, were essential for the resistance to the inactivation of the kinase activity by NAM, whereas the first cysteine of c-Yes, which is absent in Lyn, was less important. FAK has similar CC-motifs with two cysteines and both cysteines were again essential for the resistance to the inactivation of the kinase activity by NAM. Taken together, modification of cysteine residues of the CC-motif causes a repressor effect on the catalytic activity of the Src family kinases and FAK.

  10. Mechanisms of mitochondrial holocytochrome c synthase and the key roles played by cysteines and histidine of the heme attachment site, Cys-XX-Cys-His.

    PubMed

    Babbitt, Shalon E; San Francisco, Brian; Mendez, Deanna L; Lukat-Rodgers, Gudrun S; Rodgers, Kenton R; Bretsnyder, Eric C; Kranz, Robert G

    2014-10-17

    Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19.

  11. Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

    SciTech Connect

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.; Elsen, Nathaniel L.; Phillips, Jr., George N.; Fox, Brian G.

    2014-10-02

    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential

  12. Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

    PubMed

    Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

    2016-06-28

    Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes. PMID:27259687

  13. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

    SciTech Connect

    Yip, Wing-Kin; Dong, Jian-Guo; Yang, S.F. ); Kenny, J.W.; Thompson, G.A. )

    1990-10-01

    The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB{sup 3}H{sub 4} or Ado({sup 14}C)Met. Peptide sequencing of both {sup 3}H- and {sup 14}C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the {sup 3}H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the {sup 14}C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado ({sup 14}C)Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6.

  14. Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana.

    PubMed

    Romero, Luis C; Aroca, M Ángeles; Laureano-Marín, Ana M; Moreno, Inmaculada; García, Irene; Gotor, Cecilia

    2014-02-01

    Cysteine occupies a central position in plant metabolism because it is a reduced sulfur donor molecule involved in the synthesis of essential biomolecules and defense compounds. Moreover, cysteine per se and its derivative molecules play roles in the redox signaling of processes occurring in various cellular compartments. Cysteine is synthesized during the sulfate assimilation pathway via the incorporation of sulfide to O-acetylserine, catalyzed by O-acetylserine(thiol)lyase (OASTL). Plant cells contain OASTLs in the mitochondria, chloroplasts, and cytosol, resulting in a complex array of isoforms and subcellular cysteine pools. In recent years, significant progress has been made in Arabidopsis, in determining the specific roles of the OASTLs and the metabolites produced by them. Thus, the discovery of novel enzymatic activities of the less-abundant, like DES1 with L-cysteine desulfhydrase activity and SCS with S-sulfocysteine synthase activity, has provided new perspectives on their roles, besides their metabolic functions. Thereby, the research has been demonstrated that cytosolic sulfide and chloroplastic S-sulfocysteine act as signaling molecules regulating autophagy and protecting the photosystems, respectively. In the cytosol, cysteine plays an essential role in plant immunity; in the mitochondria, this molecule plays a central role in the detoxification of cyanide, which is essential for root hair development and plant responses to pathogens.

  15. The Cysteine Proteome

    PubMed Central

    Go, Young-Mi; Chandler, Joshua D.; Jones, Dean P.

    2015-01-01

    The cysteine (Cys) proteome is a major component of the adaptive interface between the genome and the exposome. The thiol moiety of Cys undergoes a range of biologic modifications enabling biological switching of structure and reactivity. These biological modifications include sulfenylation and disulfide formation, formation of higher oxidation states, S-nitrosylation, persulfidation, metallation, and other modifications. Extensive knowledge about these systems and their compartmentalization now provides a foundation to develop advanced integrative models of Cys proteome regulation. In particular, detailed understanding of redox signaling pathways and sensing networks is becoming available to discriminate network structures. This research focuses attention on the need for atlases of Cys modifications to develop systems biology models. Such atlases will be especially useful for integrative studies linking the Cys proteome to imaging and other omics platforms, providing a basis for improved redox-based therapeutics. Thus, a framework is emerging to place the Cys proteome as a complement to the quantitative proteome in the omics continuum connecting the genome to the exposome. PMID:25843657

  16. Copper inhibits the protease from human immunodeficiency virus 1 by both cysteine-dependent and cysteine-independent mechanisms.

    PubMed Central

    Karlström, A R; Levine, R L

    1991-01-01

    The protease of the human immunodeficiency virus is essential for replication of the virus, and the enzyme is therefore an attractive target for antiviral action. We have found that the viral protease is inhibited by approximately stoichiometric concentrations of copper or mercury ions. Inactivation by Cu2+ was rapid and not reversed by subsequent exposure to EDTA or dithiothreitol. Direct inhibition by Cu2+ required the presence of cysteine residue(s) in the protease. Thus, a synthetic protease lacking cysteine residues was not inhibited by exposure to copper. However, addition of dithiothreitol as an exogenous thiol rendered even the synthetic protease susceptible to inactivation by copper. Oxygen was not required for inactivation of either the wild-type or the synthetic protease. These results provide the basis for the design of novel types of protease inhibitors. PMID:2062837

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-04-21

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

  20. Orchestrating Redox Signaling Networks Through Regulatory Cysteine Switches

    PubMed Central

    Paulsen, Candice E.; Carroll, Kate S.

    2015-01-01

    Hydrogen peroxide (H2O2) acts as a second messenger that can mediate intracellular signal transduction via chemoselective oxidation of cysteine residues in signaling proteins. This Review presents current mechanistic insights into signal-mediated H2O2 production and highlights recent advances in methods to detect reactive oxygen species (ROS) and cysteine oxidation both in vitro and in cells. Selected examples from the recent literature are used to illustrate the diverse mechanisms by which H2O2 can regulate protein function. The continued development of methods to detect and quantify discrete cysteine oxoforms should further our mechanistic understanding of redox regulation of protein function and may lead to the development of new therapeutic strategies. PMID:19957967

  1. The spectrum character of photoreaction of Hypocrellin A and cysteine

    NASA Astrophysics Data System (ADS)

    Zhang, Jucheng; Liu, Wei; Li, Ying; Zhang, Pei; Yi, Zhongzhou; Min, Yong; Huang, Zhaolong; Yao, Lihua; Lu, Haiju

    2008-12-01

    In the current work, Hypocrellin A (HA) is one of the nature photosensitizer was recognized by researchers, and it used as a probe to research the molecular recognition and interaction with protein, the work suggested the HA can as the medicine to treat some disease. This paper study the spectrum character of photoreaction of Hypocrellin A and cysteine in different pH value, the spectrum show an isosbestic point at 495nm, and the absorption peak at 478nm was red-shifted to about 500nm. The result suggested the HA can react with cysteine in this condition, and farther illuminated the cysteine residue may is one of the target of the interaction of HA or HB with protein.

  2. Orchestrating redox signaling networks through regulatory cysteine switches.

    PubMed

    Paulsen, Candice E; Carroll, Kate S

    2010-01-15

    Hydrogen peroxide (H(2)O(2)) acts as a second messenger that can mediate intracellular signal transduction via chemoselective oxidation of cysteine residues in signaling proteins. This Review presents current mechanistic insights into signal-mediated H(2)O(2) production and highlights recent advances in methods to detect reactive oxygen species (ROS) and cysteine oxidation both in vitro and in cells. Selected examples from the recent literature are used to illustrate the diverse mechanisms by which H(2)O(2) can regulate protein function. The continued development of methods to detect and quantify discrete cysteine oxoforms should further our mechanistic understanding of redox regulation of protein function and may lead to the development of new therapeutic strategies.

  3. Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response.

    PubMed

    Saito, Ryota; Suzuki, Takafumi; Hiramoto, Keiichiro; Asami, Soichiro; Naganuma, Eriko; Suda, Hiromi; Iso, Tatsuro; Yamamoto, Hirotaka; Morita, Masanobu; Baird, Liam; Furusawa, Yuki; Negishi, Takaaki; Ichinose, Masakazu; Yamamoto, Masayuki

    2015-11-02

    The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1's ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.

  4. The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase*

    PubMed Central

    Tchesnokov, Egor P.; Fellner, Matthias; Siakkou, Eleni; Kleffmann, Torsten; Martin, Lois W.; Aloi, Sekotilani; Lamont, Iain L.; Wilbanks, Sigurd M.; Jameson, Guy N. L.

    2015-01-01

    Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed that p3MDO is expressed in low levels, suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid in vitro, with a marked preference for 3-mercaptopropionic acid. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mössbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates that each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site. PMID:26272617

  5. Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations

    SciTech Connect

    Radom,C.; Banerjee, A.; Verdine, G.

    2007-01-01

    The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5 to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is 'hardwired'. Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F{sup *149}) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F{sup *292}) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

  6. Caught in the act: the crystal structure of cleaved cathepsin L bound to the active site of Cathepsin L.

    PubMed

    Sosnowski, Piotr; Turk, Dušan

    2016-04-01

    Cathepsin L is a ubiquitously expressed papain-like cysteine protease involved in the endosomal degradation of proteins and has numerous roles in physiological and pathological processes, such as arthritis, osteoporosis, and cancer. Insight into the specificity of cathepsin L is important for elucidating its physiological roles and drug discovery. To study interactions with synthetic ligands, we prepared a presumably inactive mutant and crystallized it. Unexpectedly, the crystal structure determined at 1.4 Å revealed that the cathepsin L molecule is cleaved, with the cleaved region trapped in the active site cleft of the neighboring molecule. Hence, the catalytic mutant demonstrated low levels of catalytic activity. PMID:26992470

  7. Caught in the act: the crystal structure of cleaved cathepsin L bound to the active site of Cathepsin L.

    PubMed

    Sosnowski, Piotr; Turk, Dušan

    2016-04-01

    Cathepsin L is a ubiquitously expressed papain-like cysteine protease involved in the endosomal degradation of proteins and has numerous roles in physiological and pathological processes, such as arthritis, osteoporosis, and cancer. Insight into the specificity of cathepsin L is important for elucidating its physiological roles and drug discovery. To study interactions with synthetic ligands, we prepared a presumably inactive mutant and crystallized it. Unexpectedly, the crystal structure determined at 1.4 Å revealed that the cathepsin L molecule is cleaved, with the cleaved region trapped in the active site cleft of the neighboring molecule. Hence, the catalytic mutant demonstrated low levels of catalytic activity.

  8. The intrinsic cysteine and histidine residues of the anti-Salmonella antibody Se155-4: a model for the introduction of new functions into antibody-binding sites.

    PubMed

    Young, N Martin; Watson, David C; Cunningham, Anna M; MacKenzie, C Roger

    2014-10-01

    New functions can be incorporated into anti-hapten or anti-protein antibodies by mutating selected residues in the binding-site region either to Cys, to allow alkylation with reagents bearing the desired functional groups, or to His, to create metal-binding sites or to make antigen binding pH-sensitive. However, choosing suitable sites for these mutations has been hampered by the lack of antibodies with these features, to serve as models. Remarkably, the anti-carbohydrate antibody Se155-4, specific for the Salmonella group B lipopolysaccharide, already has a Cys and two pairs of His residues close to the antigen-binding pocket in its structure, and shows pH-dependent antigen binding. We therefore investigated modification of its Cys94L in an scFv version of the antibody with the aims of creating a 'reagentless' fluorescent sensor and attaching a metal-binding group that might confer lyase activity. These groups were successfully introduced, as judged by mass spectrometry, and had only slightly reduced antigen binding in enzyme-linked immunosorbent assay. The fluorescent product was sensitive to addition of antigen in a solution format, unlike a modification of a more distant Cys introduced into the VH CDR4 loop. Two other routes to modulate antigen binding were also explored, metal binding by the His pair alongside the antigen-binding pocket and insertions into CDR4 to extend the antigen-contact area. His residues adjacent to the antigen-binding pocket bound copper, causing a 5-fold decrease in antigen binding. In CDR4 of the VH domain, the preferred insert length was four residues, which gave stable antigen-binding products but did not improve overall antigen affinity.

  9. Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

    PubMed Central

    Chakraborty, Sandeep; Minda, Renu; Salaye, Lipika; Bhattacharjee, Swapan K.; Rao, Basuthkar J.

    2011-01-01

    Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - CataLytic Active Site Prediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro. PMID

  10. Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

    SciTech Connect

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong; Chen, Shoudeng; Zhou, Jie; He, Xiaojing; Lv, Zongyang; Ge, Ruowen; Li, Xuemei; Deng, Tao; Fodor, Ervin; Rao, Zihe; Liu, Yingfang

    2009-11-10

    The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.

  11. 1-Methylpyridinium-4-(4-phenylmethanethiosulfonate) iodide, MTS-MPP+, a novel scanning cysteine accessibility method (SCAM) reagent for monoamine transporter studies.

    PubMed

    Gallardo-Godoy, Alejandra; Torres-Altoro, Melissa I; White, Kellie J; Barker, Eric L; Nichols, David E

    2007-01-01

    A novel substituted cysteine accessibility method (SCAM) reagent was developed for monoamine uptake transporters. The new reagent, MTS-MPP(+), was a derivative of the neurotoxin and transporter substrate MPP(+). MTS-MPP(+) labeled cysteine residues introduced into the serotonin transporter protein. Although it did not prove to be a substrate, as is MPP(+), it appears to label cysteine residues lining the permeation pore of the transporter more readily than currently available nonspecific SCAM reagents.

  12. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

    PubMed Central

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

    2015-01-01

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  13. Covalent Inhibition of Ubc13 Affects Ubiquitin Signaling and Reveals Active Site Elements Important for Targeting.

    PubMed

    Hodge, Curtis D; Edwards, Ross A; Markin, Craig J; McDonald, Darin; Pulvino, Mary; Huen, Michael S Y; Zhao, Jiyong; Spyracopoulos, Leo; Hendzel, Michael J; Glover, J N Mark

    2015-07-17

    Ubc13 is an E2 ubiquitin conjugating enzyme that functions in nuclear DNA damage signaling and cytoplasmic NF-κB signaling. Here, we present the structures of complexes of Ubc13 with two inhibitors, NSC697923 and BAY 11-7082, which inhibit DNA damage and NF-κB signaling in human cells. NSC697923 and BAY 11-7082 both inhibit Ubc13 by covalent adduct formation through a Michael addition at the Ubc13 active site cysteine. The resulting adducts of both compounds exploit a binding groove unique to Ubc13. We developed a Ubc13 mutant which resists NSC697923 inhibition and, using this mutant, we show that the inhibition of cellular DNA damage and NF-κB signaling by NSC697923 is largely due to specific Ubc13 inhibition. We propose that unique structural features near the Ubc13 active site could provide a basis for the rational development and design of specific Ubc13 inhibitors. PMID:25909880

  14. Covalent Inhibition of Ubc13 Affects Ubiquitin Signaling and Reveals Active Site Elements Important for Targeting.

    PubMed

    Hodge, Curtis D; Edwards, Ross A; Markin, Craig J; McDonald, Darin; Pulvino, Mary; Huen, Michael S Y; Zhao, Jiyong; Spyracopoulos, Leo; Hendzel, Michael J; Glover, J N Mark

    2015-07-17

    Ubc13 is an E2 ubiquitin conjugating enzyme that functions in nuclear DNA damage signaling and cytoplasmic NF-κB signaling. Here, we present the structures of complexes of Ubc13 with two inhibitors, NSC697923 and BAY 11-7082, which inhibit DNA damage and NF-κB signaling in human cells. NSC697923 and BAY 11-7082 both inhibit Ubc13 by covalent adduct formation through a Michael addition at the Ubc13 active site cysteine. The resulting adducts of both compounds exploit a binding groove unique to Ubc13. We developed a Ubc13 mutant which resists NSC697923 inhibition and, using this mutant, we show that the inhibition of cellular DNA damage and NF-κB signaling by NSC697923 is largely due to specific Ubc13 inhibition. We propose that unique structural features near the Ubc13 active site could provide a basis for the rational development and design of specific Ubc13 inhibitors.

  15. Covalent Inhibition of Ubc13 Affects Ubiquitin Signaling and Reveals Active Site Elements Important for Targeting

    PubMed Central

    Hodge, Curtis D.; Edwards, Ross A.; Markin, Craig J.; McDonald, Darin; Pulvino, Mary; Huen, Michael S. Y.; Zhao, Jiyong; Spyracopoulos, Leo; Hendzel, Michael J.; Glover, J.N. Mark

    2015-01-01

    Ubc13 is an E2 ubiquitin conjugating enzyme that functions in nuclear DNA damage signaling and cytoplasmic NF-κB signaling. Here we present the structures of complexes of Ubc13 with two inhibitors, NSC697923 and BAY 11-7082, which inhibit DNA damage and NF-κB signaling in human cells. NSC697923 and BAY 11-7082 both inhibit Ubc13 by covalent adduct formation through a Michael addition at the Ubc13 active site cysteine. The resulting adducts of both compounds exploit a binding groove unique to Ubc13. We developed a Ubc13 mutant which resists NSC697923 inhibition and, using this mutant, we show that the inhibition of cellular DNA damage and NF-κB signaling by NSC697923 is largely due to specific Ubc13 inhibition. We propose that unique structural features near the Ubc13 active site could provide a basis for the rational development and design of specific Ubc13 inhibitors. PMID:25909880

  16. The Cys-Tyr cross-link of cysteine dioxygenase changes the optimal pH of the reaction without a structural change.

    PubMed

    Davies, Casey G; Fellner, Matthias; Tchesnokov, Egor P; Wilbanks, Sigurd M; Jameson, Guy N L

    2014-12-23

    Cysteine dioxygenase (CDO) is a non-heme monoiron enzyme with an unusual posttranslational modification in the proximity of the ferrous iron active site. This modification, a cysteine to tyrosine thioether bond, cross-links two β-strands of the β-barrel. We have investigated its role in catalysis through a combined crystallographic and kinetic approach. The C93G variant lacks the cross-link and shows little change in structure from that of the wild type, suggesting that the cross-link does not stabilize an otherwise unfavorable conformation. A pH-dependent kinetic study shows that both cross-linked and un-cross-linked CDO are active but the optimal pH decreases with the presence of the cross-link. This result reflects the effect of the thioether bond on the pKa of Y157 and this residue's role in catalysis. At higher pH values, kcat is also higher for the cross-linked form, extending the pH range of activity. We therefore propose that the cross-link also increases activity by controlling deleterious interactions involving the thiol/ate of C93.

  17. The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

    PubMed Central

    Bradshaw, William J.; Kirby, Jonathan M.; Thiyagarajan, Nethaji; Chambers, Christopher J.; Davies, Abigail H.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi

    2014-01-01

    Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA) into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Å resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33–497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism. PMID:25004975

  18. Catalytic roles of flexible regions at the active site of ribulose-bisphosphate carboxylase/oxygenase (Rubisco)

    SciTech Connect

    Hartman, F.C.; Harpel, M.R.; Chen, Yuh-Ru; Larson, E.M.; Larimer, F.W.

    1995-12-31

    Chemical and mutagenesis studies of Rubisco have identified Lys329 and Glu48 as active-site residues that are located in distinct, interacting domains from adjacent subunits. Crystallographic analyses have shown that Lys329 is the apical residue in a 12-residue flexible loop (loop 6) of the {Beta},{alpha}-barrel domain of the active site and that Glu48 resides at the end of helix B of the N-terminal domain of the active site. When phosphorylated ligands are bound by the enzyme, loop 6 adopts a closed conformation and, in concert with repositioning of helix B, thereby occludes the active site from the external environment. In this closed conformation, the {gamma}-carboxylate of Glu48 and the {epsilon}-amino group of Lys329 engage in intersubunit electrostatic interaction. By use of appropriate site-directed mutants of Rhodospirillum rubrum Rubisco, we are addressing several issues: the catalytic roles of Lys329 and Glu48, the functional significance of the intersubunit salt bridge comprised of these two residues, and the roles of loop 6 and helix B in stabilizing labile reaction intermediates. Characterization of novel products derived from misprocessing of D-ribulose-1,5-bisphosphate (RuBP) by the mutant proteins have illuminated the structure of the key intermediate in the normal oxygenase pathway.

  19. Structural basis for the active site inhibition mechanism of human kidney-type glutaminase (KGA).

    PubMed

    Thangavelu, K; Chong, Qing Yun; Low, Boon Chuan; Sivaraman, J

    2014-01-01

    Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions.

  20. Structural Basis for the Active Site Inhibition Mechanism of Human Kidney-Type Glutaminase (KGA)

    PubMed Central

    Thangavelu, K.; Chong, Qing Yun; Low, Boon Chuan; Sivaraman, J.

    2014-01-01

    Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions. PMID:24451979

  1. Free energy simulations of active-site mutants of dihydrofolate reductase.

    PubMed

    Doron, Dvir; Stojković, Vanja; Gakhar, Lokesh; Vardi-Kilshtain, Alexandra; Kohen, Amnon; Major, Dan Thomas

    2015-01-22

    This study employs hybrid quantum mechanics-molecular mechanics (QM/MM) simulations to investigate the effect of mutations of the active-site residue I14 of E. coli dihydrofolate reductase (DHFR) on the hydride transfer. Recent kinetic measurements of the I14X mutants (X = V, A, and G) indicated slower hydride transfer rates and increasingly temperature-dependent kinetic isotope effects (KIEs) with systematic reduction of the I14 side chain. The QM/MM simulations show that when the original isoleucine residue is substituted in silico by valine, alanine, or glycine (I14V, I14A, and I14G DHFR, respectively), the free energy barrier height of the hydride transfer reaction increases relative to the wild-type enzyme. These trends are in line with the single-turnover rate measurements reported for these systems. In addition, extended dynamics simulations of the reactive Michaelis complex reveal enhanced flexibility in the mutants, and in particular for the I14G mutant, including considerable fluctuations of the donor-acceptor distance (DAD) and the active-site hydrogen bonding network compared with those detected in the native enzyme. These observations suggest that the perturbations induced by the mutations partly impair the active-site environment in the reactant state. On the other hand, the average DADs at the transition state of all DHFR variants are similar. Crystal structures of I14 mutants (V, A, and G) confirmed the trend of increased flexibility of the M20 and other loops. PMID:25382260

  2. A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase

    PubMed Central

    Morgan, Hugh P.; Walsh, Martin J.; Blackburn, Elizabeth A.; Wear, Martin A.; Boxer, Matthew B.; Shen, Min; Mcnae, Iain W.; Nowicki, Matthew W.; Michels, Paul A. M.; Auld, Douglas S.; Fothergill-Gilmore, Linda A.; Walkinshaw, Malcolm D.

    2012-01-01

    SYNOPSIS Pyruvate kinase (PYK) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit Leishmania mexicana PYK (LmPYK) activity in a time- (and dose-) dependent manner; a characteristic of irreversible inhibition. The crystal structure of 4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid (DBS) complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors. PMID:22906073

  3. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  4. Cloning and characterization of a novel nuclease from shrimp hepatopancreas, and prediction of its active site.

    PubMed

    Wang, W Y; Liaw, S H; Liao, T H

    2000-03-15

    Approximately 95% of the amino acid sequence of a shrimp (Penaeus japonicus) nuclease was derived from protease-digested peptides. A 1461-base cDNA for the nuclease was amplified and sequenced with degenerate primers based on the amino acid sequence and then specific primers by 3' and 5' RACE (rapid amplification of cDNA ends). It contains an open reading frame encoding a putative 21-residue signal peptide and a 381-residue mature protein. The N-terminus of the enzyme is pyroglutamate, deduced from composition and matrix-assisted laser desorption ionization-time-of-flight MS analyses, and confirmed by a glutamine residue in the cDNA sequence. The enzyme has 11 Cys residues, forming five intramolecular disulphides. The eleventh Cys residue was linked to a thiol compound with an estimated molecular mass of between 500 and 700 Da. A sequence similarity search revealed no homologous proteins but residues 205-255 shared a conserved active-site motif within a distinct group of nucleases. His(211) in this conserved motif was shown to be very important in catalysis by site-specific modification with (14)C-labelled iodoacetate. The shrimp nuclease, previously designated DNase I, does indeed possess a low level of hydrolytic activity towards RNA in the presence of Mg(2+) and Ca(2+). The conservation of functionally important residues during distant evolution might imply that the catalytic mechanisms are similar in these nucleases, which should be classified in one subfamily. Finally, an active-site structure for shrimp nuclease was proposed on the basis of published structural data and the results of mutational and biochemical analyses of Serratia nuclease.

  5. L-Cysteine and L-AP4 microinjections in the rat caudal ventrolateral medulla decrease arterial blood pressure.

    PubMed

    Takemoto, Yumi

    2014-12-01

    The thiol amino acid L-cysteine increases arterial blood pressure (ABP) when injected into the cerebrospinal fluid space in conscious rats, indicating a pressor response to centrally acting L-cysteine. A prior synaptic membrane binding assay suggests that L-cysteine has a strong affinity for the L-2-amino-4-phosphonobutyric acid (L-AP4) binding site. The central action of L-cysteine may be vial-AP4 sensitive receptors. The present study investigated cardiovascular responses to L-cysteine and L-ap4 microinjected into the autonomic area of the caudal ventrolateral medulla (CVLM) where inhibitory neurons regulate ABP via pre-sympathetic vasomotor neurons. Both the injection of L-cysteine and L-AP4 in the CVLM sites identified with L-glutamate produced the same depressor and bradycardic responses in urethane-anesthetized rats. Neither a prior antagonist microinjection of MK801 for the N-methyl-D-aspartate (NMDA) receptor nor CNQX for the non-NMDA receptor attenuated the responses to L-cysteine, but the combination of the two receptor blocking with an additional prior injection abolished the response. In contrast, either receptor blockade alone abolished the response to L-AP4, indicating distinct mechanisms between responses to L-cysteine and L-AP4 in the CVLM. The results indicate that the CVLM is a central active site for L-cysteine's cardiovascular response. Central L-cysteine's action could be independent of the L-AP4 sensitive receptors. Cardiovascular regulation may involve endogenous L-cysteine in the CVLM. Further multidisciplinary examinations are required to elaborate on L-cysteine's functional roles in the CVLM.

  6. The crystal structure of the cysteine protease Xylellain from Xylella fastidiosa reveals an intriguing activation mechanism.

    PubMed

    Leite, Ney Ribeiro; Faro, Aline Regis; Dotta, Maria Amélia Oliva; Faim, Livia Maria; Gianotti, Andreia; Silva, Flavio Henrique; Oliva, Glaucius; Thiemann, Otavio Henrique

    2013-02-14

    Xylella fastidiosa is responsible for a wide range of economically important plant diseases. We report here the crystal structure and kinetic data of Xylellain, the first cysteine protease characterized from the genome of the pathogenic X. fastidiosa strain 9a5c. Xylellain has a papain-family fold, and part of the N-terminal sequence blocks the enzyme active site, thereby mediating protein activity. One novel feature identified in the structure is the presence of a ribonucleotide bound outside the active site. We show that this ribonucleotide plays an important regulatory role in Xylellain enzyme kinetics, possibly functioning as a physiological mediator.

  7. Artemisolide is a typical inhibitor of I{kappa}B kinase {beta} targeting cysteine-179 residue and down-regulates NF-{kappa}B-dependent TNF-{alpha} expression in LPS-activated macrophages

    SciTech Connect

    Kim, Byung Hak; Lee, Jun-Young; Seo, Jee Hee; Lee, Hwa Young; Ryu, Shi Yong; Ahn, Byung Woo; Lee, Chong-Kil; Hwang, Bang Yeon; Han, Sang-Bae; Kim, Youngsoo

    2007-09-28

    Nuclear factor (NF)-{kappa}B regulates a central common signaling for immunity and cell survival. Artemisolide (ATM) was previously isolated as a NF-{kappa}B inhibitor from a plant of Artemisia asiatica. However, molecular basis of ATM on NF-{kappa}B activation remains to be defined. Here, we demonstrate that ATM is a typical inhibitor of I{kappa}B kinase {beta} (IKK{beta}), resulting in inhibition of lipopolysaccharide (LPS)-induced NF-{kappa}B activation in RAW 264.7 macrophages. ATM inhibited the kinase activity of highly purified IKK{beta} and also LPS-induced IKK activity in the cells. Moreover, the effect of ATM on IKK{beta} activity was completely abolished by substitution of Cys-179 residue of IKK{beta} to Ala residue, indicating direct targeting site of ATM. ATM could inhibit I{kappa}B{alpha} phosphorylation in LPS-activated RAW 264.7 cells and subsequently prevent NF-{kappa}B activation. Further, we demonstrate that ATM down-regulates NF-{kappa}B-dependent TNF-{alpha} expression. Taken together, this study provides a pharmacological potential of ATM in NF-{kappa}B-dependent inflammatory disorders.

  8. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  9. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted.

  10. Effects of L-cysteine on Ni-Cu sulfide and marmatite bioleaching by Acidithiobacillus caldus.

    PubMed

    He, Zhiguo; Gao, Fengling; Zhong, Hui; Hu, Yuehua

    2009-02-01

    The effect of L-cysteine in different concentrations on the bioleaching of Ni-Cu sulfide and marmatite were studied with a moderately thermophilic, sulfur-oxidizing bacterium, strain of Acidithiobacillus caldus. X-ray diffraction (XRD) observations showed the change of bioleached solid residues and the effect of L-cysteine on the surface charges of minerals. It was found that adding certain amounts of L-cysteine to the leaching system of Ni-Cu sulfide largely enhanced the leaching rate, while L-cysteine inhibited the bioleaching of marmatite by A. caldus. The mechanism of L-cysteine interaction with mineral surfaces was studied by means of zeta potential determination and IR spectra. PMID:18829304

  11. Mechanistic pathways of mercury removal from the organomercurial lyase active site.

    PubMed

    Silva, Pedro J; Rodrigues, Viviana

    2015-01-01

    Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H(+)-assisted cleavage of the Hg-C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg(2+) from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol(-1), determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after

  12. Mechanistic pathways of mercury removal from the organomercurial lyase active site

    PubMed Central

    Rodrigues, Viviana

    2015-01-01

    Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H+-assisted cleavage of the Hg–C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg2+ from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol−1, determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after

  13. Covalent protein modification with ISG15 via a conserved cysteine in the hinge region.

    PubMed

    Bade, Veronika N; Nickels, Jochen; Keusekotten, Kirstin; Praefcke, Gerrit J K

    2012-01-01

    The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent

  14. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    PubMed

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase. PMID:17850513

  15. SABER: A computational method for identifying active sites for new reactions

    PubMed Central

    Nosrati, Geoffrey R; Houk, K N

    2012-01-01

    A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644–1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were l-Ala d/l-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. PMID:22492397

  16. SABER: a computational method for identifying active sites for new reactions.

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-05-01

    A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644-1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L-Ala D/L-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. PMID:22492397

  17. Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

    PubMed

    Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

    2016-07-19

    The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes

  18. Cysteine-Free Proteins in the Immunobiology of Arthropod-Borne Diseases

    PubMed Central

    Mejia, J. Santiago; Arthun, Erik N.; Titus, Richard G.

    2010-01-01

    One approach to identify epitopes that could be used in the design of vaccines to control several arthropod-borne diseases simultaneously is to look for common structural features in the secretome of the pathogens that cause them. Using a novel bioinformatics technique, cysteine-abundance and distribution analysis, we found that many different proteins secreted by several arthropod-borne pathogens, including Plasmodium falciparum, Borrelia burgdorferi, and eight species of Proteobacteria, are devoid of cysteine residues. The identification of three cysteine-abundance and distribution patterns in several families of proteins secreted by pathogenic and nonpathogenic Proteobacteria, and not found when the amino acid analyzed was tryptophan, provides evidence of forces restricting the content of cysteine residues in microbial proteins during evolution. We discuss these findings in the context of protein structure and function, antigenicity and immunogenicity, and host-parasite relationships. PMID:20069123

  19. A novel approach to predict active sites of enzyme molecules.

    PubMed

    Chou, Kuo-Chen; Cai, Yu-dong

    2004-04-01

    Enzymes are critical in many cellular signaling cascades. With many enzyme structures being solved, there is an increasing need to develop an automated method for identifying their active sites. However, given the atomic coordinates of an enzyme molecule, how can we predict its active site? This is a vitally important problem because the core of an enzyme molecule is its active site from the viewpoints of both pure scientific research and industrial application. In this article, a topological entity was introduced to characterize the enzymatic active site. Based on such a concept, the covariant discriminant algorithm was formulated for identifying the active site. As a paradigm, the serine hydrolase family was demonstrated. The overall success rate by jackknife test for a data set of 88 enzyme molecules was 99.92%, and that for a data set of 50 independent enzyme molecules was 99.91%. Meanwhile, it was shown through an example that the prediction algorithm can also be used to find any typographic error of a PDB file in annotating the constituent amino acids of catalytic triad and to suggest a possible correction. The very high success rates are due to the introduction of a covariance matrix in the prediction algorithm that makes allowance for taking into account the coupling effects among the key constituent atoms of active site. It is anticipated that the novel approach is quite promising and may become a useful high throughput tool in enzymology, proteomics, and structural bioinformatics. PMID:14997541

  20. Different cysteine proteinases involved in bone resorption and osteoclast formation.

    PubMed

    Brage, M; Abrahamson, M; Lindström, V; Grubb, A; Lerner, U H

    2005-06-01

    Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106 did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on cathepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase.

  1. A Hydrophobic Pocket in the Active Site of Glycolytic Aldolase Mediates Interactions with Wiskott-Aldrich Syndrome Protein

    SciTech Connect

    St-Jean,M.; Izard, T.; Sygusch, J.

    2007-01-01

    Aldolase plays essential catalytic roles in glycolysis and gluconeogenesis. However, aldolase is a highly abundant protein that is remarkably promiscuous in its interactions with other cellular proteins. In particular, aldolase binds to highly acidic amino acid sequences, including the C-terminus of the Wiskott-Aldrich syndrome protein, an actin nucleation promoting factor. Here we report the crystal structure of tetrameric rabbit muscle aldolase in complex with a C-terminal peptide of Wiskott-Aldrich syndrome protein. Aldolase recognizes a short, 4-residue DEWD motif (residues 498-501), which adopts a loose hairpin turn that folds about the central aromatic residue, enabling its tryptophan side chain to fit into a hydrophobic pocket in the active site of aldolase. The flanking acidic residues in this binding motif provide further interactions with conserved aldolase active site residues, Arg-42 and Arg-303, aligning their side chains and forming the sides of the hydrophobic pocket. The binding of Wiskott-Aldrich syndrome protein to aldolase precludes intramolecular interactions of its C-terminus with its active site, and is competitive with substrate as well as with binding by actin and cortactin. Finally, based on this structure a novel naphthol phosphate-based inhibitor of aldolase was identified and its structure in complex with aldolase demonstrated mimicry of the Wiskott-Aldrich syndrome protein-aldolase interaction. The data support a model whereby aldolase exists in distinct forms that regulate glycolysis or actin dynamics.

  2. Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog

    PubMed Central

    Englert, Markus; Nakamura, Akiyoshi; Wang, Yane-Shih; Eiler, Daniel; Söll, Dieter; Guo, Li-Tao

    2015-01-01

    Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins. PMID:26582921

  3. Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations

    PubMed Central

    Sun, Zhizeng; Mehta, Shrenik C.; Adamski, Carolyn J.; Gibbs, Richard A.; Palzkill, Timothy

    2016-01-01

    CphA is a Zn2+-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function. PMID:27616327

  4. Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations.

    PubMed

    Sun, Zhizeng; Mehta, Shrenik C; Adamski, Carolyn J; Gibbs, Richard A; Palzkill, Timothy

    2016-01-01

    CphA is a Zn(2+)-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function. PMID:27616327

  5. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS... § 582.5271 Cysteine. (a) Product. Cysteine (L-forms). (b) Conditions of use. This substance is...

  6. Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry.

    PubMed

    Mason, Alexander F; Thordarson, Pall

    2016-01-01

    The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation. We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers. PMID:27501061

  7. Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

    SciTech Connect

    Nicholas, R.A.; Suzuki, H.; Hirota, Y.; Strominger, J.L.

    1985-07-02

    This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with (/sup 14/C)penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the (/sup 14/C)penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.

  8. Regeneration of active enzyme by formation of hybrids from inactive derivatives: implications for active sites shared between polypeptide chains of aspartate transcarbamoylase.

    PubMed Central

    Robey, E A; Schachman, H K

    1985-01-01

    Crystallographic studies of Escherichia coli aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) in conjunction with chemical modification experiments have led to the suggestion that the active sites of the enzyme are at the interfaces between adjacent polypeptide chains of the catalytic trimers and involve joint participation of amino acid residues from the adjoining chains. However, the precise locations of the active sites and of the residues involved in catalysis are not known. To test the hypothesis that the active sites are shared between chains, we constructed hybrid trimers in which two chains were modified at one presumed active site residue and the third chain was altered at a different active site residue. One parental trimer was a reduced pyridoxal phosphate derivative in which lysine-84 was modified and the other was a mutant protein in which tyrosine-165 was converted to serine by site-directed mutagenesis. Incubating mixtures of these two virtually inactive derivatives under conditions promoting interchain exchange led to a large increase in enzyme activity corresponding approximately to the formation of one active site per trimer. The purified hybrid trimers, containing either two pyridoxylated and one mutant chain or vice versa, had 23% and 28%, respectively, the activity of native wild-type catalytic trimers, compared to 5% and 3% for the parental trimers. The most likely explanation for this large increase in activity is the formation of one "native" active site in each of the hybrid trimers. The results constitute strong evidence for shared active sites in aspartate transcarbamoylase. Images PMID:3881763

  9. Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA-sequestering peripheral protein domain.

    PubMed

    Hoang, Charmaine; Hamilton, Christopher S; Mueller, Eugene G; Ferré-D'Amaré, Adrian R

    2005-08-01

    The pseudouridine synthase TruB is responsible for the universally conserved post-transcriptional modification of residue 55 of elongator tRNAs. In addition to the active site, the "thumb", a peripheral domain unique to the TruB family of enzymes, makes extensive interactions with the substrate. To coordinate RNA binding and release with catalysis, the thumb may be able to sense progress of the reaction in the active site. To establish whether there is a structural correlate of communication between the active site and the RNA-sequestering thumb, we have solved the structure of a catalytically inactive point mutant of TruB in complex with a substrate RNA, and compared it to the previously determined structure of an active TruB bound to a reaction product. Superposition of the two structures shows that they are extremely similar, except in the active site and, intriguingly, in the relative position of the thumb. Because the two structures were solved using isomorphous crystals, and because the thumb is very well ordered in both structures, the displacement of the thumb we observe likely reflects preferential propagation of active site perturbations to this RNA-binding domain. One of the interactions between the active site and the thumb involves an active site residue whose hydrogen-bonding status changes during the reaction. This may allow the peripheral RNA-binding domain to monitor progress of the pseudouridylation reaction.

  10. Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.

    PubMed

    Gajadeera, Chathurada S; Zhang, Xinyi; Wei, Yinan; Tsodikov, Oleg V

    2015-02-01

    Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg(2+)-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn(2+)-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn(2+) at 2.1Å resolution. The active site contains two catalytic Mn(2+) binding sites, each half-occupied, reconciling the previously observed 1:1 Mn(2+):enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295-298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme. PMID:25576794

  11. Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant; Patel, Krunal; Vishwakarma, Rishi Kishore; Srivastava, Sameer; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M

    2013-09-01

    Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1. PMID:23688416

  12. Conformational Transitions in Human AP Endonuclease 1 and Its Active Site Mutant during Abasic Site Repair†

    PubMed Central

    Kanazhevskaya, Lyubov Yu.; Koval, Vladimir V.; Zharkov, Dmitry O.; Strauss, Phyllis R.; Fedorova, Olga S.

    2010-01-01

    AP endonuclease 1 (APE 1) is a crucial enzyme of the base excision repair pathway (BER) in human cells. APE1 recognizes apurinic/apyrimidinic (AP) sites and makes a nick in the phosphodiester backbone 5′ to them. The conformational dynamics and presteady-state kinetics of wild-type APE1 and its active site mutant, Y171F-P173L-N174K, have been studied. To observe conformational transitions occurring in the APE1 molecule during the catalytic cycle, we detected intrinsic tryptophan fluorescence of the enzyme under single turnover conditions. DNA duplexes containing a natural AP site, its tetrahydrofuran analogue, or a 2′-deoxyguanosine residue in the same position were used as specific substrates or ligands. The stopped-flow experiments have revealed high flexibility of the APE1 molecule and the complexity of the catalytic process. The fluorescent traces indicate that wild-type APE1 undergoes at least four conformational transitions during the processing of abasic sites in DNA. In contrast, nonspecific interactions of APE1 with undamaged DNA can be described by a two-step kinetic scheme. Rate and equilibrium constants were extracted from the stopped-flow and fluorescence titration data for all substrates, ligands, and products. A replacement of three residues at the enzymatic active site including the replacement of tyrosine 171 with phenylalanine in the enzyme active site resulted in a 2 × 104-fold decrease in the reaction rate and reduced binding affinity. Our data indicate the important role of conformational changes in APE1 for substrate recognition and catalysis. PMID:20575528

  13. Functional significance of Glu-77 and Tyr-137 within the active site of isoaspartyl dipeptidase.

    PubMed

    Martí-Arbona, Ricardo; Thoden, James B; Holden, Hazel M; Raushel, Frank M

    2005-12-01

    Isoaspartyl dipeptidase (IAD) is a binuclear metalloenzyme and a member of the amidohydrolase superfamily. This enzyme catalyzes the hydrolytic cleavage of beta-aspartyl dipeptides. The pH-rate profiles for the hydrolysis of beta-Asp-Leu indicates that catalysis is dependent on the ionization of two groups; one that ionizes at a pH approximately 6 and the other approximately 9. The group that must be ionized for catalysis is directly dependent on the identity of the metal ion bound to the active site. This result is consistent with the ionization of the hydroxide that bridges the two divalent cations. In addition to the residues that interact directly with the divalent cations there are two other residues that are highly conserved and found within the active site: Glu-77 and Tyr-137. Mutation of Tyr-137 to phenylalanine reduced the rate of catalysis by three orders of magnitude. The three dimensional X-ray structure of the Y137F mutant did not show any significant conformation changes relative to the three dimensional structure of the wild-type enzyme. The positioning of the side-chain phenolic group of Tyr-137 in the active site of IAD is consistent with the stabilization of the tetrahedral adduct concomitant with nucleophilic attack by the hydroxide that bridges the two divalent cations. Mutation of Glu-77 resulted in the reduction of catalytic activity by five orders of magnitude. The three dimensional structure of the E77Q mutant did not show any significant conformational changes in the mutant relative to the three dimensional structure of the wild-type enzyme. The positioning of the side-chain carboxylate of Glu-77 is consistent with the formation of an ion pair interaction with the free alpha-amino group of the substrate.

  14. Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.

    PubMed

    Qiu, Huawei; Edmunds, Tim; Baker-Malcolm, Jennifer; Karey, Kenneth P; Estes, Scott; Schwarz, Cordula; Hughes, Heather; Van Patten, Scott M

    2003-08-29

    One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

  15. The antihypertensive effect of cysteine

    PubMed Central

    Vasdev, Sudesh; Singal, Pawan; Gill, Vicki

    2009-01-01

    Hypertension is a leading cause of morbidity and mortality worldwide. Individuals with hypertension are at an increased risk for stroke, heart disease and kidney failure. Essential hypertension results from a combination of genetic and lifestyle factors. One such lifestyle factor is diet, and its role in the control of blood pressure has come under much scrutiny. Just as increased salt and sugar are known to elevate blood pressure, other dietary factors may have antihypertensive effects. Studies including the Optimal Macronutrient Intake to Prevent Heart Disease (OmniHeart) study, Multiple Risk Factor Intervention Trial (MRFIT), International Study of Salt and Blood Pressure (INTERSALT) and Dietary Approaches to Stop Hypertension (DASH) study have demonstrated an inverse relationship between dietary protein and blood pressure. One component of dietary protein that may partially account for its antihypertensive effect is the nonessential amino acid cysteine. Studies in hypertensive humans and animal models of hypertension have shown that N-acetylcysteine, a stable cysteine analogue, lowers blood pressure, which substantiates this idea. Cysteine may exert its antihypertensive effects directly or through its storage form, glutathione, by decreasing oxidative stress, improving insulin resistance and glucose metabolism, lowering advanced glycation end products, and modulating levels of nitric oxide and other vasoactive molecules. Therefore, adopting a balanced diet containing cysteine-rich proteins may be a beneficial lifestyle choice for individuals with hypertension. An example of such a diet is the DASH diet, which is low in salt and saturated fat; includes whole grains, poultry, fish and nuts; and is rich in vegetables, fruits and low-fat dairy products. PMID:22477470

  16. Multiple, Ligand-Dependent Routes from the Active Site of Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Winn, Peter J.; Wade, Rebecca C.

    2012-02-13

    The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

  17. A type IV translocated Legionella cysteine phytase counteracts intracellular growth restriction by phytate.

    PubMed

    Weber, Stephen; Stirnimann, Christian U; Wieser, Mara; Frey, Daniel; Meier, Roger; Engelhardt, Sabrina; Li, Xiaodan; Capitani, Guido; Kammerer, Richard A; Hilbi, Hubert

    2014-12-01

    The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique "Legionella-containing vacuole." The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different "effector" proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys(231)) and arginine (Arg(237)) residues. The structure of LppA at 1.4 Å resolution revealed a mainly α-helical globular protein stabilized by four antiparallel β-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens. PMID:25339170

  18. A Type IV Translocated Legionella Cysteine Phytase Counteracts Intracellular Growth Restriction by Phytate

    PubMed Central

    Weber, Stephen; Stirnimann, Christian U.; Wieser, Mara; Frey, Daniel; Meier, Roger; Engelhardt, Sabrina; Li, Xiaodan; Capitani, Guido; Kammerer, Richard A.; Hilbi, Hubert

    2014-01-01

    The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique “Legionella-containing vacuole.” The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different “effector” proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys231) and arginine (Arg237) residues. The structure of LppA at 1.4 Å resolution revealed a mainly α-helical globular protein stabilized by four antiparallel β-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens. PMID:25339170

  19. A type IV translocated Legionella cysteine phytase counteracts intracellular growth restriction by phytate.

    PubMed

    Weber, Stephen; Stirnimann, Christian U; Wieser, Mara; Frey, Daniel; Meier, Roger; Engelhardt, Sabrina; Li, Xiaodan; Capitani, Guido; Kammerer, Richard A; Hilbi, Hubert

    2014-12-01

    The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique "Legionella-containing vacuole." The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different "effector" proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys(231)) and arginine (Arg(237)) residues. The structure of LppA at 1.4 Å resolution revealed a mainly α-helical globular protein stabilized by four antiparallel β-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens.

  20. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  1. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets

    PubMed Central

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C.

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein–protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases. PMID:27199750

  2. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets.

    PubMed

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases. PMID:27199750

  3. X-ray structures of Nfs2, the plastidial cysteine desulfurase from Arabidopsis thaliana

    PubMed Central

    Roret, Thomas; Pégeot, Henri; Couturier, Jérémy; Mulliert, Guillermo; Rouhier, Nicolas; Didierjean, Claude

    2014-01-01

    The chloroplastic Arabidopsis thaliana Nfs2 (AtNfs2) is a group II pyridoxal 5′-phosphate-dependent cysteine desulfurase that is involved in the initial steps of iron–sulfur cluster biogenesis. The group II cysteine desulfurases require the presence of sulfurtransferases such as SufE proteins for optimal activity. Compared with group I cysteine desulfurases, proteins of this group contains a smaller extended lobe harbouring the catalytic cysteine and have a β-hairpin constraining the active site. Here, two crystal structures of AtNfs2 are reported: a wild-type form with the catalytic cysteine in a persulfide-intermediate state and a C384S variant mimicking the resting state of the enzyme. In both structures the well conserved Lys241 covalently binds pyridoxal 5′-phosphate, forming an internal aldimine. Based on available homologous bacterial complexes, a model of a complex between AtNfs2 and the SufE domain of its biological partner AtSufE1 is proposed, revealing the nature of the binding sites. PMID:25195888

  4. Mechanistic and bioinformatic investigation of a conserved active site helix in α-isopropylmalate synthase from Mycobacterium tuberculosis, a member of the DRE-TIM metallolyase superfamily.

    PubMed

    Casey, Ashley K; Hicks, Michael A; Johnson, Jordyn L; Babbitt, Patricia C; Frantom, Patrick A

    2014-05-13

    The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily.

  5. Discovering mechanisms of signaling-mediated cysteine oxidation.

    PubMed

    Poole, Leslie B; Nelson, Kimberly J

    2008-02-01

    Accumulating evidence reveals hydrogen peroxide as a key player both as a damaging agent and, from emerging evidence over the past decade, as a second messenger in intracellular signaling. This rather mild oxidant acts upon downstream targets within signaling cascades to modulate the activity of a host of enzymes (e.g. phosphatases and kinases) and transcriptional regulators through chemoselective oxidation of cysteine residues. With the recent development of specific detection reagents for hydrogen peroxide and new chemical tools to detect the generation of the initial oxidation product, sulfenic acid, on reactive cysteines within target proteins, the scene is set to gain a better understanding of the mechanisms through which hydrogen peroxide acts as a second messenger in cell signaling.

  6. Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

    PubMed

    Rajaonarivony, J I; Gershenzon, J; Miyazaki, J; Croteau, R

    1992-11-15

    (4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes. PMID:1444454

  7. Active site nanospace of aminoacyl tRNA synthetase: difference between the class I and class II synthetases.

    PubMed

    Dutta, Saheb; Choudhury, Kaberi; Banik, Sindrila Dutta; Nandi, Nilashis

    2014-03-01

    The present work is aimed at understanding the origin of the difference in the molecular organization of the active site nanospaces of the class I and class II aminoacyl tRNA synthetases (aaRSs) which are tunnel-like structures. The active site encloses the cognate amino acid (AA) and the adenosine triphosphate (ATP) to carry out aminoacylation reaction. Comparison of the structures of the active site of the class I and class II (aaRSs) shows that the nanodimensional tunnels are curved in opposite directions in the two classes. We investigated the origin of this difference using quantum mechanical computation of electrostatic potential (ESP) of substrates, surrounding residues and ions, using Atoms in Molecule (AIM) Theory and charge population analysis. We show that the difference is principally due to the variation in the spatial charge distribution of ATP in the two classes which correspond to extended and bent conformations of ATP. The present computation shows that the most feasible pathway for nucleophilic attack to alphaP is oppositely directed for class I and class II aaRSs. The available crystal structures show that the cognate AA is indeed located along the channel favorable for nucleophilic attack as predicted by the ESP analysis. It is also shown that the direction of the channel changes its orientation when the orientation of ATP is changed from extended to a bent like structure. We further used the AIM theory to confirm the direction of the approach of AA in each case and the results corroborate the results from the ESP analysis. The opposite curvatures of the active site nanospaces in class I and class II aaRSs are related with the influence of the charge distributions of the extended and bent conformations of ATP, respectively. The results of the computation of electrostatic potential by successive addition of active site residues show that their roles on the reaction are similar in both classes despite the difference in the organization of the

  8. Crystal structure of the cysteine desulfurase DndA from Streptomyces lividans which is involved in DNA phosphorothioation.

    PubMed

    Chen, Fukun; Zhang, Zhenyi; Lin, Kui; Qian, Tianle; Zhang, Yan; You, Delin; He, Xinyi; Wang, Zhijun; Liang, Jingdan; Deng, Zixin; Wu, Geng

    2012-01-01

    DNA phosphorothioation is widespread among prokaryotes, and might function to restrict gene transfer among different kinds of bacteria. There has been little investigation into the structural mechanism of the DNA phosphorothioation process. DndA is a cysteine desulfurase which is involved in the first step of DNA phosphorothioation. In this study, we determined the crystal structure of Streptomyces lividans DndA in complex with its covalently bound cofactor PLP, to a resolution of 2.4 Å. Our structure reveals the molecular mechanism that DndA employs to recognize its cofactor PLP, and suggests the potential binding site for the substrate L-cysteine on DndA. In contrast to previously determined structures of cysteine desulfurases, the catalytic cysteine of DndA was found to reside on a β strand. This catalytic cysteine is very far away from the presumable location of the substrate, suggesting that a conformational change of DndA is required during the catalysis process to bring the catalytic cysteine close to the substrate cysteine. Moreover, our in vitro enzymatic assay results suggested that this conformational change is unlikely to be a simple result of random thermal motion, since moving the catalytic cysteine two residues forward or backward in the primary sequence completely disabled the cysteine desulfurase activity of DndA.

  9. Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

    PubMed Central

    Stec, Boguslaw

    2012-01-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO2. We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O2 and CO2 bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO2 defines an elusive, preactivation complex that contains a metal cation Mg2+ surrounded by three H2O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming. PMID:23112176

  10. Paired natural cysteine mutation mapping: aid to constraining models of protein tertiary structure.

    PubMed Central

    Kreisberg, R.; Buchner, V.; Arad, D.

    1995-01-01

    This paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural-cysteine-mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide-bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using PileUp algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three-dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur. PMID:8563638

  11. NMR crystallography of enzyme active sites: probing chemically detailed, three-dimensional structure in tryptophan synthase.

    PubMed

    Mueller, Leonard J; Dunn, Michael F

    2013-09-17

    NMR crystallography--the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry--offers unprecedented insight into three-dimensional, chemically detailed structure. Initially, researchers used NMR crystallography to refine diffraction data from organic and inorganic solids. Now we are applying this technique to explore active sites in biomolecules, where it reveals chemically rich detail concerning the interactions between enzyme site residues and the reacting substrate. Researchers cannot achieve this level of detail from X-ray, NMR,or computational methodologies in isolation. For example, typical X-ray crystal structures (1.5-2.5 Å resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate but do not directly identify the protonation states. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but they rely on researcher-specified chemical details. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which scientists can develop models of the active site using computational chemistry; they can then distinguish these models by comparing calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at the highest possible resolution. In this Account, we detail our first steps in the development of

  12. Papain labelled with fluorescent thiol-specific reagents as a probe for characterization of interactions between cysteine proteinases and their protein inhibitors by competitive titrations.

    PubMed

    Lindahl, P; Raub-Segall, E; Olson, S T; Björk, I

    1991-06-01

    Papain was labelled by attachment of the fluorescent groups 2-(4'-acetamidoanilino)naphthalene-6-sulphonic acid (AANS) or N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid (AEDANS) to the active-site cysteine residue, with the aim of using the labelled papains as probes in competitive titrations of unlabelled cysteine proteinases with their inhibitors. The interaction between the labelled papains and cystatins was accompanied by an increase in fluorescence emission of up to 38-fold for AANS-papain and approximately 3.5-fold for AEDANS-papain. Fluorescence titrations gave dissociation equilibrium constants of 3.1 and 0.6 microM for the binding of chicken cystatin and recombinant human cystatin C respectively to AANS-papain and of 11.9 microM for the binding of chicken cystatin to AEDANS-papain. The kinetics of interaction of chicken cystatin with AANS-papain showed an unusual biphasic dependence of the observed pseudo-first-order rate constant on inhibitor concentration, consistent with the reaction occurring via both pathways of a general two-step binding mechanism. AANS-papain was selected as the most suitable probe for competitive titrations of unlabelled active or inactivated cysteine proteinases with inhibitors. This technique, which provides stoichiometries and dissociation constants for the interaction between unlabelled enzyme and inhibitor, allows monitoring of the interactions by a large fluorescent signal in a wavelength region where the interacting proteins do not contribute to the observed fluorescence. Such competitive titrations of active papain or actinidin with chicken cystatin or recombinant human cystatin C all gave inhibitor/enzyme stoichiometries of close to 1.0. A dissociation constant of 1.8 microM for the reaction of chicken cystatin with a papain derivative, S-[N-(3-carboxypropyl)succinimidyl]-papain, was also determined by the same technique. These results show the usefulness of the fluorescent papains for the characterization of

  13. Growth exponents in surface models with non-active sites

    NASA Astrophysics Data System (ADS)

    Santos, M.; Figueiredo, W.; Aarão Reis, F. D. A.

    2006-11-01

    In this work, we studied the role played by the inactive sites present on the substrate of a growing surface. In our model, one particle sticks at the surface if the site where it falls is an active site. However, we allow the deposited particle to diffuse along the surface in accordance with some mechanism previously defined. Using Monte Carlo simulations, and some analytical results, we have investigated the model in (1+1) and (2+1) dimensions considering different relaxation mechanisms. We show that the consideration of non-active sites is a crucial point in the model. In fact, we have seen that the saturation regime is not observed for any value of the density of inactive sites. Besides, the growth exponent β turns to be one, at long times, whatever the mechanism of diffusion we consider in one and two dimensions.

  14. Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

    HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.

  15. Active sites in char gasification: Final technical report

    SciTech Connect

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  16. Brownian aggregation rate of colloid particles with several active sites

    SciTech Connect

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V.; Polshchitsin, Alexey A.; Yakovleva, Galina E.; Maltsev, Valeri P.

    2014-08-14

    We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.

  17. Active-Site-Accessible, Porphyrinic Metal;#8722;Organic Framework Materials

    SciTech Connect

    Farha, Omar K.; Shultz, Abraham M.; Sarjeant, Amy A.; Nguyen, SonBinh T.; Hupp, Joseph T.

    2012-02-06

    On account of their structural similarity to cofactors found in many metallo-enzymes, metalloporphyrins are obvious potential building blocks for catalytically active, metal-organic framework (MOF) materials. While numerous porphyrin-based MOFs have already been described, versions featuring highly accessible active sites and permanent microporosity are remarkably scarce. Indeed, of the more than 70 previously reported porphyrinic MOFs, only one has been shown to be both permanently microporous and contain internally accessible active sites for chemical catalysis. Attempts to generalize the design approach used in this single successful case have failed. Reported here, however, is the synthesis of an extended family of MOFs that directly incorporate a variety of metalloporphyrins (specifically Al{sup 3+}, Zn{sup 2+}, Pd{sup 2+}, Mn{sup 3+}, and Fe{sup 3+} complexes). These robust porphyrinic materials (RPMs) feature large channels and readily accessible active sites. As an illustrative example, one of the manganese-containing RPMs is shown to be catalytically competent for the oxidation of alkenes and alkanes.

  18. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O2 binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies. PMID:22725967

  19. Inhibition and active-site modelling of prolidase.

    PubMed

    King, G F; Crossley, M J; Kuchel, P W

    1989-03-15

    Consideration of the active-site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N-benzyloxycarbonyl-L-proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki = 90 microM); its rapid protein-mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N-benzyloxycarbonyl-L-pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captopril and enalaprilat, were shown to have mild and no inhibitory effects, respectively. Analysis of inhibitor action and consideration of X-ray crystallographic data of relevant Mn2+ complexes allowed the active-site model of prolidase to be further refined; a new model is presented in which the substrate acts as a bidentate ligand towards the active-site manganous ion. Various aspects of the new model help to explain why Mn2+ has been 'chosen' by the enzyme in preference to other biologically available metal ions. PMID:2924773

  20. The Basics of Thiols and Cysteines in Redox Biology and Chemistry

    PubMed Central

    Poole, Leslie B.

    2014-01-01

    Cysteine is one of the least abundant amino acids, yet it is frequently found as a highly conserved residue within functional (regulatory, catalytic or binding) sites in proteins. It is the unique chemistry of the thiol or thiolate group of cysteine that imparts functional sites with their specialized properties (e.g., nucleophilicity, high affinity metal binding, and/or ability to form disulfide bonds). Highlighted in this review are some of the basic biophysical and biochemical properties of cysteine groups and the equations that apply to them, particularly with respect to pKa and redox potential. Also summarized are the types of low molecular weight thiols present in high concentrations in most cells, as well as the ways in which modifications of cysteinyl residues can impart or regulate molecular functions important to cellular processes including signal transduction. PMID:25433365

  1. Cysteine metabolism and metal toxicity.

    PubMed

    Quig, D

    1998-08-01

    Chronic, low level exposure to toxic metals is an increasing global problem. The symptoms associated with the slow accumulation of toxic metals are multiple and rather nondescript, and overt expression of toxic effects may not appear until later in life. The sulfhydryl-reactive metals (mercury, cadmium, lead, arsenic) are particularly insidious and can affect a vast array of biochemical and nutritional processes. The primary mechanisms by which the sulfhydryl-reactive metals elicit their toxic effects are summarized. The pro-oxidative effects of the metals are compounded by the fact that the metals also inhibit antioxidative enzymes and deplete intracellular glutathione. The metals also have the potential to disrupt the metabolism and biological activities of many proteins due to their high affinity for free sulfhydryl groups. Cysteine has a pivotal role in inducible, endogenous detoxication mechanisms in the body, and metal exposure taxes cysteine status. The protective effects of glutathione and the metallothioneins are discussed in detail. Basic research pertaining to the transport of toxic metals into the brain is summarized, and a case is made for the use of hydrolyzed whey protein to support metal detoxification and neurological function. Metal exposure also affects essential element status, which can further decrease antioxidation and detoxification processes. Early detection and treatment of metal burden is important for successful detoxification, and optimization of nutritional status is paramount to the prevention and treatment of metal toxicity.

  2. Cwp84, a Clostridium difficile cysteine protease, exhibits conformational flexibility in the absence of its propeptide

    SciTech Connect

    Bradshaw, William J.; Roberts, April K.; Shone, Clifford C.; Acharya, K. Ravi

    2015-02-19

    Two structures of Cwp84, a cysteine protease from the S-layer of C. difficile, are presented after propeptide cleavage. They reveal the movement of three loops, two in the active-site groove and one on the surface of the lectin-like domain, exposing a hydrophobic pocket. In recent decades, the global healthcare problems caused by Clostridium difficile have increased at an alarming rate. A greater understanding of this antibiotic-resistant bacterium, particularly with respect to how it interacts with the host, is required for the development of novel strategies for fighting C. difficile infections. The surface layer (S-layer) of C. difficile is likely to be of significant importance to host–pathogen interactions. The mature S-layer is formed by a proteinaceous array consisting of multiple copies of a high-molecular-weight and a low-molecular-weight S-layer protein. These components result from the cleavage of SlpA by Cwp84, a cysteine protease. The structure of a truncated Cwp84 active-site mutant has recently been reported and the key features have been identified, providing the first structural insights into the role of Cwp84 in the formation of the S-layer. Here, two structures of Cwp84 after propeptide cleavage are presented and the three conformational changes that are observed are discussed. These changes result in a reconfiguration of the active site and exposure of the hydrophobic pocket.

  3. Probing Oxygen Activation Sites in Two Flavoprotein Oxidases Using Chloride as an Oxygen Surrogate

    SciTech Connect

    Kommoju, Phaneeswara-Rao; Chen, Zhi-wei; Bruckner, Robert C.; Mathews, F. Scott; Jorns, Marilyn Schuman

    2011-08-16

    A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX-chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX-chloride complex and a ternary MSOX-chloride-MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.

  4. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    D Gallagher; S Kim; H Robinson; P Reddy

    2011-12-31

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV) - two with substrate analogs and one with product. Mn{sup 2+} binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3' on {alpha}-phosphate (distance {approx} 4 {angstrom}). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2' oxygen and ribose 3' oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears to ionize the O3' nucleophile. Different syn/anti conformations suggest that the mechanism involves a conformational transition, and further evidence suggests a role for ribosyl pseudorotation. With resolutions of 1.6-1.7 {angstrom}, these are the most detailed active-site ligand complexes for any class of this ubiquitous signaling enzyme.

  5. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    Gallagher, D.T.; Robinson, H.; Kim, S.-K.; Reddy, P. T.

    2011-01-21

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV)-two with substrate analogs and one with product. Mn{sup 2+} binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3' on {alpha}-phosphate (distance {approx} 4 {angstrom}). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2' oxygen and ribose 3' oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears to ionize the O3' nucleophile. Different syn/anti conformations suggest that the mechanism involves a conformational transition, and further evidence suggests a role for ribosyl pseudorotation. With resolutions of 1.6-1.7 {angstrom}, these are the most detailed active-site ligand complexes for any class of this ubiquitous signaling enzyme.

  6. Aminothienopyridazines and Methylene Blue Affect Tau Fibrillization via Cysteine Oxidation*

    PubMed Central

    Crowe, Alex; James, Michael J.; Lee, Virginia M.-Y.; Smith, Amos B.; Trojanowski, John Q.; Ballatore, Carlo; Brunden, Kurt R.

    2013-01-01

    Alzheimer disease and several other neurodegenerative disorders are characterized by the accumulation of intraneuronal fibrils comprised of the protein Tau. Tau is normally a soluble protein that stabilizes microtubules, with splice isoforms that contain either three (3-R) or four (4-R) microtubule binding repeats. The formation of Tau fibrils is thought to result in neuronal damage, and inhibitors of Tau fibrillization may hold promise as therapeutic agents. The process of Tau fibrillization can be replicated in vitro, and a number of small molecules have been identified that inhibit Tau fibril formation. However, little is known about how these molecules affect Tau fibrillization. Here, we examined the mechanism by which the previously described aminothieno pyridazine (ATPZ) series of compounds inhibit Tau fibrillization. Active ATPZs were found to promote the oxidation of the two cysteine residues within 4-R Tau by a redox cycling mechanism, resulting in the formation of a disulfide-containing compact monomer that was refractory to fibrillization. Moreover, the ATPZs facilitated intermolecular disulfide formation between 3-R Tau monomers, leading to dimers that were capable of fibrillization. The ATPZs also caused cysteine oxidation in molecules unrelated to Tau. Interestingly, methylene blue, an inhibitor of Tau fibrillization under evaluation in Alzheimer disease clinical trials, caused a similar oxidation of cysteines in Tau and other molecules. These findings reveal that the ATPZs and methylene blue act by a mechanism that may affect their viability as potential therapeutic agents. PMID:23443659

  7. Characterization of cysteine string protein in rat parotid acinar cells.

    PubMed

    Shimomura, Hiromi; Imai, Akane; Nashida, Tomoko

    2013-10-01

    Cysteine string proteins (CSPs) are secretory vesicle chaperone proteins that contain: (i) a heavily palmitoylated cysteine string (comprised of 14 cysteine residues, responsible for the localization of CSP to secretory vesicle membranes), (ii) an N-terminal J-domain (DnaJ domain of Hsc70, 70kDa heat-shock cognate protein family of co-chaperones), and (iii) a linker domain (important in mediating CSP effects on secretion). In this study, we investigated the localization of CSP1 in rat parotid acinar cells and evaluated the role of CSP1 in parotid secretion. RT-PCR and western blotting revealed that CSP1 was expressed and associated with Hsc70 in rat parotid acinar cells. Further, CSP1 associated with syntaxin 4, but not with syntaxin 3, on the apical plasma membrane. Introduction of anti-CSP1 antibody into SLO-permeabilized acinar cells enhanced isoproterenol (IPR)-induced amylase release. Introduction of GST-CSP11-112, containing both the J-domain and the adjacent linker region, enhanced IPR-induced amylase release, whereas neither GST-CSP11-82, containing the J-domain only, nor GST-CSP183-112, containing the linker region only, did produce detectable enhancement. These results indicated that both the J-domain and the linker domain of CSP1 are necessary to function an important role in acinar cell exocytosis.

  8. X-ray structure at 1.75 resolution of a norovirus 3C protease linked to an active site-directed peptide inhibitor

    SciTech Connect

    Cooper, Jon; Coates, Leighton; Hussey, Robert

    2010-01-01

    Noroviruses are recognized universally as the most important cause of human epidemic non-bacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.75 resolution, following initial MAD phasing with a selenomethionine derivative. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, based on a 3C protease cleavage recognition sequences in the 200kDa polyprotein substrate, reacts covalently through its propenylethylester group (X) with the active site nucleophile, Cys 139. The 3C protease-inhibitor structure permits, for the first time, the identification of substrate recognition and binding groups and provides important new information for the development of antiviral prophylactics.

  9. NMR structure of the A730 loop of the Neurospora VS ribozyme: insights into the formation of the active site

    PubMed Central

    Bonneau, Eric; Girard, Nicolas; Boisbouvier, Jérôme; Legault, Pascale

    2011-01-01

    The Neurospora VS ribozyme is a small nucleolytic ribozyme with unique primary, secondary and global tertiary structures, which displays mechanistic similarities to the hairpin ribozyme. Here, we determined the high-resolution NMR structure of a stem–loop VI fragment containing the A730 internal loop, which forms part of the active site. In the presence of magnesium ions, the A730 loop adopts a structure that is consistent with existing biochemical data and most likely reflects its conformation in the VS ribozyme prior to docking with the cleavage site internal loop. Interestingly, the A730 loop adopts an S-turn motif that is also present in loop B within the hairpin ribozyme active site. The S-turn appears necessary to expose the Watson–Crick edge of a catalytically important residue (A756) so that it can fulfill its role in catalysis. The A730 loop and the cleavage site loop of the VS ribozyme display structural similarities to internal loops found in the active site of the hairpin ribozyme. These similarities provided a rationale to build a model of the VS ribozyme active site based on the crystal structure of the hairpin ribozyme. PMID:21266483

  10. QM/MM Analysis of Cellulase Active Sites and Actions of the Enzymes on Substrates

    SciTech Connect

    Saharay, Moumita; Guo, Hao-Bo; Smith, Jeremy C; Guo, Hong

    2010-01-01

    Biodegradation of cellulosic biomass requires the actions of three types of secreted enzymes; endoglucanase (EC 3.2.1.4), cellobiohydrolase or exoglucanase (EC 3.2.1.91), and -glucosidase (EC 4.2.1.21). These enzymes act synergistically to hydrolyse the -1,4 bonds of cellulose and converts it into simple sugar. Hydrolysis of the glycosidic bond can occur either by net retention or by inversion of anomeric configuration at the anomeric center. QM/MM simulations are useful tools to study the energetics of the reactions and analyze the active-site structures at different states of the catalysis, including the formation of unstable transition states. Here, a brief description of previous work on glycoside hydrolases is first given. The results of the QM/MM potential energy and free energy simulations corresponding to glycosylation and deglycosylation processes are then provided for two retaining endoglucanases, Cel12A and Cel5A. The active-site structural features are analyzed based on the QM/MM results. The role of different residues and hydrogen bonding interactions during the catalysis and the importance of the sugar ring distortion are discussed for these two enzymes.

  11. Mechanism of Oxygen Reduction in Cytochrome c Oxidase and the Role of the Active Site Tyrosine.

    PubMed

    Blomberg, Margareta R A

    2016-01-26

    Cytochrome c oxidase, the terminal enzyme in the respiratory chain, reduces molecular oxygen to water and stores the released energy through electrogenic chemistry and proton pumping across the membrane. Apart from the heme-copper binuclear center, there is a conserved tyrosine residue in the active site (BNC). The tyrosine delivers both an electron and a proton during the O-O bond cleavage step, forming a tyrosyl radical. The catalytic cycle then occurs in four reduction steps, each taking up one proton for the chemistry (water formation) and one proton to be pumped. It is here suggested that in three of the reduction steps the chemical proton enters the center of the BNC, leaving the tyrosine unprotonated with radical character. The reproprotonation of the tyrosine occurs first in the final reduction step before binding the next oxygen molecule. It is also suggested that this reduction mechanism and the presence of the tyrosine are essential for the proton pumping. Density functional theory calculations on large cluster models of the active site show that only the intermediates with the proton in the center of the BNC and with an unprotonated tyrosyl radical have a high electron affinity of similar size as the electron donor, which is essential for the ability to take up two protons per electron and thus for the proton pumping. This type of reduction mechanism is also the only one that gives a free energy profile in accordance with experimental observations for the amount of proton pumping in the working enzyme.

  12. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  13. The Active Site of Oligogalacturonate Lyase Provides Unique Insights into Cytoplasmic Oligogalacturonate β-Elimination*

    PubMed Central

    Abbott, D. Wade; Gilbert, Harry J.; Boraston, Alisdair B.

    2010-01-01

    Oligogalacturonate lyases (OGLs; now also classified as pectate lyase family 22) are cytoplasmic enzymes found in pectinolytic members of Enterobacteriaceae, such as the enteropathogen Yersinia enterocolitica. OGLs utilize a β-elimination mechanism to preferentially catalyze the conversion of saturated and unsaturated digalacturonate into monogalacturonate and the 4,5-unsaturated monogalacturonate-like molecule, 5-keto-4-deoxyuronate. To provide mechanistic insights into the specificity of this enzyme activity, we have characterized the OGL from Y. enterocolitica, YeOGL, on oligogalacturonides and determined its three-dimensional x-ray structure to 1.65 Å. The model contains a Mn2+ atom in the active site, which is coordinated by three histidines, one glutamine, and an acetate ion. The acetate mimics the binding of the uronate group of galactourono-configured substrates. These findings, in combination with enzyme kinetics and metal supplementation assays, provide a framework for modeling the active site architecture of OGL. This enzyme appears to contain a histidine for the abstraction of the α-proton in the −1 subsite, a residue that is highly conserved throughout the OGL family and represents a unique catalytic base among pectic active lyases. In addition, we present a hypothesis for an emerging relationship observed between the cellular distribution of pectate lyase folding and the distinct metal coordination chemistries of pectate lyases. PMID:20851883

  14. Mapping protein cysteine sulfonic acid modifications with specific enrichment and mass spectrometry: an integrated approach to explore the cysteine oxidation.

    PubMed

    Chang, Yuan-Chang; Huang, Chien-Ning; Lin, Chia-Hung; Chang, Huan-Cheng; Wu, Chih-Che

    2010-08-01

    Oxidation of thiol proteins, which results in conversion of cysteine residues to cysteine sulfenic, sulfinic or sulfonic acids, is an important posttranslational control of protein function in cells. To facilitate the analysis of this process with MALDI-MS, we have developed a method for selective enrichment and identification of peptides containing cysteine sulfonic acid (sulfopeptides) in tryptic digests of proteins based on ionic affinity capture using polyarginine-coated nanodiamonds as high-affinity probes. The method was applied to selectively concentrate sulfopeptides from either a highly dilute solution or a complex peptide mixture in which the abundance of the sulfonated analyte is as low as 0.02%. The polyarginine-coated probes exhibit a higher affinity for peptides containing multiple sulfonic acids than peptides containing single sulfonic acid. The limit of the detection is in the femtomole range, with the MALDI-TOF mass spectrometer operating in the negative ion mode. The results show that the new approach has good specificity even in the presence of phosphopeptides. An application of this method for selective enrichment and structural identification of sulfopeptides is demonstrated with the tryptic digests of performic-acid-oxidized BSA.

  15. Development of Novel Sugar Isomerases by Optimization of Active Sites in Phosphosugar Isomerases for Monosaccharides

    PubMed Central

    Yeom, Soo-Jin; Kim, Yeong-Su

    2013-01-01

    Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a d-ribose isomerase, an l-ribose isomerase, and an l-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases. PMID:23204422

  16. Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence.

    PubMed

    Jones, M L; Larsen, P B; Woodson, W R

    1995-06-01

    The senescence of carnation (Dianthus caryophyllus L.) flower petals is regulated by the phytohormone ethylene and is associated with considerable catabolic activity including the loss of protein. In this paper we present the molecular cloning of a cysteine proteinase and show that its expression is regulated by ethylene and associated with petal senescence. A 1600 bp cDNA was amplified by polymerase chain reaction using a 5'-specific primer and 3'-nonspecific primer designed to amplify a 1-aminocyclopropane-1-carboxylate synthase cDNA from reverse-transcribed stylar RNA. The nucleotide sequence of the cloned product (pDCCP1) was found to share significant homology to several cysteine proteinases rather than ACC synthase. A single open reading frame of 428 amino acids was shown to share significant homology with other plant cysteine proteinases including greater than 70% identity with a cysteine proteinase from Arabidopsis thaliana. Amino acids in the active site of cysteine proteinases were conserved in the pDCCP1 peptide. RNA gel blot analysis revealed that the expression of pDCCP1 increased substantially with the onset of ethylene production and senescence of petals. Increased pDCCP1 expression was also associated with ethylene production in other senescing floral organs including ovaries and styles. The pDCCP1 transcript accumulated in petals treated with exogenous ethylene within 3 h and treatment of flowers with 2,5-norbornadiene, an inhibitor of ethylene action, prevented the increase in pDCCP1 expression in petals. The temporal and spatial patterns of pDCCP1 expression suggests a role for cysteine proteinase in the loss of protein during floral senescence.

  17. Improving Functional Annotation in the DRE-TIM Metallolyase Superfamily through Identification of Active Site Fingerprints.

    PubMed

    Kumar, Garima; Johnson, Jordyn L; Frantom, Patrick A

    2016-03-29

    Within the DRE-TIM metallolyase superfamily, members of the Claisen-like condensation (CC-like) subgroup catalyze C-C bond-forming reactions between various α-ketoacids and acetyl-coenzyme A. These reactions are important in the metabolic pathways of many bacterial pathogens and serve as engineering scaffolds for the production of long-chain alcohol biofuels. To improve functional annotation and identify sequences that might use novel substrates in the CC-like subgroup, a combination of structural modeling and multiple-sequence alignments identified active site residues on the third, fourth, and fifth β-strands of the TIM-barrel catalytic domain that are differentially conserved within the substrate-diverse enzyme families. Using α-isopropylmalate synthase and citramalate synthase from Methanococcus jannaschii (MjIPMS and MjCMS), site-directed mutagenesis was used to test the role of each identified position in substrate selectivity. Kinetic data suggest that residues at the β3-5 and β4-7 positions play a significant role in the selection of α-ketoisovalerate over pyruvate in MjIPMS. However, complementary substitutions in MjCMS fail to alter substrate specificity, suggesting residues in these positions do not contribute to substrate selectivity in this enzyme. Analysis of the kinetic data with respect to a protein similarity network for the CC-like subgroup suggests that evolutionarily distinct forms of IPMS utilize residues at the β3-5 and β4-7 positions to affect substrate selectivity while the different versions of CMS use unique architectures. Importantly, mapping the identities of residues at the β3-5 and β4-7 positions onto the protein similarity network allows for rapid annotation of probable IPMS enzymes as well as several outlier sequences that may represent novel functions in the subgroup. PMID:26935545

  18. Proton nuclear Overhauser effect study of the heme active site structure of Coprinus macrorhizus peroxidase.

    PubMed

    Dugad, L B; Goff, H M

    1992-07-13

    Proton nuclear Overhauser effect and paramagnetic relaxation measurements have been used to define more extensively the heme active site structure of Coprinus macrorhizus peroxidase, CMP (previously known as Coprinus cinereus peroxidase), as the ferric low-spin cyanide ligated complex. The results are compared with other well-characterized peroxidase enzymes. The NMR spectrum of CMPCN shows changes in the paramagnetically shifted resonances as a function of time, suggesting a significant heme disorder for CMP. The presence of proximal and distal histidine amino acid residues are common to the heme environments of both CMPCN and HRPCN. However, the upfield distal arginine signals of HRPCN are not evident in the 1H-NMR spectra of CMPCN.

  19. Identification of the active-site serine in human lecithin: cholesterol acyltransferase

    SciTech Connect

    Farooqui, J.; Wohl, R.C.; Kezdy, F.J.; Scanu, A.M.

    1987-05-01

    Lecithin:cholesterol acyltransferase (LCAT) from human plasma reacts stoichiometrically with diisopropylphosphorofluoridate (DFP) resulting in the complete loss of transacylase activity. Purified LCAT was covalently labeled with (TH) DFP and the labeled protein was reduced and carboxymethylated. Cyanogen bromide cleavage followed by gel permeation chromatography yielded a peptide of 4-5 KDa (LCAT CNBr-III) containing most of the radioactive label. Preliminary studies comparing the amino acid composition of the LCAT-CNBr-III with the sequence of LCAT indicate that this peptide corresponds to fragment 168-220. Automated Edman degradation of the radioactive peptide recovered a radioactive PTC-amino acid at cycle 14. Of all predicted CNBr fragments only peptide 168-220 contained a serine at residue 14 from the amino terminus of the peptide. The authors conclude that serine 181 is the active site serine of LCAT.

  20. Cysteine S-conjugate β-lyases: Important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents

    PubMed Central

    Cooper, Arthur J. L.; Krasnikov, Boris F.; Niatsetskaya, Zoya V.; Pinto, John T.; Callery, Patrick S.; Villar, Maria T.; Artigues, Antonio; Bruschi, Sam A.

    2010-01-01

    Summary Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously [Cooper and Pinto, 2006]. Here we focus on more recent findings regarding: 1) the identification of enzymes associated with high-Mr cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; 2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); 3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; 4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and 5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated. PMID:20306345

  1. Cysteine S-conjugate β-lyases: important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents.

    PubMed

    Cooper, Arthur J L; Krasnikov, Boris F; Niatsetskaya, Zoya V; Pinto, John T; Callery, Patrick S; Villar, Maria T; Artigues, Antonio; Bruschi, Sam A

    2011-06-01

    Cysteine S-conjugate β-lyases are pyridoxal 5'-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1-15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M(r) cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated.

  2. A single mutation in the hepta-peptide active site of Aspergillus niger PhyA phytase leads to myriad of biochemical changes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The active site motif of proteins belonging to ‘Histidine Acid Phosphatase’ (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs has revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger phyA phytase. However,...

  3. Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families as revealed by active-site architecture analysis

    PubMed Central

    Tian, Li; Liu, Shijia; Wang, Shuai; Wang, Lushan

    2016-01-01

    Biomass can be converted into sugars by a series of lignocellulolytic enzymes, which belong to the glycoside hydrolase (GH) families summarized in CAZy databases. Here, using a structural bioinformatics method, we analyzed the active site architecture of the main lignocellulolytic enzyme families. The aromatic amino acids Trp/Tyr and polar amino acids Glu/Asp/Asn/Gln/Arg occurred at higher frequencies in the active site architecture than in the whole enzyme structure. And the number of potential subsites was significantly different among different families. In the cellulase and xylanase families, the conserved amino acids in the active site architecture were mostly found at the −2 to +1 subsites, while in β-glucosidase they were mainly concentrated at the −1 subsite. Families with more conserved binding amino acid residues displayed strong selectivity for their ligands, while those with fewer conserved binding amino acid residues often exhibited promiscuity when recognizing ligands. Enzymes with different activities also tended to bind different hydroxyl oxygen atoms on the ligand. These results may help us to better understand the common and unique structural bases of enzyme-ligand recognition from different families and provide a theoretical basis for the functional evolution and rational design of major lignocellulolytic enzymes. PMID:27009476

  4. Cysteine sensing by plasmons of silver nanocubes

    NASA Astrophysics Data System (ADS)

    Elfassy, Eitan; Mastai, Yitzhak; Salomon, Adi

    2016-09-01

    Noble metal nanoparticles are considered to be valuable nanostructures in the field of sensors due to their spectral response sensitivity to small changes in the surrounding refractive index which enables them to detect a small amount of molecules. In this research, we use silver nanocubes of about 50 nm length to detect low concentrations of cysteine, a semi-essential amino acid. Following cysteine adsorption onto the nanocubes, a redshift in the plasmonic modes was observed, enabling the detection of cysteine down to 10 μM and high sensitivity of about 125 nm/RIU (refractive index units). Furthermore, we found that multilayer adsorption of cysteine leads to the stabilization of the silver nanocubes. The cysteine growth onto the nanocubes was also characterized by high-resolution transmission electron microscopy (HR-TEM).

  5. Control of active sites in selective flocculation: I -- Mathematical model

    SciTech Connect

    Behl, S.; Moudgil, B.M.; Prakash, T.S. . Dept. of Materials Science and Engineering)

    1993-12-01

    Heteroflocculation has been determined to be another major reason for loss in selectivity for flocculation process. In a mathematical model developed earlier, conditions for controlling heteroflocculation were discussed. Blocking active sites to control selective adsorption of a flocculant oil a desirable solid surface is discussed. It has been demonstrated that the lower molecular weight fraction of a flocculant which is incapable of flocculating the particles is an efficient site blocking agent. The major application of selective flocculation has been in mineral processing but many potential uses exist in biological and other colloidal systems. These include purification of ceramic powders, separating hazardous solids from chemical waste, and removal of deleterious components from paper pulp.

  6. Catalytic stimulation by restrained active-site floppiness--the case of high density lipoprotein-bound serum paraoxonase-1.

    PubMed

    Ben-David, Moshe; Sussman, Joel L; Maxwell, Christopher I; Szeler, Klaudia; Kamerlin, Shina C L; Tawfik, Dan S

    2015-03-27

    Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168--a key catalytic residue residing >15Å from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design.

  7. Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

    PubMed Central

    Yi, Xianwen; Mroczko, Mark; Manoj, Kelath M.; Wang, Xiaotang; Hager, Lowell P.

    1999-01-01

    Chloroperoxidase is a versatile heme enzyme which can cross over the catalytic boundaries of other oxidative hemoproteins and perform multiple functions. Chloroperoxidase, in addition to catalyzing classical peroxidative reactions, also acts as a P450 cytochrome and a potent catalase. The multiple functions of chloroperoxidase must be derived from its unique active site structure. Chloroperoxidase possesses a proximal cysteine thiolate heme iron ligand analogous to the P450 cytochromes; however, unlike the P450 enzymes, chloroperoxidase possesses a very polar environment distal to its heme prosthetic group and contains a glutamic acid residue in close proximity to the heme iron. The presence of a thiolate ligand in chloroperoxidase has long been thought to play an essential role in its chlorination and epoxidation activities; however, the research reported in this paper proves that hypothesis to be invalid. To explore the role of Cys-29, the amino acid residue supplying the thiolate ligand in chloroperoxidase, Cys-29 has been replaced with a histidine residue. Mutant clones of the chloroperoxidase genome have been expressed in a Caldariomyces fumago expression system by using gene replacement rather than gene insertion technology. C. fumago produces wild-type chloroperoxidase, thus requiring gene replacement of the wild type by the mutant gene. To the best of our knowledge, this is the first time that gene replacement has been reported for this type of fungus. The recombinant histidine mutants retain most of their chlorination, peroxidation, epoxidation, and catalase activities. These results downplay the importance of a thiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse activities of this enzyme. PMID:10535936

  8. Identification and Quantification of S-Nitrosylation by Cysteine Reactive Tandem Mass Tag Switch Assay*

    PubMed Central

    Murray, Christopher I.; Uhrigshardt, Helge; O'Meally, Robert N.; Cole, Robert N.; Van Eyk, Jennifer E.

    2012-01-01

    Redox-switches are critical cysteine thiols that are modified in response to changes in the cell's environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT6, is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT6 quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 μm; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches. PMID:22126794

  9. Estimation of protein function using template-based alignment of enzyme active sites

    PubMed Central

    2014-01-01

    Background The accumulation of protein structural data occurs more rapidly than it can be characterized by traditional laboratory means. This has motivated widespread efforts to predict enzyme function computationally. The most useful/accurate strategies employed to date are based on the detection of motifs in novel structures that correspond to a specific function. Functional residues are critical components of predictively useful motifs. We have implemented a novel method, to complement current approaches, which detects motifs solely on the basis of distance restraints between catalytic residues. Results ProMOL is a plugin for the PyMOL molecular graphics environment that can be used to create active site motifs for enzymes. A library of 181 active site motifs has been created with ProMOL, based on definitions published in the Catalytic Site Atlas (CSA). Searches with ProMOL produce better than 50% useful Enzyme Commission (EC) class suggestions for level 1 searches in EC classes 1, 4 and 5, and produce some useful results for other classes. 261 additional motifs automatically translated from Jonathan Barker’s JESS motif set [Bioinformatics 19:1644–1649, 2003] and a set of NMR motifs is under development. Alignments are evaluated by visual superposition, Levenshtein distance and root-mean-square deviation (RMSD) and are reasonably consistent with related search methods. Conclusion The ProMOL plugin for PyMOL provides ready access to template-based local alignments. Recent improvements to ProMOL, including the expanded motif library, RMSD calculations and output selection formatting, have greatly increased the program’s usability and speed, and have improved the way that the results are presented. PMID:24669788

  10. Ligand-dependent dynamics of the active-site lid in bacterial dimethylarginine dimethylaminohydrolase.

    PubMed

    Rasheed, Masooma; Richter, Christine; Chisty, Liisa T; Kirkpatrick, John; Blackledge, Martin; Webb, Martin R; Driscoll, Paul C

    2014-02-18

    The dimethylarginine dimethylaminohydrolase (DDAH) enzyme family has been the subject of substantial investigation as a potential therapeutic target for the regulation of vascular tension. DDAH enzymes catalyze the conversion of asymmetric N(η),N(η)-dimethylarginine (ADMA) to l-citrulline. Here the influence of substrate and product binding on the dynamic flexibility of DDAH from Pseudomonas aeruginosa (PaDDAH) has been assessed. A combination of heteronuclear NMR spectroscopy, static and time-resolved fluorescence measurements, and atomistic molecular dynamics simulations was employed. A monodisperse monomeric variant of the wild-type enzyme binds the reaction product l-citrulline with a low millimolar dissociation constant. A second variant, engineered to be catalytically inactive by substitution of the nucleophilic Cys249 residue with serine, can still convert the substrate ADMA to products very slowly. This PaDDAH variant also binds l-citrulline, but with a low micromolar dissociation constant. NMR and molecular dynamics simulations indicate that the active site "lid", formed by residues Gly17-Asp27, exhibits a high degree of internal motion on the picosecond-to-nanosecond time scale. This suggests that the lid is open in the apo state and allows substrate access to the active site that is otherwise buried. l-Citrulline binding to both protein variants is accompanied by an ordering of the lid. Modification of PaDDAH with a coumarin fluorescence reporter allowed measurement of the kinetic mechanism of the PaDDAH reaction. A combination of NMR and kinetic data shows that the catalytic turnover of the enzyme is not limited by release of the l-citrulline product. The potential to develop the coumarin-PaDDAH adduct as an l-citrulline sensor is discussed. PMID:24484052

  11. Shifting redox states of the iron center partitions CDO between crosslink formation or cysteine oxidation.

    PubMed

    Njeri, Catherine W; Ellis, Holly R

    2014-09-15

    Cysteine dioxygenase (CDO) is a mononuclear iron-dependent enzyme that catalyzes the oxidation of L-cysteine to L-cysteine sulfinic acid. The mammalian CDO enzymes contain a thioether crosslink between Cys93 and Tyr157, and purified recombinant CDO exists as a mixture of the crosslinked and non crosslinked isoforms. The current study presents a method of expressing homogenously non crosslinked CDO using a cell permeative metal chelator in order to provide a comprehensive investigation of the non crosslinked and crosslinked isoforms. Electron paramagnetic resonance analysis of purified non crosslinked CDO revealed that the iron was in the EPR silent Fe(II) form. Activity of non crosslinked CDO monitoring dioxygen utilization showed a distinct lag phase, which correlated with crosslink formation. Generation of homogenously crosslinked CDO resulted in an ∼5-fold higher kcat/Km value compared to the enzyme with a heterogenous mixture of crosslinked and non crosslinked CDO isoforms. EPR analysis of homogenously crosslinked CDO revealed that this isoform exists in the Fe(III) form. These studies present a new perspective on the redox properties of the active site iron and demonstrate that a redox switch commits CDO towards either formation of the Cys93-Tyr157 crosslink or oxidation of the cysteine substrate.

  12. Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation.

    PubMed

    Rhee, S G; Bae, Y S; Lee, S R; Kwon, J

    2000-10-10

    Ligand-receptor interactions can generate the production of hydrogen peroxide (H(2)O(2)) in cells, the implications of which are becoming appreciated. Fluctuations in H(2)O(2) levels can affect the intracellular activity of key signaling components including protein kinases and protein phosphatases. Rhee et al. discuss recent findings on the role of H(2)O(2) in signal transduction. Specifically, H(2)O(2) appears to oxidize active site cysteines in phosphatases, thereby inactivating them. H(2)O(2) also can activate protein kinases; however, although the mechanism of activation for some kinases appears to be similar to that of phosphatase inactivation (cysteine oxidation), it is unclear how H(2)O(2) promotes increased activation of other kinases. Thus, the higher levels of intracellular phosphoproteins observed in cells most likely occur because of the concomitant inhibition of protein phosphatases and activation of protein kinases.

  13. Femtomolar Zn(II) affinity in a peptide-based ligand designed to model thiolate-rich metalloprotein active sites.

    PubMed

    Petros, Amy K; Reddi, Amit R; Kennedy, Michelle L; Hyslop, Alison G; Gibney, Brian R

    2006-12-11

    Metal-ligand interactions are critical components of metalloprotein assembly, folding, stability, electrochemistry, and catalytic function. Research over the past 3 decades on the interaction of metals with peptide and protein ligands has progressed from the characterization of amino acid-metal and polypeptide-metal complexes to the design of folded protein scaffolds containing multiple metal cofactors. De novo metalloprotein design has emerged as a valuable tool both for the modular synthesis of these complex metalloproteins and for revealing the fundamental tenets of metalloprotein structure-function relationships. Our research has focused on using the coordination chemistry of de novo designed metalloproteins to probe the interactions of metal cofactors with protein ligands relevant to biological phenomena. Herein, we present a detailed thermodynamic analysis of Fe(II), Co(II), Zn(II), and[4Fe-4S]2(+/+) binding to IGA, a 16 amino acid peptide ligand containing four cysteine residues, H2N-KLCEGG-CIGCGAC-GGW-CONH2. These studies were conducted to delineate the inherent metal-ion preferences of this unfolded tetrathiolate peptide ligand as well as to evaluate the role of the solution pH on metal-peptide complex speciation. The [4Fe-4S]2(+/+)-IGA complex is both an excellent peptide-based synthetic analogue for natural ferredoxins and is flexible enough to accommodate mononuclear metal-ion binding. Incorporation of a single ferrous ion provides the FeII-IGA complex, a spectroscopic model of a reduced rubredoxin active site that possesses limited stability in aqueous buffers. As expected based on the Irving-Williams series and hard-soft acid-base theory, the Co(II) and Zn(II) complexes of IGA are significantly more stable than the Fe(II) complex. Direct proton competition experiments, coupled with determinations of the conditional dissociation constants over a range of pH values, fully define the thermodynamic stabilities and speciation of each MII-IGA complex. The

  14. Druggability analysis and classification of protein tyrosine phosphatase active sites

    PubMed Central

    Ghattas, Mohammad A; Raslan, Noor; Sadeq, Asil; Al Sorkhy, Mohammad; Atatreh, Noor

    2016-01-01

    Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability. PMID:27757011

  15. Supported oligomethionine sulfoxide and Ellman's reagent for cysteine bridges formation.

    PubMed

    Ronga, Luisa; Verdié, Pascal; Sanchez, Pierre; Enjabal, Christine; Maurras, Amélie; Jullian, Magalie; Puget, Karine; Martinez, Jean; Subra, Gilles

    2013-02-01

    A large number of bioactive peptides are cyclized through a disulfide bridge. This structural feature is very important for both bioactivity and stability. The oxidation of cysteine side chains is challenging not only to avoid intermolecular reaction leading to oligomers and oxidation of other residues but also to remove solvents and oxidant such as dimethyl sulfoxide. Supported reagents advantageously simplify the work-up of such disulfide bond formation, but may lead to a significant decrease in yield of the oxidized product. In this study, two resins working through different mechanisms were evaluated: Clear-Ox, a supported version of Ellman's reagent and Oxyfold, consisting in a series of oxidized methionine residues. The choice of the supported reagent is discussed on the light of reaction speed, side-products formation and yield considerations.

  16. Non-empirical analysis of the nature of the inhibitor active-site interactions in leucine aminopeptidase

    NASA Astrophysics Data System (ADS)

    Grembecka, J.; K ȩdzierski, P.; Sokalski, W. A.

    1999-11-01

    Non-empirical analysis of the physical nature of the intermolecular interactions between several leucine aminopeptidase inhibitors and various constituents of the enzyme active site has been performed using a direct version of the hybrid variation-perturbation decomposition of SCF and MP2 interaction energies. The interaction energy terms obtained at different theory levels have been correlated with experimentally measured activities of the inhibitors, indicating that the more advanced the quantum-chemical method and, the larger the active-site model, the better is the correlation between calculated and measured binding energies. The electrostatic multipole term constitutes the dominant contribution in the total interaction energy, whereas Zn 2+488 and Lys +262 enzyme residues play the crucial role in the binding of these inhibitors by leucine aminopeptidase.

  17. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

    PubMed

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

  18. S-sulfhydration: a cysteine posttranslational modification in plant systems.

    PubMed

    Aroca, Ángeles; Serna, Antonio; Gotor, Cecilia; Romero, Luis C

    2015-05-01

    Hydrogen sulfide is a highly reactive molecule that is currently accepted as a signaling compound. This molecule is as important as carbon monoxide in mammals and hydrogen peroxide in plants, as well as nitric oxide in both eukaryotic systems. Although many studies have been conducted on the physiological effects of hydrogen sulfide, the underlying mechanisms are poorly understood. One of the proposed mechanisms involves the posttranslational modification of protein cysteine residues, a process called S-sulfhydration. In this work, a modified biotin switch method was used for the detection of Arabidopsis (Arabidopsis thaliana) proteins modified by S-sulfhydration under physiological conditions. The presence of an S-sulfhydration-modified cysteine residue on cytosolic ascorbate peroxidase was demonstrated using liquid chromatography-tandem mass spectrometry analysis, and a total of 106 S-sulfhydrated proteins were identified. Immunoblot and enzyme activity analyses of some of these proteins showed that the sulfide added through S-sulfhydration reversibly regulates the functions of plant proteins in a manner similar to that described in mammalian systems.

  19. Salt Effect Accelerates Site-Selective Cysteine Bioconjugation

    PubMed Central

    2016-01-01

    Highly efficient and selective chemical reactions are desired. For small molecule chemistry, the reaction rate can be varied by changing the concentration, temperature, and solvent used. In contrast for large biomolecules, the reaction rate is difficult to modify by adjusting these variables because stringent biocompatible reaction conditions are required. Here we show that adding salts can change the rate constant over 4 orders of magnitude for an arylation bioconjugation reaction between a cysteine residue within a four-residue sequence (π-clamp) and a perfluoroaryl electrophile. Biocompatible ammonium sulfate significantly enhances the reaction rate without influencing the site-specificity of π-clamp mediated arylation, enabling the fast synthesis of two site-specific antibody–drug conjugates that selectively kill HER2-positive breast cancer cells. Computational and structure–reactivity studies indicate that salts may tune the reaction rate through modulating the interactions between the π-clamp hydrophobic side chains and the electrophile. On the basis of this understanding, the salt effect is extended to other bioconjugation chemistry, and a new regioselective alkylation reaction at π-clamp cysteine is developed. PMID:27725962

  20. S-sulfhydration: a cysteine posttranslational modification in plant systems.

    PubMed

    Aroca, Ángeles; Serna, Antonio; Gotor, Cecilia; Romero, Luis C

    2015-05-01

    Hydrogen sulfide is a highly reactive molecule that is currently accepted as a signaling compound. This molecule is as important as carbon monoxide in mammals and hydrogen peroxide in plants, as well as nitric oxide in both eukaryotic systems. Although many studies have been conducted on the physiological effects of hydrogen sulfide, the underlying mechanisms are poorly understood. One of the proposed mechanisms involves the posttranslational modification of protein cysteine residues, a process called S-sulfhydration. In this work, a modified biotin switch method was used for the detection of Arabidopsis (Arabidopsis thaliana) proteins modified by S-sulfhydration under physiological conditions. The presence of an S-sulfhydration-modified cysteine residue on cytosolic ascorbate peroxidase was demonstrated using liquid chromatography-tandem mass spectrometry analysis, and a total of 106 S-sulfhydrated proteins were identified. Immunoblot and enzyme activity analyses of some of these proteins showed that the sulfide added through S-sulfhydration reversibly regulates the functions of plant proteins in a manner similar to that described in mammalian systems. PMID:25810097

  1. Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase

    PubMed Central

    2015-01-01

    The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity. PMID:24517311

  2. Coulombic effects of remote subsites on the active site of ribonuclease A.

    PubMed

    Fisher, B M; Schultz, L W; Raines, R T

    1998-12-15

    The active-site cleft of bovine pancreatic ribonuclease A (RNase A) is lined with cationic residues that interact with a bound nucleic acid. Those residues interacting with the phosphoryl groups comprise the P0, P1, and P2 subsites, with the scissile P-O5' bond residing in the P1 subsite. Coulombic interactions between the P0 and P2 subsites and phosphoryl groups of the substrate were characterized previously [Fisher, B. M., Ha, J.-H., and Raines, R. T. (1998) Biochemistry 37, 12121-12132]. Here, the interactions between these subsites and the active-site residues His12 and His119 are described in detail. A protein variant in which the cationic residues in these subsites (Lys66 in the P0 subsite and Lys7 and Arg10 in the P2 subsite) were replaced with alanine was crystallized, both free and with bound 3'-uridine monophosphate (3'-UMP). Structures of K7A/R10A/K66A RNase A and the K7A/R10A/K66A RNase A.3'-UMP complex were determined by X-ray diffraction analysis to resolutions of 2.0 and 2.1 A, respectively. There is little observable change between these structures and that of wild-type RNase A, either free or with bound 3'-cytidine monophosphate. K7A/R10A/K66A RNase A was evaluated for its ability to cleave UpA, a dinucleotide substrate that does not span the P0 or the P2 subsites. In comparison to the wild-type enzyme, the value of kcat was decreased by 5-fold and that of kcat/Km was decreased 10-fold, suggesting that these remote subsites interact with the active site. These interactions were characterized by determining the pKa values of His12 and His119 at 0.018 and 0.142 M Na+, both in wild-type RNase A and the K7A/R10A/K66A variant. The side chains of Lys7, Arg10, and Lys66 depress the pKa values of these histidine residues, and this depression is sensitive to the salt concentration. In addition, the P0 and P2 subsites influence the interaction of His12 and His119 with each other, as demonstrated by changes in the cooperativity that gives rise to microscopic

  3. Hydride binding to the active site of [FeFe]-hydrogenase.

    PubMed

    Chernev, Petko; Lambertz, Camilla; Brünje, Annika; Leidel, Nils; Sigfridsson, Kajsa G V; Kositzki, Ramona; Hsieh, Chung-Hung; Yao, Shenglai; Schiwon, Rafael; Driess, Matthias; Limberg, Christian; Happe, Thomas; Haumann, Michael

    2014-11-17

    [FeFe]-hydrogenase from green algae (HydA1) is the most efficient hydrogen (H2) producing enzyme in nature and of prime interest for (bio)technology. Its active site is a unique six-iron center (H-cluster) composed of a cubane cluster, [4Fe4S]H, cysteine-linked to a diiron unit, [2Fe]H, which carries unusual carbon monoxide (CO) and cyanide ligands and a bridging azadithiolate group. We have probed the molecular and electronic configurations of the H-cluster in functional oxidized, reduced, and super-reduced or CO-inhibited HydA1 protein, in particular searching for intermediates with iron-hydride bonds. Site-selective X-ray absorption and emission spectroscopy were used to distinguish between low- and high-spin iron sites in the two subcomplexes of the H-cluster. The experimental methods and spectral simulations were calibrated using synthetic model complexes with ligand variations and bound hydride species. Distinct X-ray spectroscopic signatures of electronic excitation or decay transitions in [4Fe4S]H and [2Fe]H were obtained, which were quantitatively reproduced by density functional theory calculations, thereby leading to specific H-cluster model structures. We show that iron-hydride bonds are absent in the reduced state, whereas only in the super-reduced state, ligand rotation facilitates hydride binding presumably to the Fe-Fe bridging position at [2Fe]H. These results are in agreement with a catalytic cycle involving three main intermediates and at least two protonation and electron transfer steps prior to the H2 formation chemistry in [FeFe]-hydrogenases. PMID:25369169

  4. Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.

    PubMed

    Pandey, Alok; Gordon, Donna M; Pain, Jayashree; Stemmler, Timothy L; Dancis, Andrew; Pain, Debkumar

    2013-12-27

    For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the "buried" substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.

  5. Cysteine Proteases from Bloodfeeding Arthropod Ectoparasites

    PubMed Central

    Sojka, Daniel; Francischetti, Ivo M. B.; Calvo, Eric; Kotsyfakis, Michalis

    2012-01-01

    Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion, and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells. PMID:21660665

  6. Blends of cysteine-containing proteins

    NASA Astrophysics Data System (ADS)

    Barone, Justin

    2005-03-01

    Many agricultural wastes are made of proteins such as keratin, lactalbumin, gluten, and albumin. These proteins contain the amino acid cysteine. Cysteine allows for the formation of inter-and intra-molecular sulfur-sulfur bonds. Correlations are made between the properties of films made from the proteins and the amino acid sequence. Blends of cysteine-containing proteins show possible synergies in physical properties at intermediate concentrations. FT-IR spectroscopy shows increased hydrogen bonding at intermediate concentrations suggesting that this contributes to increased physical properties. DSC shows limited miscibility and the formation of new crystalline phases in the blends suggesting that this too contributes.

  7. Part I. Cobalt thiolate complexes modeling the active site of cobalt nitrile hydratase. Part II. Formation of inorganic nanoparticles on protein scaffolding in Escherichia coli glutamine synthetase

    NASA Astrophysics Data System (ADS)

    Kung, Irene Yuk Man

    Part I. A series of novel cobalt dithiolate complexes with mixed imine/amine ligand systems is presented here as electronic and structural models for the active site in the bacterial enzyme class, nitrile hydratase (NHase). Pentadentate cobalt(II) complexes with S2N 3 ligand environments are first studied as precursors to the more relevant cobalt(III) complexes. Adjustment of the backbone length by removal of a methylene group increases the reactivity of the system; whereas reduction of the two backbone imine bonds to allow free rotation about those bonds may decrease reactivity. Reactivity change due to the replacement of the backbone amine proton with a more sterically challenging methyl group is not yet clear. Upon oxidation, the monocationic pentadentate cobalt(III) complex, 1b, shows promising reactivity similar to that of NHase. The metal's open coordination site allows reversible binding of the endogenous, monoanionic ligands, N 3- and NCS-. Oxygenation of the thiolate sulfur atoms by exposure to O2 and H2O 2 produces sulfenate and sulfinate ligands in complex 8, which resembles the crystal structure of "deactivated" Fe NHase. However, its lack of reactivity argues against the oxygenated enzyme structure as the active form. Six-coordinate cobalt(III) complexes with S2N4 amine/amine ligand systems are also presented as analogues of previously reported iron(III) compounds, which mimic the spectroscopic properties of Fe NHase. The cobalt complexes do not seem to similarly model Co NHase. However, the S = 0 cobalt(III) center can be spectroscopically silent and difficult to detect, making comparison with synthetic models using common techniques hard. Part II. Dodecameric Escherichia coli glutamine synthetase mutant, E165C, stacks along its six-fold axis to produce tubular nanostructures in the presence of some divalent metal ions, as does the wild type enzyme. The centrally located, engineered Cys-165 residues appear to bind to various species and may serve as

  8. Studies on the active site of pig plasma amine oxidase.

    PubMed Central

    Collison, D; Knowles, P F; Mabbs, F E; Rius, F X; Singh, I; Dooley, D M; Cote, C E; McGuirl, M

    1989-01-01

    Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity. PMID:2559715

  9. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  10. Metavanadate at the active site of the phosphatase VHZ.

    PubMed

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  11. Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

    SciTech Connect

    Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

    1986-05-01

    Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with (/sup 3/H)-NaBH/sub 4/, and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P.

  12. Distinguishing two groups of flavin reductases by analyzing the protonation state of an active site carboxylic acid.

    PubMed

    Dumit, Verónica I; Cortez, Néstor; Matthias Ullmann, G

    2011-07-01

    Flavin-containing reductases are involved in a wide variety of physiological reactions such as photosynthesis, nitric oxide synthesis, and detoxification of foreign compounds, including therapeutic drugs. Ferredoxin-NADP(H)-reductase (FNR) is the prototypical enzyme of this family. The fold of this protein is highly conserved and occurs as one domain of several multidomain enzymes such as the members of the diflavin reductase family. The enzymes of this family have emerged as fusion of a FNR and a flavodoxin. Although the active sites of these enzymes are very similar, different enz