ABSTRACT
Infectious diseases have been jeopardized problem that threaten public health over a long period of time. The growing prevalence of drug-resistant pathogens and infectious cases have led to a decrease in the number of effective antibiotics, which highlights the urgent need for the development of new antibacterial agents. Serine acetyltransferase (SAT), also known as CysE in certain bacterial species, and O-acetylserine sulfhydrylase (OASS), also known as CysK in select bacteria, are indispensable enzymes within the cysteine biosynthesis pathway of various pathogenic microorganisms. These enzymes play a crucial role in the survival of these pathogens, making SAT and OASS promising targets for the development of novel anti-infective agents. In this comprehensive review, we present an introduction to the structure and function of SAT and OASS, along with an overview of existing inhibitors for SAT and OASS as potential antibacterial agents. Our primary focus is on elucidating the inhibitory activities, structure-activity relationships, and mechanisms of action of these inhibitors. Through this exploration, we aim to provide insights into promising strategies and prospects in the development of antibacterial agents that target these essential enzymes.
Subject(s)
Anti-Bacterial Agents , Cysteine Synthase , Cysteine , Enzyme Inhibitors , Serine O-Acetyltransferase , Serine O-Acetyltransferase/metabolism , Serine O-Acetyltransferase/chemistry , Serine O-Acetyltransferase/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/metabolism , Cysteine/metabolism , Cysteine/chemistry , Cysteine/biosynthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/biosynthesis , Cysteine Synthase/metabolism , Cysteine Synthase/antagonists & inhibitors , Structure-Activity Relationship , Humans , Bacteria/enzymology , Bacteria/drug effects , Bacteria/metabolismABSTRACT
l-cysteine, a primary building block of mycothiol, plays an essential role in the defense mechanism of Mycobacterium tuberculosis (Mtb). However, it is unclear how Mtb regulates cysteine biosynthesis as no study has reported the cysteine regulatory complex (CRC) in Mtb. Serine acetyltransferase (SAT) and cysteine synthase (CS) interact to form CRC. Although MtCS has been characterized well, minimal information is available on MtSAT, which synthesizes, O-acetylserine (OAS), the precursor of cysteine. This study fills the gap and provides experimental evidence for the presence of MtCRC and a non-canonical multi-oligomeric MtSAT. We employed multiple analytical methods to characterize the oligomeric and kinetic properties of MtSAT and MtCRC. Results show that MtSAT, lacking >75 N-terminal amino acids exists in three different assembly states; trimer, hexamer, and dodecamer, compared to the single hexameric state of SAT of other bacteria. While hexamers display the highest catalytic turnover, the trimer is the least active. The predominance of trimers at low physiologically relevant concentrations suggests that MtSAT displays the lowest catalytic potential known. Further, the catalytic potential of MtSAT is also significantly reduced in CRC state, in contrast to enhanced activity of SAT in CRC of other organisms. Our study provides insights into multi-oligomeric MtSAT with reduced catalytic potential and demonstrates that both MtSAT and MtCS of Mycobacterium interact to form CRC, although with altered catalytic properties. We discuss our results in light of the altered biochemistry of the last step of canonical sulfate-dependent cysteine biosynthesis of Mycobacterium.
Subject(s)
Bacterial Proteins , Cysteine Synthase , Cysteine , Mycobacterium tuberculosis , Serine O-Acetyltransferase , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Serine O-Acetyltransferase/metabolism , Serine O-Acetyltransferase/genetics , Serine O-Acetyltransferase/chemistry , Cysteine/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Cysteine Synthase/metabolism , Cysteine Synthase/genetics , Protein Multimerization , KineticsABSTRACT
Antibiotics are the cornerstone of modern medicine and agriculture, and rising antibiotic resistance is one the biggest threats to global health and food security. Identifying new and different druggable targets for the development of new antibiotics is absolutely crucial to overcome resistance. Adjuvant strategies that either enhance the activity of existing antibiotics or improve clearance by the host immune system provide another mechanism to combat antibiotic resistance. Targeting a combination of essential and non-essential enzymes that play key roles in bacterial metabolism is a promising strategy to develop new antimicrobials and adjuvants, respectively. The enzymatic synthesis of L-cysteine is one such strategy. Cysteine plays a key role in proteins and is crucial for the synthesis of many biomolecules important for defense against the host immune system. Cysteine synthesis is a two-step process, catalyzed by two enzymes. Serine acetyltransferase (CysE) catalyzes the first step to synthesize the pathway intermediate O-acetylserine, and O-acetylserine sulfhydrylase (CysK/CysM) catalyzes the second step using sulfide or thiosulfate to produce cysteine. Disruption of the cysteine biosynthesis pathway results in dysregulated sulfur metabolism, altering the redox state of the cell leading to decreased fitness, enhanced susceptibility to oxidative stress and increased sensitivity to antibiotics. In this review, we summarize the structure and mechanism of characterized CysE and CysK/CysM enzymes from a variety of bacterial pathogens, and the evidence that support targeting these enzymes for the development of new antimicrobials or antibiotic adjuvants. In addition, we explore and compare compounds identified thus far that target these enzymes.
Subject(s)
Cysteine Synthase , Serine O-Acetyltransferase , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Bacteria/metabolism , Cysteine/metabolism , Cysteine Synthase/chemistry , Cysteine Synthase/genetics , Drug Resistance, Bacterial , Serine O-Acetyltransferase/chemistry , Serine O-Acetyltransferase/metabolism , Sulfides , Sulfur/metabolism , ThiosulfatesABSTRACT
Serine acetyltransferase (SAT) catalyzes the acetylation of l-serine in the first step of the two-step pathway to synthesize L-cysteine in bacteria, protozoans and plants. L-cysteine is known to be involved in feedback regulation of SAT. However, in E. histolytica, SAT exists in three isoforms where third isoform SAT3 is nearly insensitive to feedback inhibition. Here, we explored the previously unknown precise mechanism of the insensitivity of EhSAT3 to L-cysteine. The C-terminal deletion mutants of EhSAT3 were inhibited completely by L-cysteine in contrast to the wildtype EhSAT3. The crystal structure of EhSAT3ΔC22 in complex with cysteine revealed that C-terminal region swaps over the neighboring monomer in the trimer. This structure combined with the modeled C-terminal residues suggests that EhSAT3 C-terminal end interacts with the active site and play crucial role in feedback inhibition. The interacting distances between sulfur of cysteine and protein indicate cysteine is in deprotonated (S-) state, thus making stronger interactions than serine. In the full length SAT3, C-terminal tail provides an acidic environment at the active site pocket, so that cysteine can't be deprotonated and bind strongly at the active site. These results conveyed a unique role of the C-terminal region of EhSAT3 in regulating the feedback inhibition.
Subject(s)
Entamoeba histolytica , Serine O-Acetyltransferase , Cysteine/metabolism , Entamoeba histolytica/genetics , Feedback , Protein Isoforms/metabolism , Serine/metabolism , Serine O-Acetyltransferase/chemistry , Serine O-Acetyltransferase/geneticsABSTRACT
Serine acetyltransferase (SAT) catalyzes the first step in the two-step pathway to synthesize l-cysteine in bacteria and plants. SAT synthesizes O-acetylserine from substrates l-serine and acetyl coenzyme A and is a key enzyme for regulating cellular cysteine levels by feedback inhibition of l-cysteine, and its involvement in the cysteine synthase complex. We have performed extensive structural and kinetic characterization of the SAT enzyme from the antibiotic-resistant pathogen Neisseria gonorrhoeae. Using X-ray crystallography, we have solved the structures of NgSAT with the non-natural ligand, l-malate (present in the crystallization screen) to 2.01â Å and with the natural substrate l-serine (2.80â Å) bound. Both structures are hexamers, with each monomer displaying the characteristic left-handed parallel ß-helix domain of the acyltransferase superfamily of enzymes. Each structure displays both extended and closed conformations of the C-terminal tail. l-malate bound in the active site results in an interesting mix of open and closed active site conformations, exhibiting a structural change mimicking the conformation of cysteine (inhibitor) bound structures from other organisms. Kinetic characterization shows competitive inhibition of l-cysteine with substrates l-serine and acetyl coenzyme A. The SAT reaction represents a key point for the regulation of cysteine biosynthesis and controlling cellular sulfur due to feedback inhibition by l-cysteine and formation of the cysteine synthase complex. Data presented here provide the structural and mechanistic basis for inhibitor design and given this enzyme is not present in humans could be explored to combat the rise of extensively antimicrobial resistant N. gonorrhoeae.
Subject(s)
Cysteine/antagonists & inhibitors , Feedback, Physiological , Neisseria gonorrhoeae/enzymology , Serine O-Acetyltransferase/chemistry , Serine O-Acetyltransferase/metabolism , Acetyl Coenzyme A/metabolism , Amino Acid Sequence , Biocatalysis , Catalytic Domain , Cloning, Molecular/methods , Crystallization , Crystallography, X-Ray/methods , Cysteine/biosynthesis , Cysteine/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Kinetics , Ligands , Malates/chemistry , Malates/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Serine/chemistry , Serine/metabolism , Serine O-Acetyltransferase/geneticsABSTRACT
BACKGROUND: Assimilation of sulfur to cysteine (Cys) occurs in presence of serine acetyltransferase (SAT). Drought and salt stresses are known to be regulated by abscisic acid, whose biosynthesis is limited by Cys. Cys is formed by cysteine synthase complex depending on SAT and OASTL enzymes. Functions of some SAT genes were identified in Arabidopsis; however, it is not known how SAT genes are regulated in rice (Oryza sativa) under salt stress. METHODS AND RESULTS: Sequence, protein domain, gene structure, nucleotide, phylogenetic, selection, gene duplication, motif, synteny, digital expression and co-expression, secondary and tertiary protein structures, and binding site analyses were conducted. The wet-lab expressions of OsSAT genes were also tested under salt stress. OsSATs have underwent purifying selection. Segmental and tandem duplications may be driving force of structural and functional divergences of OsSATs. The digital expression analyses of OsSATs showed that jasmonic acid (JA) was the only hormone inducing the expressions of OsSAT1;1, OsSAT2;1, and OsSAT2;2 whereas auxin and ABA only triggered OsSAT1;1 expression. Leaf blade is the only plant organ where all OsSATs but OsSAT1;1 were expressed. Wet-lab expressions of OsSATs indicated that OsSAT1;1, OsSAT1;2 and OsSAT1;3 genes were upregulated at different exposure times of salt stress. CONCLUSIONS: OsSAT1;1, expressed highly in rice roots, may be a hub gene regulated by cross-talk of JA, ABA and auxin hormones. The cross-talk of the mentioned hormones and the structural variations of OsSAT proteins may also explain the different responses of OsSATs to salt stress.
Subject(s)
Gene Expression Regulation, Plant/drug effects , Genes, Plant , Oryza/enzymology , Oryza/genetics , Plant Proteins/genetics , Salt Stress/genetics , Serine O-Acetyltransferase/genetics , Amino Acid Sequence , Arabidopsis/enzymology , Arabidopsis/genetics , Binding Sites , Cyclopentanes/pharmacology , Cysteine/metabolism , Gene Duplication , Oxylipins/pharmacology , Phylogeny , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Proteins/chemistry , Plant Proteins/metabolism , Plant Roots/enzymology , Plant Roots/genetics , Protein Domains , Protein Structure, Secondary , Protein Structure, Tertiary , Serine O-Acetyltransferase/chemistry , Serine O-Acetyltransferase/metabolism , SyntenyABSTRACT
Multiple sequence alignment of homoserine-acetyltransferases, serine-acetyltransferases and homoserine-succinyltransferases show they all belong to MetX family, having evolved from a common ancestor by conserving the catalytic site and substrate binding residues. The discrimination in the substrate selection arises due to the presence of substrate-specific residues lining the substrate-binding pocket. Mutation of Ala59 and Gly62 to Gly and Pro respectively in homoserine-acetyltransferase from M. tuberculosis resulted in a serine-acetyltransferase like enzyme as it acetylated both l-homoserine and l-serine. Homoserine-acetyltransferase from M. tuberculosis when mutated at positon 322 where Leu was converted to Arg, resulted in succinylation over acetylation of l-homoserine. Our studies establish the importance of the substrate binding residues in determining the type of activity possessed by MetX family, despite all of them having the same catalytic triad Ser-Asp-His. Hence key residues at the substrate binding pocket dictate whether the given enzyme shows predominant transferase or hydrolase activity.
Subject(s)
Acetyltransferases/chemistry , Bacterial Proteins/chemistry , Homoserine O-Succinyltransferase/chemistry , Mycobacterium tuberculosis/enzymology , Serine O-Acetyltransferase/chemistry , Acetyltransferases/genetics , Arginine/chemistry , Arginine/genetics , Bacterial Proteins/genetics , Homoserine O-Succinyltransferase/genetics , Leucine/chemistry , Leucine/genetics , Mycobacterium tuberculosis/genetics , Serine O-Acetyltransferase/geneticsABSTRACT
The emergence and spread of multidrug-resistant strains of Klebsiella pneumoniae is a major concern that necessitates the development of unique therapeutics. The essential requirement of serine acetyltransferase (SAT/CysE) for survival of several human pathogens makes it a very promising target for inhibitor designing and drug discovery. In this study, as an initial step to structure-based drug discovery, CysE from K. pneumonia was structurally and biochemically characterized. Subsequently, blind docking of selected natural products into the X-ray crystallography determined 3D structure of the target was carried out. Experimental validation of the inhibitory potential of the top-scorers established quercetin as an uncompetitive inhibitor of Kpn CysE. Molecular dynamics simulations carried out to elucidate the binding mode of quercetin reveal that this small molecule binds at the trimer-trimer interface of hexameric CysE, a site physically distinct from the active site of the enzyme. Detailed analysis of conformational differences incurred in Kpn CysE structure on binding to quercetin provides mechanistic understanding of allosteric modulation. Binding of quercetin to CysE leads to conformation changes in the active site loops and proximal loops that affect its internal dynamics and consequently its affinity for substrate/co-factor binding, justifying the reduced enzyme activity.
Subject(s)
Anti-Bacterial Agents/chemistry , Klebsiella pneumoniae/enzymology , Serine O-Acetyltransferase/chemistry , Allosteric Regulation/drug effects , Anti-Bacterial Agents/pharmacology , Cloning, Molecular , Enzyme Stability , Gene Expression , Humans , Kinetics , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Denaturation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Serine O-Acetyltransferase/antagonists & inhibitors , Serine O-Acetyltransferase/genetics , Serine O-Acetyltransferase/isolation & purification , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Bacteria/enzymology , Cysteine Synthase/chemistry , Cysteine Synthase/metabolism , Serine O-Acetyltransferase/chemistry , Serine O-Acetyltransferase/metabolism , Bacteria/chemistry , Bacteria/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Cysteine Synthase/genetics , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Models, Molecular , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Mutagenesis, Site-Directed , Protein Interaction Maps , Scattering, Small Angle , Serine O-Acetyltransferase/genetics , X-Ray DiffractionABSTRACT
The emergence of drug resistance in Streptococcus pneumoniae (Spn) is a global health threat and necessitates discovery of novel therapeutics. The serine acetyltransferase (also known as CysE) is an enzyme of cysteine biosynthesis pathway and is reported to be essential for the survival of several pathogenic bacteria. Therefore, it appears to be a very attractive target for structure-function understanding and inhibitor design. This study describes the molecular cloning of cysE from Spn in the pET21c vector and efforts carried out for expression and purification of active recombinant CysE. Significant expression of recombinant Spn cysE could be achieved in codon optimized BL21(DE3)-RIL strain as opposed to conventional BL21(DE3) strain. Analysis of codon adaptation index (CAI) with levels of eukaryotic genes and prokaryotic cysEs expressed in heterologous E. coli host suggests that codon optimized E. coli BL21(DE3)-RIL may be a better host for expressing genes with low CAI. Here, an efficient protocol has been developed for recovery of recombinant Spn CysE in soluble and biologically active form by the usage of nonionic detergent Triton X-100 at a concentration as low as 1%. Altogether, this study reports a simple strategy for producing functionally active Spn CysE in E. coli.
Subject(s)
Cloning, Molecular/methods , Serine O-Acetyltransferase/biosynthesis , Streptococcus pneumoniae/enzymology , Base Sequence , Codon , Detergents/chemistry , Escherichia coli/genetics , Octoxynol/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Serine O-Acetyltransferase/chemistry , Serine O-Acetyltransferase/genetics , Serine O-Acetyltransferase/isolation & purificationABSTRACT
Drug resistance to almost all antibiotics of Shigella flexneri, a major cause of shigellosis in developing countries, necessitates continuous discovery of novel therapeutics. This study reports a structure-function analysis of a potential drug target serine acetyltransferase (CysE), an enzyme of de novo cysteine biosynthesis pathway that is absent in humans. Analysis of CysE sequences of S. flexneri species and serotypes displayed only two variants that differed by a single amino acid substitution at position 241. Structural inspection of the available crystal structure disclosed this site to be distinct from the substrate/cofactor binding pockets or dimer/trimer interfaces. This study discovers that V241 variant of S. flexneri CysE has nearly null enzymatic activity. The observation is explained by molecular dynamic studies which reveal that the disorder generated by A241V substitution is the basis of dissociation of the quaternary assembly of S. flexneri CysE leading to loss of enzymatic activity and stability. The study provides the first evidence that position 241 of CysE, affects the catalytic efficiency of enzyme and suggests this locus as a 'hot spot' for the propagation of conformational changes. It may be postulated that transient quaternary structure of CysE maybe another mechanism for regulating the intracellular level of cysteine.
Subject(s)
Cysteine/biosynthesis , Dysentery, Bacillary/enzymology , Serine O-Acetyltransferase/chemistry , Shigella flexneri/enzymology , Amino Acid Sequence , Cloning, Molecular , Cysteine/genetics , Drug Resistance, Bacterial/genetics , Enzyme Stability , Escherichia coli/genetics , Humans , Molecular Dynamics Simulation , Mutation , Protein Structure, Quaternary , Serine O-Acetyltransferase/genetics , Shigella flexneri/genetics , Shigella flexneri/pathogenicityABSTRACT
By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (104-106) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.
Subject(s)
Alanine/analogs & derivatives , Bacterial Proteins/antagonists & inhibitors , Cysteine Synthase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Haemophilus influenzae/enzymology , Models, Molecular , Salmonella enterica/metabolism , Acetyl Coenzyme A/chemistry , Acetyl Coenzyme A/metabolism , Alanine/chemistry , Alanine/genetics , Alanine/metabolism , Alanine/pharmacology , Allosteric Regulation , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Binding, Competitive , Catalytic Domain , Crystallography, X-Ray , Cysteine Synthase/chemistry , Cysteine Synthase/genetics , Cysteine Synthase/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Haemophilus influenzae/metabolism , Kinetics , Ligands , Molecular Conformation , Oligopeptides/chemistry , Oligopeptides/genetics , Oligopeptides/metabolism , Oligopeptides/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Salmonella enterica/enzymology , Serine/chemistry , Serine/metabolism , Serine O-Acetyltransferase/chemistry , Serine O-Acetyltransferase/genetics , Serine O-Acetyltransferase/metabolism , Serine O-Acetyltransferase/pharmacologyABSTRACT
Contact-dependent growth inhibition (CDI) is a wide-spread mechanism of inter-bacterial competition. CDI+ bacteria deliver CdiA-CT toxins into neighboring bacteria and produce specific immunity proteins that protect against self-intoxication. The CdiA-CT toxin from uropathogenic Escherichia coli 536 is a latent tRNase that is only active when bound to the cysteine biosynthetic enzyme CysK. Remarkably, the CysK:CdiA-CT binding interaction mimics the 'cysteine synthase' complex of CysK:CysE. The C-terminal tails of CysE and CdiA-CT each insert into the CysK active-site cleft to anchor the respective complexes. The dissociation constant for CysK:CdiA-CT (K d ~ 11 nM) is comparable to that of the E. coli cysteine synthase complex (K d ~ 6 nM), and both complexes bind through a two-step mechanism with a slow isomerization phase after the initial encounter. However, the second-order rate constant for CysK:CdiA-CT binding is two orders of magnitude slower than that of the cysteine synthase complex, suggesting that CysE should outcompete the toxin for CysK occupancy. However, we find that CdiA-CT can effectively displace CysE from pre-formed cysteine synthase complexes, enabling toxin activation even in the presence of excess competing CysE. This adventitious binding, coupled with the very slow rate of CysK:CdiA-CT dissociation, ensures robust nuclease activity in target bacteria.
Subject(s)
Bacterial Toxins/antagonists & inhibitors , Cysteine Synthase/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Cysteine Synthase/chemistry , Models, Biological , Protein Binding , Protein Multimerization , Serine O-Acetyltransferase/chemistry , Serine O-Acetyltransferase/metabolismABSTRACT
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.
Subject(s)
Cysteine Synthase/chemistry , Cysteine/chemistry , Gene Expression Regulation, Bacterial , Salmonella typhimurium/enzymology , Serine O-Acetyltransferase/chemistry , Binding Sites , Cloning, Molecular , Cysteine/biosynthesis , Cysteine Synthase/genetics , Cysteine Synthase/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Kinetics , Molecular Dynamics Simulation , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Salmonella typhimurium/genetics , Serine O-Acetyltransferase/genetics , Serine O-Acetyltransferase/metabolism , Substrate SpecificityABSTRACT
Serine acetyltransferase (CysE) belongs to the hexapeptide acetyltransferase family and is involved in the biosynthesis of Lcysteine in microorganisms. Mycobacterium tuberculosis CysE is regarded as a potential target for antituberculosis (TB) drugs; however, the structure and active sites of M. tuberculosis CysE remain unknown. The present study aimed to predict the secondary structure and to construct a 3D model for M. tuberculosis CysE using bioinformatics analysis. To determine the essential amino acids that are associated with CysE enzymatic activity, amino acid sequences from several microorganisms were compared, and a consensus sequence was identified. Subsequently, sitedirected mutagenesis was used to generate mutant M. tuberculosis CysE proteins. Enzyme assays demonstrated that D67A, H82A and H117A mutants abolished ~75% activity of M. tuberculosis CysE. Prediction of the protein structure and identification of the active amino acids for M. tuberculosis CysE is essential for designing inhibitors, which may aid the discovery of effective antiTB drugs.
Subject(s)
Amino Acids/chemistry , Catalytic Domain , Models, Molecular , Mycobacterium tuberculosis , Protein Conformation , Serine O-Acetyltransferase/chemistry , Amino Acid Sequence , Catalysis , Mutagenesis, Site-Directed , Mutation , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Protein Structure, Secondary , Serine O-Acetyltransferase/genetics , Serine O-Acetyltransferase/metabolism , Structure-Activity RelationshipABSTRACT
Cysteine biosynthesis takes place via a two-step pathway in bacteria, fungi, plants and protozoan parasites, but not in humans, and hence, the machinery of cysteine biosynthesis is an opportune target for therapeutics. The decameric cysteine synthase complex (CSC) is formed when the C-terminal tail of serine acetyltransferase (SAT) binds in the active site of O-acetylserine sulfydrylase (OASS), playing a role in the regulation of this pathway. Here, we show that OASS from Brucella abortus (BaOASS) does not interact with its cognate SAT C-terminal tail. Crystal structures of native BaOASS showed that residues Gln96 and Tyr125 occupy the active-site pocket and interfere with the entry of the SAT C-terminal tail. The BaOASS (Q96A-Y125A) mutant showed relatively strong binding (Kd = 32.4â µM) to BaSAT C-terminal peptides in comparison with native BaOASS. The mutant structure looks similar except that the active-site pocket has enough space to bind the SAT C-terminal end. Surface plasmon resonance results showed a relatively strong (7.3â µM Kd) interaction between BaSAT and the BaOASS (Q96A-Y125A), but no interaction with native BaOASS. Taken together, our observations suggest that the CSC does not form in B. abortus.
Subject(s)
Bacterial Proteins/chemistry , Brucella abortus/chemistry , Cysteine Synthase/chemistry , Cysteine/biosynthesis , Serine O-Acetyltransferase/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Brucella abortus/enzymology , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Cysteine Synthase/genetics , Cysteine Synthase/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Kinetics , Models, Molecular , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Serine O-Acetyltransferase/genetics , Serine O-Acetyltransferase/metabolism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
In contrast to animals, which release the signal molecule sulfide in small amounts from cysteine and its derivates, phototrophic eukaryotes generate sulfide as an essential intermediate of the sulfur assimilation pathway. Additionally, iron-sulfur cluster turnover and cyanide detoxification might contribute to the release of sulfide in mitochondria. However, sulfide is a potent inhibitor of cytochrome c oxidase in mitochondria. Thus, efficient sulfide detoxification mechanisms are required in mitochondria to ensure adequate energy production and consequently survival of the plant cell. Two enzymes have been recently described to catalyze sulfide detoxification in mitochondria of Arabidopsis thaliana, O-acetylserine(thiol)lyase C (OAS-TL C), and the sulfur dioxygenase (SDO) ethylmalonic encephalopathy protein 1 (ETHE1). Biochemical characterization of sulfide producing and consuming enzymes in mitochondria of plants is fundamental to understand the regulatory network that enables mitochondrial sulfide homeostasis under nonstressed and stressed conditions. In this chapter, we provide established protocols to determine the activity of the sulfide releasing enzyme ß-cyanoalanine synthase as well as sulfide-consuming enzymes OAS-TL and SDO. Additionally, we describe a reliable and efficient method to purify OAS-TL proteins from plant material.
Subject(s)
Arabidopsis Proteins/metabolism , Carbon-Oxygen Lyases/metabolism , Dioxygenases/metabolism , Hydrogen Sulfide/metabolism , Lyases/metabolism , Mitochondria/enzymology , Arabidopsis/chemistry , Arabidopsis/enzymology , Arabidopsis Proteins/isolation & purification , Carbon-Oxygen Lyases/isolation & purification , Dioxygenases/isolation & purification , Enzyme Assays , Kinetics , Lyases/isolation & purification , Serine O-Acetyltransferase/chemistryABSTRACT
O-acetylserine sulfhydrylase A (CysK) is the pyridoxal 5'-phosphate-dependent enzyme that catalyzes the final reaction of cysteine biosynthesis in bacteria. CysK was initially identified in a complex with serine acetyltransferase (CysE), which catalyzes the penultimate reaction in the synthetic pathway. This "cysteine synthase" complex is stabilized by insertion of the CysE C-terminus into the active-site of CysK. Remarkably, the CysK/CysE binding interaction is conserved in most bacterial and plant systems. For the past 40years, CysK was thought to function exclusively in cysteine biosynthesis, but recent studies have revealed a repertoire of additional "moonlighting" activities for this enzyme. CysK and its paralogs influence transcription in both Gram-positive bacteria and the nematode Caenorhabditis elegans. CysK also activates an antibacterial nuclease toxin produced by uropathogenic Escherichia coli. Intriguingly, each moonlighting activity requires a binding partner that invariably mimics the C-terminus of CysE to interact with the CysK active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.
Subject(s)
Cysteine Synthase/physiology , Bacteria/metabolism , Binding Sites , Cysteine/biosynthesis , Cysteine Synthase/chemistry , Serine O-Acetyltransferase/chemistry , Serine O-Acetyltransferase/physiology , Transcription, GeneticABSTRACT
Serine acetyltransferase (CysE) is involved in L-cysteine biosynthesis in Mycobacterium, and it is important for the self-defense mechanism of the bacteria. Mycobacterium tuberculosis CysE (Rv2335) has been identified as a serine acetyltransferase, and it is orthologous to Mycobacterium smegmatis MSMEG_5947. In this study, the MSMEG_5947 gene was cloned, expressed, and identified as a serine acetyltransferase. To investigate the function of M. smegmatis CysE, a MSMEG_5947 knockout mutant strain (M. sm-ΔM_5947) was generated through homologous recombination. The growth and morphological characteristics of this strain were studied using growth curves and electron microscopy, respectively. M. sm-ΔM_5947 grew slower than M. smegmatis mc(2) 155. Electron microscopy revealed that the lack of the M. smegmatis CysE protein caused drastic morphological changes. Therefore, deletion of the serine acetyltransferase retards the growth of the Mycobacterium, but serine acetyltransferase expression is not essential for the survival of the bacteria.
Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Mycobacterium smegmatis/enzymology , Serine O-Acetyltransferase/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cysteine/biosynthesis , Gene Knockout Techniques , Homologous Recombination , Microbial Viability , Molecular Sequence Data , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/growth & development , Mycobacterium smegmatis/ultrastructure , Sequence Alignment , Serine O-Acetyltransferase/chemistry , Serine O-Acetyltransferase/geneticsABSTRACT
Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase. Formation of the cysteine regulatory complex (CRC) is a critical biochemical control feature in plant sulfur metabolism. Here we present the 1.75-3.0 Å resolution x-ray crystal structures of soybean (Glycine max) SAT (GmSAT) in apoenzyme, serine-bound, and CoA-bound forms. The GmSAT-serine and GmSAT-CoA structures provide new details on substrate interactions in the active site. The crystal structures and analysis of site-directed mutants suggest that His(169) and Asp(154) form a catalytic dyad for general base catalysis and that His(189) may stabilize the oxyanion reaction intermediate. Glu(177) helps to position Arg(203) and His(204) and the ß1c-ß2c loop for serine binding. A similar role for ionic interactions formed by Lys(230) is required for CoA binding. The GmSAT structures also identify Arg(253) as important for the enhanced catalytic efficiency of SAT in the CRC and suggest that movement of the residue may stabilize CoA binding in the macromolecular complex. Differences in the effect of cold on GmSAT activity in the isolated enzyme versus the enzyme in the CRC were also observed. A role for CRC formation as a molecular chaperone to maintain SAT activity in response to an environmental stress is proposed for this multienzyme complex in plants.