Step Of Cysteine Biosynthesis Research Articles

A Competitive O-Acetylserine Sulfhydrylase Inhibitor Modulates the Formation of Cysteine Synthase Complex

Cysteine is the main precursor of sulfur-containing biological molecules in bacteria and contributes to the control of the cell redox state. Hence, this amino acid plays an essential role in microbial survival and pathogenicity and the reductive sulfate assimilation pathway is considered a promising target for the development of new antibacterials. Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS-A), the enzymes catalyzing the last two steps of cysteine biosynthesis, engage in the formation of the cysteine synthase (CS) complex. The interaction between SAT and OASS-A finely tunes cysteine homeostasis, and the development of inhibitors targeting either protein–protein interaction or the single enzymes represents an attractive strategy to undermine bacterial viability. Given the peculiar mode of interaction between SAT and OASS-A, which exploits the insertion of SAT C-terminal sequence into OASS-A active site, we tested whether a recently developed competitive inhibitor of OASS-A exhibited any effect on the CS stability. Through surface plasmon resonance spectroscopy, we (i) determined the equilibrium constant for the Salmonella Typhimurium CS complex formation and (ii) demonstrated that the inhibitor targeting OASS-A active site affects CS complex formation. For comparison, the Escherichia coli CS complex was also investigated, with the aim of testing the potential broad-spectrum activity of the candidate antimicrobial compound.

Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action.

The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein–protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS.

Characterization of the O-acetylserine(thiol)lyase gene family in Solanum lycopersicum L.

This research demonstrated the conservation and diversification of the functions of the O-acetylserine-(thiol) lyase gene family genes in Solanum lycopersicum L. Cysteine is the first sulfur-containing organic molecule generated by plants and is the precursor of many important biomolecules and defense compounds. Cysteine and its derivatives are also essential in various redox signaling-related processes. O-acetylserine(thiol)lyase (OASTL) proteins catalyze the last step of cysteine biosynthesis. Previously, researches focused mainly on OASTL proteins which were the most abundant or possessed the authentic OASTL activity, whereas few studies have ever given a comprehensive view of the functions of all the OASTL members in one specific species. Here, we characterized 8 genes belonging to the OASTL gene family from tomato genome (SlOAS2 to SlOAS9), including the sequence analyses, subcellular localization, enzymatic activity assays, expression patterns, as well as the interaction property with SATs. Apart from SlOAS3, all the other genes encoded OASTL-like proteins. Tomato OASTLs were differentially expressed during the development of tomato plants, and their encoded proteins had diverse compartmental distributions and functions. SlOAS5 and SlOAS6 catalyzed the biogenesis of cysteine in chloroplasts and in the cytosol, respectively, and this was in consistent with their interaction abilities with SlSATs. SlOAS4 catalyzed the generation of hydrogen sulfide, similar to its Arabidopsis ortholog, DES1. SlOAS2 also functioned as an L-cysteine desulfhydrase, but its expression pattern was very different from that of SlOAS4. Additionally, SlOAS8 might be a β-cyanoalanine synthase in mitochondria, and the S-sulfocysteine synthase activity appeared lost in tomato plants. SlOAS7 exhibited a transactivational ability in yeast; while the subcellular localization of SlOAS9 was in the peroxisome and correlated with the process of leaf senescence, indicating that these two genes might have novel roles.

Two Distinct Assembly States of the Cysteine Regulatory Complex of Salmonella typhimurium Are Regulated by Enzyme–Substrate Cognate Pairs

Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), which catalyze the last two steps of cysteine biosynthesis, interact and form the cysteine regulatory complex (CRC). The current model of Salmonella typhimurium predicts that CRC is composed of one [SAT]hexamer unit and two molecules of [OASS]dimer. However, it is not clear why [SAT]hexamer cannot engage all of its six high-affinity binding sites. We examined the assembly state(s) of CRC by size exclusion chromatography, analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) approaches. We show that CRC exists in two major assembly states, low-molecular weight (CRC1; 1[SAT]hexamer + 2[OASS]dimer) and high-molecular weight (CRC2; 1[SAT]hexamer + 4[OASS]dimer) states. Along with AUC results, ITC and SPR studies show that [OASS]dimer binds to [SAT]hexamer in a stepwise manner but the formation of fully saturated CRC3 (1[SAT]hexamer + 6[OASS]dimer) is not favorable. The fraction of CRC2 increases as the [OASS]dimer/[SAT]hexamer ratio increases to >4-fold, but CRC2 can be selectively dissociated into either CRC1 or free enzymes, in the presence of OAS and sulfide, in a concentration-dependent manner. Together, we show that CRC is a regulatable multienzyme assembly, sensitive to OASS-substrate(s) levels but subject to negative cooperativity and steric hindrance. Our results constitute the first report of the dual-assembly-state nature of CRC and suggest that physiological conditions, which limit sulfate uptake, would favor CRC1 over CRC2.

Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical investigation of O-acetylserine sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR

Cysteine is a building block for many biomolecules that are crucial for living organisms. O-Acetylserine sulfhydrylase (OASS), present in bacteria and plants but absent in mammals, catalyzes the last step of cysteine biosynthesis. This enzyme has been deeply investigated because, beside the biosynthesis of cysteine, it exerts a series of “moonlighting” activities in bacteria. We have previously reported a series of molecules capable of inhibiting Salmonella typhimurium (S. typhymurium) OASS isoforms at nanomolar concentrations, using a combination of computational and spectroscopic approaches. The cyclopropane-1,2-dicarboxylic acids presented herein provide further insights into the binding mode of small molecules to OASS enzymes. Saturation transfer difference NMR (STD-NMR) was used to characterize the molecule/enzyme interactions for both OASS-A and B. Most of the compounds induce a several fold increase in fluorescence emission of the pyridoxal 5′-phosphate (PLP) coenzyme upon binding to either OASS-A or OASS-B, making these compounds excellent tools for the development of competition-binding experiments.

Structural insight into the interaction of O-acetylserine sulfhydrylase with competitive, peptidic inhibitors by saturation transfer difference-NMR.

O-acetylserine sulfhydrylase (OASS), the enzyme catalysing the last step of cysteine biosynthesis in bacteria, is involved in antibiotic resistance and biofilm formation. Since mammals lack OASS, it is a potential target for antimicrobials. However, a limited number of inhibitors has been developed and crystallized in complex with OASS. STD-NMR was applied to study the interaction of the inhibitory pentapeptide MNYDI with the CysK and CysM OASS isozymes from Salmonella Typhimurium. Results are in excellent agreement with docking and SAR studies and confirm the important role played by the C-terminal Ile5 and the arylic moiety at P3 in dictating affinity.

Rational Design, Synthesis, and Preliminary Structure-Activity Relationships of α-Substituted-2-Phenylcyclopropane Carboxylic Acids as Inhibitors of Salmonella typhimurium O-Acetylserine Sulfhydrylase.

Cysteine is a building block for several biomolecules that are crucial for living organisms. The last step of cysteine biosynthesis is catalyzed by O-acetylserine sulfydrylase (OASS), a highly conserved pyridoxal 5'-phosphate (PLP)-dependent enzyme, present in different isoforms in bacteria, plants, and nematodes, but absent in mammals. Beside the biosynthesis of cysteine, OASS exerts a series of "moonlighting" activities in bacteria, such as transcriptional regulation, contact-dependent growth inhibition, swarming motility, and induction of antibiotic resistance. Therefore, the discovery of molecules capable of inhibiting OASS would be a valuable tool to unravel how this protein affects the physiology of unicellular organisms. As a continuation of our efforts toward the synthesis of OASS inhibitors, in this work we have used a combination of computational and spectroscopic approaches to rationally design, synthesize, and test a series of substituted 2-phenylcyclopropane carboxylic acids that bind to the two S. typhymurium OASS isoforms at nanomolar concentrations.

Isozyme-Specific Ligands for O-acetylserine sulfhydrylase, a Novel Antibiotic Target

The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.

Computational Insights into the Mechanism of Inhibition of OASS-A by a Small Molecule Inhibitor.

O-Acetylserine sulfhydrylase (isoform A, OASS-A) is a PLP-dependent enzyme involved in the last step of cysteine biosynthesis in many pathogens. Many microorganisms use cysteine as the main building block for sulfur-containing antioxidants, and cysteine depletion in several pathogens resulted in a reduced antibiotic resistance, thus leading to the identification of OASS as novel suitable molecular targets to overcome antimicrobial resistances. The precise molecular mechanism of OASS-A inhibition by small peptides or by small molecule inhibitors is still unclear. To shed more lights on the structural basis underlying the inhibition mechanism for OASS, we engaged ourselves in studying the dynamic properties of this enzyme. In this paper, we describe a computational study involving unbiased MD simulations of OASS-A from Haemophilus influenzae (HiOASS) in its inhibitor free, PLP-bound form, and in complex with a pentapeptide inhibitor and with UPAR40, a small molecule which we have recently reported as a potent OASS-A inhibitors. We proposed that UPAR40 inhibits HiOASS-A through the stabilization of a closed conformation. Moreover, preliminary docking studies and sequence analysis allow us to speculate about the non-specificity of UPAR40 toward a particular OASS enzyme species or isoforms.

Overexpression of a soybean O-acetylserine (thiol) lyase-encoding gene GmOASTL4 in tobacco increases cysteine levels and enhances tolerance to cadmium stress

O-Acetylserine (thiol) lyase (OASTL, EC 2.5.1.47) catalyzes the final step in cysteine biosynthesis. The soybean GmOASTL4 gene was overexpressed in tobacco. Transgenic plants showed markedly increased accumulation of transcripts and higher cysteine content compared with the wild-type. Upon exposure to cadmium stress, OASTL activity and cysteine levels increased significantly in transgenic plants. Cadmium accumulation and the activity of both superoxide dismutase and catalase enzymes were enhanced in transformants. These results demonstrate that overexpression of GmOASTL4 in tobacco can enhance cysteine levels and increase tolerance to cadmium stress.

Bacillus subtilis Cysteine Synthetase Is a Global Regulator of the Expression of Genes Involved in Sulfur Assimilation

The synthesis of L-cysteine, the major mechanism by which sulfur is incorporated into organic compounds in microorganisms, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis the cysH operon, encoding several proteins involved in cysteine biosynthesis, is induced by sulfur starvation and tightly repressed by cysteine. We show that a null mutation in the cysK gene encoding an O-acetylserine-(thiol)lyase, the enzyme that catalyzes the final step in cysteine biosynthesis, results in constitutive expression of the cysH operon. Using DNA microarrays we found that, in addition to cysH, almost all of the genes required for sulfate assimilation are constitutively expressed in cysK mutants. These results indicate that CysK, besides its enzymatic role in cysteine biosynthesis, is a global negative regulator of genes involved in sulfur metabolism.

Overproduction of SAT and/or OASTL in transgenic plants: a survey of effects.

The last steps of cysteine biosynthesis are catalysed by a bi-enzyme complex composed of serine acetyltransferase (SAT) and cysteine synthase, also called O-acetyl-serine (thiol) lyase (OASTL). SAT is responsible for the production of O-acetyl-serine (OAS) from serine and acetyl-coenzyme A, while OASTL catalyses the formation of cysteine from OAS and hydrogen sulphide. Several distinct nuclear genes for SAT and OASTL enzymes exist in plants. Products of these genes are targeted into at least three cellular compartments: cytosol, chloroplasts, and mitochondria. The SAT and OASTL enzymes are strongly evolutionary conserved, both structurally and functionally. Therefore, isoenzymes from various cellular compartments can be substituted, not only by their plant counterparts from the other cellular compartments but also by their bacterial homologues. During the last decade transgenic plants overproducing SAT, OASTL or both enzymes simultaneously were obtained independently by several research groups. These manipulations led not only to the elevated levels of the respective products, namely OAS and cysteine, but also to increased amounts of glutathione and changes in the levels of other metabolites and enzymatic activities. In several cases, the transgenic plants were also shown to be less susceptible to applied abiotic stresses. In this review, all published and some unpublished results from this laboratory related to heterologous overproduction of SAT and OASTL in transgenic plants are discussed and summarized.

Surface-exposed Tryptophan Residues Are Essential for O-Acetylserine Sulfhydrylase Structure, Function, and Stability

O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5'-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions.

Production of cysteine for bacterial and plant biotechnology: application of cysteine feedback-insensitive isoforms of serine acetyltransferase.

The first step of cysteine biosynthesis in bacteria and plants consists in the formation of O-acetylserine catalyzed by serine acetyltransferase (SAT). SAT is highly sensitive to feedback inhibition by cysteine as part of the regulatory circuit of cysteine biosynthesis und thus hampers over-expression and fermentation of cysteine in biotechnological production processes. Since plants contain multiple SAT isoforms with different cysteine feedback sensitivity, this resource was exploited to demonstrate the suitability of plant SATs for the production of cysteine in both bacteria and plants. Three new cDNAs encoding SATs were isolated from Nicotiana tabacum. The catalytic activity of SAT4 was insensitive up to 0.6 mM cysteine. Expression of SAT4 in a newly constructed Escherichia coli host strain without endogenous SAT activity yielded a significant accumulation of cysteine in the culture medium compared to expression of cysteine sensitive SATs in the same strain. The application of a similarly insensitive SAT isoform from A. thaliana demonstrated the suitability of this approach to increase cysteine levels in transgenic tobacco plants.

Molecular and biochemical analysis of serine acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants.

Serine acetyltransferase (SATase) and cysteine synthase (O-acetylserine (thiol)-lyase) (CSase) are committed in the final step of cysteine biosynthesis. Six cDNA clones encoding SATase have been isolated from several plants, e.g. watermelon, spinach, Chinese chive and Arabidopsis thaliana. Feedback-inhibition pattern and subcellular localization of plant SATases were evaluated. Two types of SATase that differ in their sensitivity to the feedback inhibition by L-cysteine were found in plants. In Arabidopsis, cytosolic SATase was inhibited by L-cysteine at a physiological concentration in an allosteric manner, but the plastidic and mitochondrial forms were not subjected to this feedback regulation. These results suggest that the regulation of cysteine biosynthesis through feedback inhibition may differ depending on the subcellular compartment. The allosteric domain responsible for L-cysteine inhibition was characterized, using several SATase mutants. The single change of amino acid residue, glycine-277 to cysteine, in the C-terminal region of watermelon SATase caused a significant decrease of the feedback-inhibition sensitivity of watermelon SATase. We made the transgenic Arabidopsis overexpressing point-mutated watermelon SATase gene whose product was not inhibited by L-cysteine. The contents of OAS, cysteine, and glutathione in transgenic Arabidopsis were significantly increased as compared to the wild-type Arabidopsis. Transgenic tobacco (Nicotiana tabacum) (F1) plants with enhanced CSase activities both in the cytosol and in the chloroplasts were generated by cross-fertilization of two transgenic tobacco expressing either cytosolic CSase or chloroplastic CSase. Upon fumigation with 0.1 microLL(-1) sulfur dioxide, both the cysteine and glutathione contents in leaves of F1 plants were increased significantly, but not in leaves of non-transformed control plants. These results indicated that both SATase and CSase play important roles in cysteine biosynthesis and its regulation in plants.

Cysteine synthesis and cysteine desulfuration in Arabidopsis plants at different developmental stages and light conditions

The last step in cysteine biosynthesis is catalysed by the pyridoxal 5’-phosphate-dependent enzyme O-acetyl-L-serine(thiol)lyase (OAS-TL). Several isoforms are localized in different compartments of the cell. OAS-TL and OAS-TL-like proteins also catalyse the formation of β-cyanoalanine and sulfide; the release of sulfide and of an unknown product was also observed. In Arabidopsis plants of different age, the OAS-TL activity decreased with increasing age whereas the enzymatical sulfide release from cysteine increased in older plants. The β-cyanoalanine synthase showed two activity peaks during the time course investigated. During a 12-h light/12-h dark rhythm, the OAS-TL and the sulfide-releasing activities ran parallel with a maximum in the light phase and a decrease of enzyme activity in the dark phase. β-Cyanoalanine synthase activities did not follow a special pattern. Three genes coding for the OAS-TL isoforms A, B, and C, which are presumably localized in the cytoplasm, in plastids, and in mitochondria, respectively, were differentially expressed. The mRNA steady-state levels of the three proteins varied at different developmental stages. The results are discussed with respect to the physiological relevance in the plant organism.

Characterization of Cysteine Synthase in Echinochloa crus-galli L. and Its Inhibition by Substrate Analogues

Abstract Cysteine synthase [CS; EC 4.2.99.8] catalyzes the terminal step of cysteine biosynthesis whereby cysteine is formed from O -acetylserine (OAS) and sulfide. CS was extracted from the leaves of Echinochloa crus-galli L., fractionated with ammonium sulfate, and then characterized. The optimum assay temperature of CS was 50°C, suggesting thermal stability. The activity was relatively low at pH 6.0, but increased sharply when the pH increased from 6.4 to 7.4. Maximal activity seemed between pH 7.4 and 9.0. The apparent K m value was 2.53 mM for OAS and 0.88 mM for sulfide, which indicates that CS has a low affinity for OAS. Two inhibitors of pyridoxal-5′-phosphate (PLP)-dependent enzymes, amino-oxyacetate (AOA) and cycloserine, inhibited this enzyme by 76 and 42%, respectively, at 10 mM. An analogue of OAS, O -phosphoserine, inhibited CS slightly, but other analogues (e.g., O -phosphothreonine) were not significantly inhibitory, even at 30 mM. The I 50 value of AOA was about 3 mM. When CS and AOA were preincubated together at 30°C, AOA inhibited CS in a time-dependent manner, by 51% with 0.1 mM AOA after a 4-h incubation. CS was stable during this incubation period. AOA and cycloserine inhibited the shoot elongation of E. crus-galli by 72 and 31%, respectively, at 5 kg a.i./ha postemergence. Growth inhibition by other compounds was less than 10%. These data suggest a correlation between CS inhibition and growth reduction; however, the supplementation of exogenous cysteine failed to reverse this inhibition in a test tube bioassay. This indicates that the growth inhibition could not be entirely attributed to the starvation of cysteine which might be caused by the inhibitor.

Genomic and functional characterization of the oas gene family encoding O-acetylserine (thiol) lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana

The final step of cysteine biosynthesis in plants is catalyzed by O-acetylserine (thiol) lyase (OAS-TL), which occurs as several isoforms found in the cytosol, the plastids and the mitochondria. Genomic DNA blot hybridization and isolation of genomic clones indicate single copy genes ( oasA1, oasA2, oasB and oasC) that encode the activities of OAS-TL A, B and C found in separate subcellular compartments in the model plant Arabidopsis thaliana. Sequence analysis reveals that the newly discovered oasA2 gene represents a pseudogene that is still transcribed, but is not functionally translated. The comparison of gene structures suggests that oasA1/ oasA2 and oasB/ oasC are closely related and may be derived from a common ancestor by subsequent duplications. OAS-TL A, B and C were overexpressed in an Escherichia coli mutant lacking cysteine synthesis and exhibited bifunctional OAS-TL and β-cyanoalanine synthase (CAS) activities. However, all three proteins represent true OAS-TLs according to kinetic analysis and are unlikely to function in cyanide detoxification or secondary metabolism. In addition, it was demonstrated that the mitochondrial OAS-TL C exhibits in vivo protein–protein interaction capabilities with respect to cysteine synthase complex formation similar to cytosolic OAS-TL A and plastid OAS-TL B. Multiple database accessions for each of the A. thaliana OAS-TL isoforms can thus be attributed to a specified number of oas genes to which functionally defined gene products are assigned, and which are responsible for compartment-specific cysteine synthesis.

Three-dimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium

The last step in cysteine biosynthesis in enteric bacteria is catalyzed by the pyridoxal 5′-phosphate-dependent enzyme O-acetylserine sulfhydrylase. Here we report the crystal structure at 2.2 Å resolution of the A-isozyme of O-acetylserine sulfhydrylase isolated from Salmonella typhimurium. O-acetylserine sulfhydrylase shares the same fold with tryptophan synthase-β from Salmonella typhimurium but the sequence identity level is below 20%. There are some major structural differences: the loops providing the interface to the α-subunit in tryptophan synthase-β and two surface helices of tryptophan synthase-β are missing in O-acetylserine sulfhydrylase. The hydrophobic channel for indole transport from the α to the β active site of tryptophan synthase-β is, not unexpectedly, also absent in O-acetylserine sulfhydrylase. The dimer interface, on the other hand, is more or less conserved in the two enzymes. The active site cleft of O-acetylserine sulfhydrylase is wider and therefore more exposed to the solvent. A possible binding site for the substrate O-acetylserine is discussed.

Assembly of Iron-Sulfur Clusters: IDENTIFICATION OF AN iscSUA-hscBA-fdx GENE CLUSTER FROM AZOTOBACTER VINELANDII

An enzyme having the same L-cysteine desulfurization activity previously described for the NifS protein was purified from a strain of Azotobacter vinelandii deleted for the nifS gene. This protein was designated IscS to indicate its proposed role in iron-sulfur cluster assembly. Like NifS, IscS is a pyridoxal-phosphate containing homodimer. Information gained from microsequencing of oligopeptides obtained by tryptic digestion of purified IscS was used to design a strategy for isolation and DNA sequence analysis of a 7,886-base pair A. vinelandii genomic segment that includes the iscS gene. The iscS gene is contained within a gene cluster that includes homologs to nifU and another gene contained within the major nif cluster of A. vinelandii previously designated orf6. These genes have been designated iscU and iscA, respectively. Information available from complete genome sequences of Escherichia coli and Hemophilus influenzae reveals that they also encode iscSUA gene clusters. A wide conservation of iscSUA genes in nature and evidence that NifU and NifS participate in the mobilization of iron and sulfur for nitrogenase-specific iron-sulfur cluster formation suggest that the products of the iscSUA genes could play a general role in the formation or repair of iron-sulfur clusters. The proposal that IscS is involved in mobilization of sulfur for iron-sulfur cluster formation in A. vinelandii is supported by the presence of a cysE-like homolog in another gene cluster located immediately upstream from the one containing the iscSUA genes. O-Acetylserine synthase is the product of the cysE gene, and it catalyzes the rate-limiting step in cysteine biosynthesis. A similar cysE-like gene is also located within the nif gene cluster of A. vinelandii. The likely role of such cysE-like gene products is to increase the cysteine pool needed for iron-sulfur cluster formation. Another feature of the iscSUA gene cluster region from A. vinelandii is that E. coli genes previously designated as hscB, hscA, and fdx are located immediately downstream from, and are probably co-transcribed with, the iscSUA genes. The hscB, hscA, and fdx genes are also located adjacent to the iscSUA genes in both E. coli and H. influenzae. The E. coli hscA and hscB gene products have previously been shown to bear primary sequence identity when respectively compared with the dnaK and dnaJ gene products and have been proposed to be members of a heat-shock-cognate molecular chaperone system of unknown function. The close proximity and apparent co-expression of iscSUA and hscBA in A. vinelandii indicate that the proposed chaperone function of the hscBA gene products could be related to the maturation of iron-sulfur cluster-containing proteins. Attempts to place non-polar insertion mutations within either A. vinelandii iscS or hscA revealed that such mutations could not be stably maintained in the absence of the corresponding wild-type allele. These results reveal a very strong selective pressure against the maintenance of A. vinelandii iscS or hscA knock-out mutations and suggest that such mutations are either lethal or highly deleterious. In contrast to iscS or hscA, a strain having a polar insertion mutation within the cysE-like gene was readily isolated and could be stably maintained. These results show that the cysE-like gene located upstream from iscS is not essential for cell growth and that the cysE-like gene and the iscSUA-hscBA-fdx genes are contained within separate transcription units.