Structural Basis for the Reaction Mechanism of S-Carbamoylation of HypE by HypF in the Maturation of [NiFe]-Hydrogenases

As a remarkable structural feature of hydrogenase active sites, [NiFe]-hydrogenases harbor one carbonyl and two cyano ligands, where HypE and HypF are involved in the biosynthesis of the nitrile group as a precursor of the cyano groups. HypF catalyzes S-carbamoylation of the C-terminal cysteine of HypE via three steps using carbamoylphosphate and ATP, producing two unstable intermediates: carbamate and carbamoyladenylate. Although the crystal structures of intact HypE homodimers and partial HypF have been reported, it remains unclear how the consecutive reactions occur without the loss of unstable intermediates during the proposed reaction scheme. Here we report the crystal structures of full-length HypF both alone and in complex with HypE at resolutions of 2.0 and 2.6 Å, respectively. Three catalytic sites of the structures of the HypF nucleotide- and phosphate-bound forms have been identified, with each site connected via channels inside the protein. This finding suggests that the first two consecutive reactions occur without the release of carbamate or carbamoyladenylate from the enzyme. The structure of HypF in complex with HypE revealed that HypF can associate with HypE without disturbing its homodimeric interaction and that the binding manner allows the C-terminal Cys-351 of HypE to access the S-carbamoylation active site in HypF, suggesting that the third step can also proceed without the release of carbamoyladenylate. A comparison of the structure of HypF with the recently reported structures of O-carbamoyltransferase revealed different reaction mechanisms for carbamoyladenylate synthesis and a similar reaction mechanism for carbamoyltransfer to produce the carbamoyl-HypE molecule.     

The complex between hydrogenase-maturation proteins HypC and HypD is an intermediate in the supply of cyanide to the active site iron of [NiFe]-hydrogenases

Carbamoylphosphate has been shown to be the educt for the synthesis of the CN ligands of the NiFe metal centre of hydrogenases from Escherichia coli. In the absence of carbamoylphosphate, cells accumulate a complex of two hydrogenase maturation proteins, namely HypC and HypD for the synthesis of hydrogenase 3. A procedure for the purification of wild-type HypD protein or of a biologically active derivative carrying the Strep-tagII((R)) at the N terminus has been developed. HypD is a monomeric protein possessing about 4 mol of iron per mol of protein. Electron paramagnetic resonance (EPR) and Mossbauer spectroscopy demonstrated that the iron is present as a diamagnetic [4Fe-4S](2+) cluster. The complex between HypC and HypD can be cross-linked by a number of thiol and primary amine-specific linkers. When HypD and HypC were overproduced side-by-side with HypE, the HypC-HypD complex contained substoichiometric amounts of HypE whose proportion in the complex could be augmented when HypF was also overproduced. HypE trapped in this complex could be carbamoylated by protein HypF and after dehydration transferred the cyano group to the HypC-HypD part of the complex. Free HypC and HypD were not cyanated by HypE-CN. An active HypC-HypD complex from anaerobic cells was inactivated by incubation with K(3)[Fe(CN)(6)] but not with K(4)[Fe(CN)(6)]. The results suggest the existence of a dynamic complex between the hydrogenase maturation proteins HypD, HypC, HypE and HypF, which is the site of ligand biosynthesis and attachment to the iron atom of the NiFe site in hydrogenase 3.     

Crystal structures of [NiFe] hydrogenase maturation proteins HypC, HypD, and HypE: insights into cyanation reaction by thiol redox signaling

[NiFe] hydrogenase maturation proteins HypC, HypD, and HypE catalyze the insertion and cyanation of the iron center of [NiFe] hydrogenases by an unknown mechanism. We have determined the crystal structures of HypC, HypD, and HypE from Thermococcus kodakaraensis KOD1 at 1.8 A, 2.07 A, and 1.55 A resolution, respectively. The structure of HypD reveals its probable iron binding and active sites for cyanation. An extended conformation of each conserved motif of HypC and HypE allows the essential cysteine residues of both proteins to interact with the active site of HypD. Furthermore, the C-terminal tail of HypE is shown to exist in an ATP-dependent dynamic equilibrium between outward and inward conformations. Unexpectedly, the [4Fe-4S] cluster environment of HypD is quite similar to that of ferredoxin:thioredoxin reductase (FTR), indicating the existence of a redox cascade similar to the FTR system. These results suggest a cyanation reaction mechanism via unique thiol redox signaling in the HypCDE complex.     

Analysis of the transcarbamoylation-dehydration reaction catalyzed by the hydrogenase maturation proteins HypF and HypE.

The hydrogenase maturation proteins HypF and HypE catalyze the synthesis of the CN ligands of the active site iron of the NiFe-hydrogenases using carbamoylphosphate as a substrate. HypE protein from Escherichia coli was purified from a transformant overexpressing the hypE gene from a plasmid. Purified HypE in gel filtration experiments behaves predominantly as a monomer. It does not contain statistically significant amounts of metals or of cofactors absorbing in the UV and visible light range. The protein displays low intrinsic ATPase activity with ADP and phosphate as the products, the apparent K(m) being 25 micro m and the k(cat) 1.7 x 10(-3) s(-1). Removal of the C-terminal cysteine residue of HypE which accepts the carbamoyl moiety from HypF affected the K(m) (47 micro m) but not significantly the k(cat) (2.1 x 10(-3) s(-1)). During the carbamoyltransfer reaction, HypE and HypF enter a complex which is rather tight at stoichiometric ratios of the two proteins. A mutant HypE variant was generated by amino acid replacements in the nucleoside triphosphate binding region, which showed no intrinsic ATPase activity. The variant was active as an acceptor in the transcarbamoylation reaction but did not dehydrate the thiocarboxamide to the thiocyanate. The results obtained with the HypE variants and also with mutant HypF forms are integrated to explain the complex reaction pattern of protein HypF.     

Structure of [NiFe] hydrogenase maturation protein HypE from Escherichia coli and its interaction with HypF

Hydrogenases are enzymes involved in hydrogen metabolism, utilizing H₂ as an electron source. [NiFe] hydrogenases are heterodimeric Fe-S proteins, with a large subunit containing the reaction center involving Fe and Ni metal ions and a small subunit containing one or more Fe-S clusters. Maturation of the [NiFe] hydrogenase involves assembly of nonproteinaceous ligands on the large subunit by accessory proteins encoded by the hyp operon. HypE is an essential accessory protein and participates in the synthesis of two cyano groups found in the large subunit. We report the crystal structure of Escherichia coli HypE at 2.0-Å resolution. HypE exhibits a fold similar to that of PurM and ThiL and forms dimers. The C-terminal catalytically essential Cys336 is internalized at the dimer interface between the N- and C-terminal domains. A mechanism for dehydration of the thiocarbamate to the thiocyanate is proposed, involving Asp83 and Glu272. The interactions of HypE and HypF were characterized in detail by surface plasmon resonance and isothermal titration calorimetry, revealing a K_d (dissociation constant) of ~400 nM. The stoichiometry and molecular weights of the complex were verified by size exclusion chromatography and gel scanning densitometry. These experiments reveal that HypE and HypF associate to form a stoichiometric, hetero-oligomeric complex predominantly consisting of a [EF]₂ heterotetramer which exists in a dynamic equilibrium with the EF heterodimer. The surface plasmon resonance results indicate that a conformational change occurs upon heterodimerization which facilitates formation of a productive complex as part of the carbamate transfer reaction.     

Structural studies of thiamin monophosphate kinase in complex with substrates and products

Thiamin monophosphate kinase (ThiL) catalyzes the ATP-dependent phosphorylation of thiamin monophosphate (TMP) to form thiamin pyrophosphate (TPP), the active form of vitamin B 1. ThiL is a member of a small ATP binding superfamily that also includes the purine biosynthetic enzymes, PurM and PurL, NiFe hydrogenase maturation protein, HypE, and selenophosphate synthase, SelD. The latter four enzymes are believed to utilize phosphorylated intermediates during catalysis. To understand the mechanism of ThiL and its relationship to the other superfamily members, we determined the structure of Aquifex aeolicus ThiL (AaThiL) with nonhydrolyzable AMP-PCP and TMP, and also with the products of the reaction, ADP and TPP. The results suggest that AaThiL utilizes a direct, inline transfer of the gamma-phosphate of ATP to TMP rather than a phosphorylated enzyme intermediate. The structure of ThiL is compared to those of PurM, PurL, and HypE, and the ATP binding site is compared to that of PurL, for which nucleotide complexes are available.     

NiFe hydrogenase active site biosynthesis: identification of Hyp protein complexes in Ralstonia eutropha

Biosynthesis of the NiFe hydrogenase active site is a complex process involving the action of the Hyp proteins: HypA-HypF. Here we investigate the mechanism of NiFe site biosynthesis in Ralstonia eutropha by examining the interactions between HypC, HypD, HypE, and HypF1. Using an affinity purification procedure based on the Strep-tag II, we purified HypC and HypE from different genetic backgrounds as complexes with other hydrogenase-related proteins and characterized them using immunological analysis. Copurification of HypC and HoxH, the active site-containing subunit of the soluble hydrogenase in R. eutropha, from several different genetic backgrounds suggests that this complex forms early in the maturation process. With respect to the Hyp proteins, it is shown that HypE and HypF1 formed a stable complex both in vivo and in vitro. Furthermore, HypC and HypD functioned as a unit. Together, they were able to interact with HypE to form a range of complexes probably varying in stoichiometry. The HypC/HypD/HypE complexes did not involve HypF1 but appeared to be more stable when HypF1 was also present in the cells. We hypothesize that HypF1 is able to modify some component of the HypC/HypD/HypE complex. Since we have also seen that HypF1 and HypE form a complex, it is likely that HypF1 modifies HypE. On the basis of these results, we propose a complete catalytic cycle for HypE. First, it is modified by HypF1, and then it can form a complex with HypC/HypD. This activated HypE/HypC/HypD complex could then decompose by donating active site components to the immature hydrogenase and regenerate unmodified HypE.     

Chemical Modification of Catalytically Essential Functional Groups of NAD-Dependent Hydrogenase from Ralstonia eutropha H16

Amino acid residues His and Cys of the NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha H16 were chemically modified with specific reagents. The modification of His residues of the nonactivated hydrogenase resulted in decrease in both hydrogenase and diaphorase activities of the enzyme. Activation of NADH hydrogenase under anaerobic conditions additionally modified a His residue (or residues) significant only for the hydrogenase activity. The rate of decrease in the diaphorase activity was unchanged. The modification of thiol groups of the nonactivated enzyme did not affect the hydrogenase activity. The effect of thiol-modifying agents on the activated hydrogenase was accompanied by inactivation of both diaphorase and hydrogenase activities. The modification degree and changes in the corresponding catalytic activities depended on conditions of the enzyme activation. Data on the modification of cysteine and histidine residues of the hydrogenase suggested that the enzyme activation should be associated with significant conformational changes in the protein globule.     

Measurements of cysteine reactivity during protein unfolding suggest the presence of competing pathways

Evidence that proteins may unfold utilizing complex competing pathways comes from a new pulse-labeling protocol in which the change in reactivity of a single cysteine residue in a protein during unfolding is measured, making use of its easily monitored reaction with the Ellman reagent, dithionitrobenzoic acid. The kinetics of unfolding of two single cysteine-containing mutant forms of the small protein barstar, C82A, which contains only Cys40, and C40A, which contains only Cys82, have been studied. The data suggest that unfolding occurs via two parallel pathways, each forming competing intermediates. In one of these early intermediates, Cys40 and Cys82 are already as reactive as they are in the fully unfolded protein, while in the other intermediate, the Cys thiol groups are unreactive. One more long-lived intermediate also needs to be included on the pathway defined by the early intermediate with unreactive Cys thiol groups to account for the difference in the rates of fluorescence change and of change in Cys40 reactivity. The demonstration of multiple intermediates and pathways for unfolding indicates that protein unfolding reactions can be as complex as protein folding reactions.     

Microenvironment of cysteine 242 in type-1 ribosome-inactivating protein from iris

IRIP is a type-1 ribosome-inactivating protein isolated from the bulbs of Iris hollandica. It is one of the few type-1 RIPs that contain Cys residue(s) in their primary sequence. IRIP contains a single Cys residue at position 242. Although IRIP is thought to be a monomeric protein, SDS-PAGE indicates that part of the IRIP molecules can exist as disulphide bridge-linked dimers. Probing of the reactivity of the unique Cys residue by 5, 5'-dithiobis(2-nitrobenzoic acid) indicates that Cys(242) in IRIP is free but is only partially accessible to modifiers. Molecular modelling of IRIP is in agreement with this conclusion. Binding of the ligands adenine and poly(A) results in little or no effect on the conformation of Cys(242) in IRIP. Chemical modification of IRIP by a specific thiol modifier does not abolish the RNA N-glycosidase activity of IRIP, suggesting that Cys(242) is not critical for the enzymatic activity of IRIP. These results suggest that IRIP has the potential to be developed as a novel immunotoxin.     

Escherichia coli mutant SELD enzymes. The cysteine 17 residue is essential for selenophosphate formation from ATP and selenide.

Synthesis of a labile selenium donor compound, selenophosphate, from selenide and ATP by the Escherichia coli SELD enzyme was reported previously from this laboratory. From the gene sequence, SELD is a 37-kDa protein that contains 7 cysteine residues, 2 of which are located at positions 17 and 19 in the sequence -Gly-Ala-Cys-Gly-Cys-Lys-Ile- (Leinfelder, W., Forchhammer, K., Veprek, B., Zehelein, E., and B?ck, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 73, 543-547). Inactivation of the enzyme by alkylation with iodoacetamide indicated that at least 1 cysteine residue in the protein is essential for enzyme activity. To test the possibility that the Cys17 and/or Cys19 residue might be essential, these were changed to serine residues by site-specific mutagenesis. The biological activities of the wild type and mutant proteins were studied using E. coli MB08 (selD-) transformed with plasmids containing the selD genes. The plasmid containing the Cys17-mutated gene failed to complement MB08, whereas the Cys19-mutated gene was indistinguishable from wild type. The mutant proteins, like the wild type enzyme, bound to an ATP-agarose matrix, showing that their affinities for ATP were unimpaired. Selenide-dependent formation of AMP from ATP was abolished by mutation of Cys17, but the Cys19 mutation had no effect on the ability of the enzyme to catalyze the reaction. These results indicate that Cys17 has an essential role in the catalytic process that leads to the formation of selenophosphate from ATP and selenide.     

Characterization of the activity and folding of the glutathione transferase from Escherichia coli and the roles of residues Cys(10) and His(106)

GSTs (glutathione transferases) are an important class of enzymes involved in cellular detoxification. GSTs are found in all classes of organisms and are implicated in resistance towards drugs, pesticides, herbicides and antibiotics. The activity, structure and folding, particularly of eukaryotic GSTs, have therefore been widely studied. The crystal structure of EGST (GST from Escherichia coli) was reported around 10 years ago and it suggested Cys(10) and His(106) as potential catalytic residues. However, the role of these residues in catalysis has not been further investigated, nor have the folding properties of the protein been described. In the present study we investigated the contributions of residues Cys(10) and His(106) to the activity and stability of EGST. We found that EGST shows a complex equilibrium unfolding profile, involving a population of at least two partially folded intermediates, one of which is dimeric. Mutation of residues Cys(10) and His(106) leads to stabilization of the protein and affects the apparent steady-state kinetic parameters for enzyme catalysis. The results suggest that the imidazole ring of His(106) plays an important role in the catalytic mechanism of the enzyme, whereas Cys(10) is involved in binding of the substrate, glutathione. Engineering of the Cys(10) site can be used to increase both the stability and GST activity of EGST. However, in addition to GST activity, we discovered that EGST also possesses thiol:disulfide oxidoreductase activity, for which the residue Cys(10) plays an essential role. Further, tryptophan quenching experiments indicate that a mixed disulfide is formed between the free thiol group of Cys(10) and the substrate, glutathione.     

Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila,with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.     

Analysis of the HypC-hycE complex, a key intermediate in the assembly of the metal center of the Escherichia coli hydrogenase 3

The formation of a complex between the specific chaperone-type protein HypC and the precursor form of the large subunit HycE in the maturation pathway of hydrogenase 3 from Escherichia coli has been studied by targeted replacement of amino acids in both proteins. HypC and its homologs contain the motif MC(L/I/V)(G/A)(L/I/V)P at the amino terminus, from which the methionine residue is post-translationally removed. The exchange of the cysteine residue led to complete loss of the ability to interact with the precursor form of HycE, but replacement of the proline residue had no effect. Site-directed replacement of the conserved cysteine residues in HycE involved in nickel binding was also performed. Exchange of Cys(241) resulted in the inability of the HycE variant to interact with HypC and to incorporate nickel. The variants of HycE in which Cys(244) and Cys(531) were replaced by alanine residues were unable to incorporate nickel, although the mutated proteins could interact with HypC. Intriguingly, the precursor of HycE in which the Cys(534) residue was exchanged could form the complex with HypC, could incorporate nickel, and was C-terminally processed, but it delivered an inactive enzyme. Our findings are in favor of a model in which binding of HypC masks Cys(241); Cys(244) and Cys(531) bind the iron and nickel moieties, respectively; and C534 closes the bridge between the two metals after C-terminal processing has taken place.     

Highly conserved cysteines of mouse core 2 beta1,6-N-acetylglucosaminyltransferase I form a network of disulfide bonds and include a thiol that affects enzyme activity

Core 2 beta1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59-Cys413, Cys100-Cys172, Cys151-Cys199, and Cys372-Cys381. The only non-conserved residue within the beta1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys217 --> Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage beta-glucosyltransferase fold.     

Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains.

Exposed thiols act as intracellular retention elements for unassembled secretory molecules. Yet, some free Ig lambda light chains are secreted despite the presence of an unpaired cysteine (Cys214). This is due largely to the presence of a flanking acidic residue: substitution of Asp213 for Gly or Lys increases pre-Golgi retention and degradation of free lambda. Secretion is restored by exogenous reducing agents or by assembly with heavy chains. In the endoplasmic reticulum (ER), lambda chains form covalent complexes with many proteins through Cys214. These complexes are absent from the Golgi. They are more abundant in transfectants expressing the lambdaGly2I3 and lambdaLys213 mutants that are poorly secreted. Radioactive N-ethylmaleimide labels some monomeric lambda chains isolated from the ER, but not from the Golgi or from the medium, indicating that the Cys214 thiol is masked during ER-Golgi transport. Mass spectrometry reveals the presence of a free cysteine residue disulfide-linked to Cys214. We suggest that thiol-mediated retention involves the formation of reversible disulfide bonds with the protein matrix of the ER. The presence of an acidic residue next to the critical cysteine may allow the masking of the thiol and transport to the Golgi.     

Structure of human thioredoxin exhibits a large conformational change

Thioredoxin is an oxidoreductase, which is ubiquitously present across phyla from humans to plants and bacteria. Thioredoxin reduces a variety of substrates through active site Cys 32, which is subsequently oxidized to form the intramolecular disulphide with Cys 35. The thioredoxin fold is known to be highly stable and conformational changes in the active site loops and residues Cys 32, Cys 35 have been characterized between ligand bound and free structures. We have determined a novel 2.0 Å resolution crystal structure for a human thioredoxin, which reveals a much larger conformational change than previously characterized. The principal change involves unraveling of a helix to form an extended loop that is linked to secondary changes in further loop regions and the wider area of the active site Cys 32. This gives rise to a more open conformation and an elongated hydrophobic pocket results in place of the helix. Buried residue Cys 62 from this helix becomes exposed in the open conformation. This provides a structural basis for observations that the Cys 62 sidechain can form mixed disulphides and be modified by thiol reactive small molecules.     

Evidence for proximal cysteine and lysine residues at or near the ative site of arginine kinase of <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

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.     

Refined structure of dienelactone hydrolase at 1.8 A

The structure of dienelactone hydrolase (DLH) from Pseudomonus sp. B13, after stereochemically restrained least-squares refinement at 1.8 A resolution, is described. The final molecular model of DLH has a conventional R value of 0.150 and includes all but the carboxyl-terminal three residues that are crystallographically disordered. The positions of 279 water molecules are included in the final model. The root-mean-square deviation from ideal bond distances for the model is 0.014 A and the error in atomic co-ordinates is estimated to be 0.15 A. DLH is a monomeric enzyme containing 236 amino acid residues and is a member of the beta-ketoadipate pathway found in bacteria and fungi. DLH is an alpha/beta protein containing seven helices and eight strands of beta-pleated sheet. A single 4-turn 3(10)-helix is seen. The active-site Cys123 residues at the N-terminal end of an alpha-helix that is peculiar in its consisting entirely of hydrophobic residues (except for a C-terminal lysine). The beta-sheet is composed of parallel strands except for strand 2, which gives rise to a short antiparallel region at the N-terminal end of the central beta-sheet. The active-site cysteine residue is part of a triad of residues consisting of Cys123, His202 and Asp171, and is reminiscent of the serine/cysteine proteases. As in papain and actinidin, the active thiol is partially oxidized during X-ray data collection. The positions of both the reduced and the oxidized sulphur are described. The active site geometry suggests that a change in the conformation of the native thiol occurs upon diffusion of substrate into the active site cleft of DLH. This enables nucleophilic attack by the gamma-sulphur to occur on the cyclic ester substrate through a ring-opening reaction.     

A Structure-Based Approach for Detection of Thiol Oxidoreductases and Their Catalytic Redox-Active Cysteine Residues

Cysteine (Cys) residues often play critical roles in proteins, for example, in the formation of structural disulfide bonds, metal binding, targeting proteins to the membranes, and various catalytic functions. However, the structural determinants for various Cys functions are not clear. Thiol oxidoreductases, which are enzymes containing catalytic redox-active Cys residues, have been extensively studied, but even for these proteins there is little understanding of what distinguishes their catalytic redox Cys from other Cys functions. Herein, we characterized thiol oxidoreductases at a structural level and developed an algorithm that can recognize these enzymes by (i) analyzing amino acid and secondary structure composition of the active site and its similarity to known active sites containing redox Cys and (ii) calculating accessibility, active site location, and reactivity of Cys. For proteins with known or modeled structures, this method can identify proteins with catalytic Cys residues and distinguish thiol oxidoreductases from the enzymes containing other catalytic Cys types. Furthermore, by applying this procedure to Saccharomyces cerevisiae proteins containing conserved Cys, we could identify the majority of known yeast thiol oxidoreductases. This study provides insights into the structural properties of catalytic redox-active Cys and should further help to recognize thiol oxidoreductases in protein sequence and structure databases.