Multi-oligomeric and catalytically compromised serine acetyltransferase and cysteine regulatory complex of Mycobacterium tuberculosis.

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.

Dynamic association of the plastid localized cysteine synthase complex is vital for efficient cysteine production, photosynthesis, and granal thylakoid formation in transgenic tobacco.

Cysteine biosynthesis is essential for translation and represents the entry point of reduced sulfur into plant metabolism. The two consecutively acting enzymes serine acetyltransferase (SAT) and O-acetylserine-thiol-lyase catalyse cysteine production and form the cysteine synthase complex, in which SAT is activated. Here we show that tobacco (Nicotiana tabacum) expressing active SAT in plastids (referred to as PSA lines) shows substantial cysteine accumulation in plastids. Remarkably, enhanced cysteine production in plastids entirely abolished granal stack formation, impaired photosynthesis capacity, and decreased the number of chloroplasts in mesophyll cells of the PSA lines. A transgenic tobacco line expressing active SAT in the cytosol accumulated comparable amounts of thiols but displayed no phenotype. To dissect the consequences of cysteine synthase complex formation from enhanced SAT activity in tobacco plastids, we expressed an enzymatically inactive SAT that can still form the cysteine synthase complex in tobacco plastids (PSI lines). The PSI lines were indistinguishable from the PSA lines, although the PSI lines displayed no increase in plastid-localized SAT activity. Neither PSA lines nor PSI lines suffered from an oxidized redox environment in plastids that could have been causative for the disturbed photosynthesis. From these findings, we infer that the association of the plastid cysteine synthase complex itself triggers a signaling cascade controlling sulfur assimilation and photosynthetic capacity in leaves.

Loss of the Rhodobacter capsulatus Serine Acetyl Transferase Gene, cysE1, Impairs Gene Transfer by Gene Transfer Agents and Biofilm Phenotypes.

Biofilms are widespread in the environment, where they allow bacterial species to survive adverse conditions. Cells in biofilms are densely packed, and this proximity is likely to increase the frequency of horizontal gene transfer. Gene transfer agents (GTAs) are domesticated viruses with the potential to spread any gene between bacteria. GTA production is normally restricted to a small subpopulation of bacteria, and regulation of GTA loci is highly coordinated, but the environmental conditions that favor GTA production are poorly understood. Here, we identified a serine acetyltransferase gene, cysE1, in Rhodobacter capsulatus that is required for optimal receipt of GTA DNA, accumulation of extracellular polysaccharide, and biofilm formation. The cysE1 gene is directly downstream of the core Rhodobacter-like GTA (RcGTA) structural gene cluster and upregulated in an RcGTA overproducer strain, although it is expressed on a separate transcript. The data we present suggest that GTA production and biofilm are coregulated, which could have important implications for the study of rapid bacterial evolution and understanding the full impact of GTAs in the environment. IMPORTANCE Direct exchange of genes between bacteria leads to rapid evolution and is the major factor underlying the spread of antibiotic resistance. Gene transfer agents (GTAs) are an unusual but understudied mechanism for genetic exchange that are capable of transferring any gene from one bacterium to another, and therefore, GTAs are likely to be important factors in genome plasticity in the environment. Despite the potential impact of GTAs, our knowledge of their regulation is incomplete. In this paper, we present evidence that elements of the cysteine biosynthesis pathway are involved in coregulation of various phenotypes required for optimal biofilm formation by Rhodobacter capsulatus and successful infection by the archetypal RcGTA. Establishing the regulatory mechanisms controlling GTA-mediated gene transfer is a key stepping stone to allow a full understanding of their role in the environment and wider impact.

Structural and functional studies of serine acetyltransferase isoform from Entamoeba histolytica reveals novel role of the C-terminal tail in loss of regulation from feedback inhibition.

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.

Single-molecule real-time sequencing of the full-length transcriptome of purple garlic (Allium sativum L. cv. Leduzipi) and identification of serine O-acetyltransferase family proteins involved in cysteine biosynthesis.

BACKGROUND: Garlic (Allium sativum L.), whose bioactive components are mainly organosulfur compounds (OSCs), is a herbaceous perennial widely consumed as a green vegetable and a condiment. Yet, the metabolic enzymes involved in the biosynthesis of OSCs are not identified in garlic. RESULTS: Here, a full-length transcriptome of purple garlic was generated via PacBio and Illumina sequencing, to characterize the garlic transcriptome and identify key proteins mediating the biosynthesis of OSCs. Overall, 22.56 Gb of clean data were generated, resulting in 454 698 circular consensus sequence (CCS) reads, of which 83.4% (379 206) were identified as being full-length non-chimeric reads - their further transcript clustering facilitated identification of 36 571 high-quality consensus reads. Once corrected, their genome-wide mapping revealed that 6140 reads were novel isoforms of known genes, and 2186 reads were novel isoforms from novel genes. We detected 1677 alternative splicing events, finding 2902 genes possessing either two or more poly(A) sites. Given the importance of serine O-acetyltransferase (SERAT) in cysteine biosynthesis, we investigated the five SERAT homologs in garlic. Phylogenetic analysis revealed a three-tier classification of SERAT proteins, each featuring a serine acetyltransferase domain (N-terminal) and one or two hexapeptide transferase motifs. Template-based modeling showed that garlic SERATs shared a common homo-trimeric structure with homologs from bacteria and other plants. The residues responsible for substrate recognition and catalysis were highly conserved, implying a similar reaction mechanism. In profiling the five SERAT genes' transcript levels, their expression pattern varied significantly among different tissues. CONCLUSION: This study's findings deepen our knowledge of SERAT proteins, and provide timely genetic resources that could advance future exploration into garlic's genetic improvement and breeding. © 2021 Society of Chemical Industry.

Serine acetyltransferase from Neisseria gonorrhoeae; structural and biochemical basis of inhibition.

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 2.01 Šand with the natural substrate l-serine (2.80 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.

Genome-wide identification of serine acetyltransferase (SAT) gene family in rice (Oryza sativa) and their expressions under salt stress.

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.

NV14 from serine O-acetyltransferase of cyanobacteria influences the antioxidant enzymes in vitro cells, gene expression against H2 O2 and other responses in vivo zebrafish larval model.

In this study, we have identified a novel peptide NV14 with antioxidative functions from serine O-acetyltransferase (SAT) of Artrospira platensis (Ap). The full sequence of ApSAT and its derived NV14 peptide "NVRIGAGSVVLRDV" (141-154) was characterized using bioinformatics tools. To address the transcriptional activity of ApSAT in response to induce generic oxidative stress, the spirulina culture was exposed to H2 O2 (10 mM). The ApSAT expression was studied using RT-PCR across various time points and it was found that the expression of the ApSAT was significantly upregulated on Day 15. The in vitro cytotoxicity assay against NV14 was performed in human dermal fibroblast cells and human blood leukocytes. Results showed that NV14 treatment was non-cytotoxic to the cells. Besides, in vivo treatment of NV14 in zebrafish larvae did not exhibit the signs of developmental toxicity. Further, the in vitro antioxidant assays enhanced the activity of the antioxidant enzymes, such as SOD and CAT, due to NV14 treatment; and also significantly reduced the MDA levels, while increasing the superoxide radical and H2 O2 scavenging activity. The expression of antioxidant enzyme genes glutathione peroxidase, γ-glutamyl cysteine synthase, and glutathione S-transferase were found to be upregulated in the NV14 peptide pretreated zebrafish larvae when induced with generic oxidative stress, H2 O2 . Overall, the study showed that NV14 peptide possessed potent antioxidant properties, which were demonstrated over both in vitro and in vivo assays. NV14 enhanced the expression of antioxidant enzyme genes at the molecular level, thereby modulating and reversing the cellular antioxidant balance disrupted due to the H2 O2 -induced oxidative stress.

Cysteine biosynthesis contributes to ß-methylamino-l-alanine tolerance in Escherichia coli.

In contrast to mammalian cells, bacteria such as Escherichia coli have been shown to display tolerance towards the neurotoxin ß-methylamino-l-alanine (BMAA) suggesting that these prokaryotes possess a way to metabolise BMAA or its products, resulting in their export, degradation, or detoxification. Single gene deletion mutants of E. coli K-12 with inactivated amino acid biosynthesis pathways were treated with 500 µg/ml BMAA and the resulting growth was monitored. Wild type E. coli and most of the gene deletion mutants displayed unaltered growth in the presence of BMAA over 12 h. Conversely, deletion of genes in the cysteine biosynthesis pathway, cysE, cysK or cysM resulted in a BMAA dose-dependent growth delay in minimal medium. Through further studies of the ΔcysE strain, we observed increased susceptibility to oxidative stress from H2O2 in minimal medium, and disruptions in glutathione levels and oxidation state. The cysteine biosynthesis pathway is therefore linked to the tolerance of BMAA and oxidative stress in E. coli, which potentially represents a mechanism of BMAA detoxification.

Genetic Variation of the Serine Acetyltransferase Gene Family for Sulfur Assimilation in Maize.

Improving sulfur assimilation in maize kernels is essential due to humans and animals' inability to synthesize methionine. Serine acetyltransferase (SAT) is a critical enzyme that controls cystine biosynthesis in plants. In this study, all SAT gene members were genome-wide characterized by using a sequence homology search. The RNA-seq quantification indicates that they are highly expressed in leaves, other than root and seeds, consistent with their biological functions in sulfur assimilation. With the recently released 25 genomes of nested association mapping (NAM) founders representing the diverse maize stock, we had the opportunity to investigate the SAT genetic variation comprehensively. The abundant transposon insertions into SAT genes indicate their driving power in terms of gene structure and genome evolution. We found that the transposon insertion into exons could change SAT gene transcription, whereas there was no significant correlation between transposable element (TE) insertion into introns and their gene expression, indicating that other regulatory elements such as promoters could also be involved. Understanding the SAT gene structure, gene expression and genetic variation involved in natural selection and species adaption could precisely guide genetic engineering to manipulate sulfur assimilation in maize and to improve nutritional quality.

MRPA-independent mechanisms of antimony resistance in Leishmania infantum.

Control of both human and canine leishmaniasis is based on a very short list of chemotherapeutic agents, headed by antimonial derivatives (Sb). The utility of these molecules is severely threatened by high rates of drug resistance. The ABC transporter MRPA is one of the few key Sb resistance proteins described to date, whose role in detoxification has been thoroughly studied in Leishmania parasites. Nonetheless, its rapid amplification during drug selection complicates the discovery of other mechanisms potentially involved in Sb resistance. In this study, stepwise drug-resistance selection and next-generation sequencing were combined in the search for novel Sb-resistance mechanisms deployed by parasites when MRPA is abolished by targeted gene disruption. The gene mrpA is not essential in L. infantum, and its disruption leads to an Sb hypersensitive phenotype in both promastigotes and amastigotes. Five independent mrpA-/- mutants were selected for antimony resistance. These mutants displayed major changes in their ploidy, as well as extrachromosomal linear amplifications of the subtelomeric region of chromosome 23, which includes the genes coding for ABCC1 and ABCC2. Overexpression of ABCC2, but not of ABCC1, resulted in increased Sb tolerance in the mrpA-/- mutant. SNP analyses revealed three different heterozygous mutations in the gene coding for a serine acetyltransferase (SAT) involved in de novo cysteine synthesis in Leishmania. Overexpression of satQ390K, satG321R and satG325R variants led to a 2-3.2 -fold increase in Sb resistance in mrpA-/- parasites. Only satG321R and satG325R induced increased Sb resistance in wild-type parasites. These results reinforce and expand knowledge on the complex nature of Sb resistance in Leishmania parasites.

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 serine acetyltransferase (CysE) from methicillin-resistant Staphylococcus aureus and inhibitory effect of two natural products on CysE.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major hospital-acquired infective pathogen that has developed resistance to many antibiotics. It is imperious to develop novel anti-MRSA drugs to control the emergence of drug resistance. The biosynthesis of cysteine in bacteria is catalyzed by CysE and CysK. CysE was predicted to be important for bacterial viability, it could be a potential drug target. The serine acetyltransferase activity of CysE was detected and its catalytic properties were also determined. CysE homology model was built to investigate interaction sites between CysE and substrate L-Ser or inhibitors by molecular docking. Docking data showed that residues Asp94 and His95 were essential for serine acetyltransferase activity of CysE, which were confirmed by site-directed mutagenesis. Colorimetric assay was used to screen natural products and six compounds which inhibited CysE activity (IC50 ranging from 29.83 µM to 203.13 µM) were found. Inhibition types of two compounds 4 (11-oxo-ebracteolatanolide B) and 30 ((4R,4aR)-dihydroxy-3-hydroxymethyl-7,7,10a-trimethyl-2,4,4a,5,6,6a,7,8,9,10,10a,l0b-dodecahydrophenanthro[3,2-b]furan-2-one) on CysE were determined. Compounds 4 and 30 showed inhibitory effect on MRSA growth (MIC at 12.5 µg/ml and 25 µg/ml) and mature biofilm. The established colorimetric assay will facilitate further high-throughput screening of CysE inhibitors from different compound libraries. The compounds 4 and 30 may offer structural basis for developing new anti-MRSA drugs.

Recombinant production of active Streptococcus pneumoniae CysE in E. coli facilitated by codon optimized BL21(DE3)-RIL and detergent.

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.

L-Cysteine production by metabolically engineered Corynebacterium glutamicum.

L-Cysteine is a commercially important amino acid. Here, we report the construction of L-cysteine-producing Corynebacterium glutamicum using a metabolic engineering approach. L-Serine O-acetyltransferase (SAT), encoded by cysE gene, is a key enzyme of L-cysteine biosynthesis, because of its feedback inhibition by L-cysteine. Therefore, we introduced a mutation into the C. glutamicum cysE gene, which appeared to desensitize SAT against feedback inhibition by L-cysteine. We successfully produced L-cysteine by overexpressing this mutant cysE gene in C. glutamicum, while the wild-type strain scarcely produced L-cysteine. To enhance the biosynthesis of L-serine (a substrate for SAT), a mutant serA gene, encoding D-3-phosphoglycerate dehydrogenase to desensitize it against feedback inhibition by L-serine, was additionally overexpressed in the mutant cysE-overexpressing strain and its L-cysteine production was indeed improved. Moreover, we disrupted the ldh gene encoding L-lactate dehydrogenase and the aecD gene encoding cysteine desulfhydrase to prevent the formation of lactic acid as a by-product and degradation of L-cysteine produced at the stationary phase, respectively, which resulted in enhanced L-cysteine production. However, since the concentration of L-cysteine produced still decreased at the stationary phase despite the aecD disruption, NCgl2463 encoding a possible cystine importer protein was further disrupted to prevent cystine import, because the produced L-cysteine is immediately oxidized to cystine. As a result, the time before the start of the decrease in L-cysteine concentration was successfully prolonged. Approximately 200 mg/L of L-cysteine production was achieved by overexpression of mutant cysE and serA genes and disruption of aecD and NCgl2463 genes in C. glutamicum.

Metabolic engineering of Corynebacterium glutamicum for L-cysteine production.

L-cysteine, a valuable sulfur-containing amino acid, has been widely used in food, agriculture, and pharmaceutical industries. Due to the toxicity and complex regulation of L-cysteine, no efficient cell factory has yet been achieved for L-cysteine industrial production. In this study, the food-grade microorganism Corynebacterium glutamicum was engineered for L-cysteine production. Through deletion of the L-cysteine desulfhydrases (CD) and overexpression of the native serine acetyltransferase (CysE), the initial L-cysteine-producing strain CYS-2 was constructed to produce 58.2 ± 5.1 mg/L of L-cysteine. Subsequently, several metabolic engineering strategies were performed to further promote L-cysteine biosynthesis, including using strong promoter tac-M to enhance expression intensity of CysE, investigating the best candidate among several heterogeneous feedback-insensitive CysEs for L-cysteine biosynthesis, overexpressing L-cysteine synthase (CysK) to drive more metabolic flux, evaluating the efflux capacity of several heterogeneous L-cysteine transporters, engineering L-serine biosynthesis module to increase the precursor L-serine level and using thiosulfate as the sulfur source. Finally, the L-cysteine concentration of the engineered strain CYS-19 could produce 947.9 ± 46.5 mg/L with addition of 6 g/L Na2S2O3, approximately 14.1-fold higher than that of the initial strain CYS-2, which was the highest titer of L-cysteine ever reported in C. glutamicum. These results indicated that C. glutamicum was a promising platform for L-cysteine production.

A Novel Mitochondrial Serine O-Acetyltransferase, OpSAT1, Plays a Critical Role in Sulfur Metabolism in the Thermotolerant Methylotrophic Yeast Ogataea parapolymorpha.

In most bacteria and plants, direct biosynthesis of cysteine from sulfide via O-acetylserine (OAS) is essential to produce sulfur amino acids from inorganic sulfur. Here, we report the functional analysis of a novel mitochondrial serine O-acetyltransferase (SAT), responsible for converting serine into OAS, in the thermotolerant methylotrophic yeast Ogataea parapolymorpha. Domain analysis of O. parapolymorpha SAT (OpSat1p) and other fungal SATs revealed that these proteins possess a mitochondrial targeting sequence (MTS) at the N-terminus and an α/ß hydrolase 1 domain at the C-terminal region, which is quite different from the classical SATs of bacteria and plants. Noticeably, OpSat1p is functionally interchangeable with Escherichia coli SAT, CysE, despite that it displays much less enzymatic activity, with marginal feedback inhibition by cysteine, compared to CysE. The Opsat1Δ-null mutant showed remarkably reduced intracellular levels of cysteine and glutathione, implying OAS generation defect. The MTS of OpSat1p directs the mitochondrial targeting of a reporter protein, thus, supporting the localization of OpSat1p in the mitochondria. Intriguingly, the OpSat1p variant lacking MTS restores the OAS auxotrophy, but not the cysteine auxotrophy of the Opsat1Δ mutant strain. This is the first study on a mitochondrial SAT with critical function in sulfur assimilatory metabolism in fungal species.

Is perturbation in the quaternary structure of bacterial CysE, another regulatory mechanism for cysteine synthesis?

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.

Genetic, metabolomic and transcriptomic analyses of the de novo L-cysteine biosynthetic pathway in the enteric protozoan parasite Entamoeba histolytica.

The de novo L-cysteine biosynthetic pathway is critical for the growth, antioxidative stress defenses, and pathogenesis of bacterial and protozoan pathogens, such as Salmonella typhimurium and Entamoeba histolytica. This pathway involves two key enzymes, serine acetyltransferase (SAT) and cysteine synthase (CS), which are absent in mammals and therefore represent rational drug targets. The human parasite E. histolytica possesses three SAT and CS isozymes; however, the specific roles of individual isoforms and significance of such apparent redundancy remains unclear. In the present study, we generated E. histolytica cell lines in which CS and SAT expression was knocked down by transcriptional gene silencing. The strain in which CS1, 2 and 3 were simultaneously silenced and the SAT3 gene-silenced strain showed impaired growth when cultured in a cysteine lacking BI-S-33 medium, whereas silencing of SAT1 and SAT2 had no effects on growth. Combined transcriptomic and metabolomic analyses revealed that, CS and SAT3 are involved in S-methylcysteine/cysteine synthesis. Furthermore, silencing of the CS1-3 or SAT3 caused upregulation of various iron-sulfur flavoprotein genes. Taken together, these results provide the first direct evidence of the biological importance of SAT3 and CS isoforms in E. histolytica and justify the exploitation of these enzymes as potential drug targets.

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism.

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.