Unveiling the role of serine o-acetyltransferase in drug resistance and oxidative stress tolerance in Leishmania donovani through the regulation of thiol-based redox metabolism.

Understanding the unique metabolic pathway of L. donovani is crucial for comprehending its biology under oxidative stress conditions. The de novo cysteine biosynthetic pathway of L. donovani is absent in humans and its product, cysteine regulates the downstream components of trypanothione-based thiol metabolism, important for maintaining cellular redox homeostasis. The role of serine o-acetyl transferase (SAT), the first enzyme of this pathway remains unexplored. In order to investigate the role of SAT protein, we cloned SAT gene into pXG-GFP+ vector for episomal expression of SAT in Amphotericin B sensitive L. donovani promastigotes. The SAT overexpression was confirmed by SAT enzymatic assay, GFP fluorescence, immunoblotting and PCR. Our study unveiled an upregulated expression of both LdSAT and LdCS of cysteine biosynthetic pathway and other downstream thiol pathway proteins in LdSAT-OE promastigotes. Additionally, there was an increase in enzymatic activities of LdSAT and LdCS proteins in LdSAT-OE, which was found similar to the Amp B resistant parasites, indicating a potential role of SAT protein in modulating drug resistance. We observed that the overexpression of SAT in Amp B sensitive parasites increases tolerance to drug pressure and oxidative stress via trypanothione-dependent antioxidant mechanism. Moreover, the in vitro J774A.1 macrophage infectivity assessment showed that SAT overexpression augments parasite infectivity. In LdSAT-OE promastigotes, antioxidant enzyme activities like APx and SOD were upregulated, intracellular reactive oxygen species were reduced with a corresponding increase in thiol level, emphasizing SAT's role in stress tolerance and enhanced infectivity. Additionally, the ROS mediated upregulation in the expression of LdSAT, LdCS, LdTryS and LdcTXNPx proteins reveals an essential cross talk between SAT and proteins of thiol metabolism in combating oxidative stress and maintaining redox homeostasis. Taken together, our results provide the first insight into the role of SAT protein in parasite infectivity and survival under drug pressure and oxidative stress.

Critical Sites of Serine Acetyltransferase in Lathyrus sativus L. Affecting Its Enzymatic Activities.

LsSAT2 (serine acetyltransferase in Lathyrus sativus) is the rate-limiting enzyme in biosynthesis of ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), a neuroactive metabolite distributed widely in several plant species including Panax notoginseng, Panax ginseng, and L. sativus. The enzymatic activity of LsSAT2 is post-translationally regulated by its involvement in the cysteine regulatory complex in mitochondria via interaction with ß-CAS (ß-cyanoalanine synthase). In this study, the binding sites of LsSAT2 with the substrate Ser were first determined as Glu290, Arg316, and His317 and the catalytic sites were determined as Asp267, Asp281, and His282 via site-directed/truncated mutagenesis, in vitro enzymatic activity assay, and functional complementation of the SAT-deficient Escherichia coli strain JM39. Furthermore, the C-terminal 10-residue peptide of LsSAT2 is confirmed to be critical to interact with LsCAS, and Ile336 in C10 peptide is the critical amino acid. These results will enhance our understanding of the regulation of LsSAT2 activities and the biosynthesis of ß-ODAP in L. sativus.

Enhanced selenocysteine biosynthesis for seleno-methylselenocysteine production in Bacillus subtilis.

Seleno-methylselenocysteine (SeMCys) is an effective component for selenium supplementation with anti-carcinogenic potential and can ameliorate neuropathology and cognitive deficits. In this study, we aimed to engineer Bacillus subtilis 168 for the microbial production of SeMCys. First, the accumulation of intracellular selenocysteine (SeCys) as the precursor of SeMCys was enhanced through overexpression of serine O-acetyltransferase, which was desensitized against feedback inhibition by cysteine. Next, the S-adenosylmethionine (SAM) synthetic pathway was optimized to improve methyl donor availability through expression of S-adenosylmethionine synthetase. Further, SeMCys was successfully produced through expression of the selenocysteine methyltransferase in SeCys and SAM-producing strain. The increased expression level of selenocysteine methyltransferase benefited the SeMCys production. Finally, all the heterologous genes were integrated into the genome of B. subtilis, and the strain produced SeMCys at a titer of 18.4 µg/L in fed-batch culture. This is the first report on the metabolic engineering of B. subtilis for microbial production of SeMCys and provides a good starting point for future pathway engineering to achieve the industrial-grade production of SeMCys. KEY POINTS: • Expression of the feedback-insensitive serine O-acetyltransferase provided B. subtilis the ability of accumulating SeCys. • SAM production was enhanced through expressing S-adenosylmethionine synthetase in B. subtilis. • Expression of selenocysteine methyltransferase in SeCys and SAM-accumulating strain facilitated SeMCys production.

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.

Combatting antimicrobial resistance via the cysteine biosynthesis pathway in bacterial pathogens.

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.

Serine O-acetyltransferase derived NV14 peptide reduces cytotoxicity in H2O2 induced MDCK cells and inhibits MCF-7 cell proliferation through caspase gene expression.

BACKGROUND: Most of the bioactive peptides exhibit antioxidant effect and do elicit inhibitory effect on proliferation of cancer cells. This study investigates the in-vitro antioxidant and anti-cancer properties of NV14 peptide, derived from serine O-acetyltransferase (SAT) of spirulina, Arthrospira platensis. METHODS: The anti-cancer effect of the peptide was evaluated using human adenocarcinoma epithelial cells (MCF-7), while the anti-oxidant potential, as in reduction in ROS concentration, has been established using the H2O2-exposed, Madin-Darby canine kidney (MDCK) cells. The outcome of the in vitro analyses has been evaluated by in silico molecular docking analyses. RESULTS: The peptide, dose-dependently, reduced oxidative stress as well as cell proliferation. Besides, based on the binding scores between NV14 peptide and the important proteins associated with apoptosis and antioxidant defense, it is evident that the peptide has antioxidant and anti-cancer effect, in vitro. CONCLUSIONS: Together, this study demonstrates that NV14 has a potent antioxidant and anti-cancer capability; however, further direction needs to be focused on clinical or pharmacodynamics aspects.

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.

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.

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.

Homology modeling, structural insights and in-silico screening for selective inhibitors of mycobacterial CysE.

Tuberculosis posses a major threat for health practitioners due to lengthy treatment regimen, increase in the drug-resistant strains of Mycobacterium tuberculosis (M. tb) and unavailability of drugs for its persistent form. Therefore, there is an urgent need for discovery of new and improved anti-tubercular drugs. In M. tb, the two step de novo biosynthesis of L-cysteine, an essential metabolic pathway is reported to be up-regulated in the persistent phase of the organism, involves two enzymes CysE and CysK. Although, structural insights for rational drug discovery are available for the later, not much information is known for the former. This study proposes a 3-dimensional model of M. tb CysE followed by in-silico screening of 67,030 anti-tuberculosis bioactive compounds. Subsequently, post-processing of 1000 best hits was carried out and top 200 compounds thus obtained were docked into the active site cleft of E. coli homologue as a control, but revealed unexpected results. Differences in the active site architectures and comparative analysis of molecular electrostatic potentials between the two CysEs provide molecular basis for the compounds C1, C3, C4 and C7 exhibiting preferential binding for M. tb CysE. In addition, shorter N-terminus along with positive and irregular trimeric base of M. tb CysE indicates its biological assembly as trimer. Based on mapping of residues involved in cysteine sensitivity on to the model structure of M. tb CysE, it is hypothesized that feedback inhibition of this homologue by cysteine may be affected.Communicated by Ramaswamy H. Sarma.

CYP20-3 deglutathionylates 2-CysPRX A and suppresses peroxide detoxification during heat stress.

In plants, growth-defense trade-offs occur because of limited resources, which demand prioritization towards either of them depending on various external and internal factors. However, very little is known about molecular mechanisms underlying their occurrence. Here, we describe that cyclophilin 20-3 (CYP20-3), a 12-oxo-phytodienoic acid (OPDA)-binding protein, crisscrosses stress responses with light-dependent electron reactions, which fine-tunes activities of key enzymes in plastid sulfur assimilations and photosynthesis. Under stressed states, OPDA, accumulates in the chloroplasts, binds and stimulates CYP20-3 to convey electrons towards serine acetyltransferase 1 (SAT1) and 2-Cys peroxiredoxin A (2CPA). The latter is a thiol-based peroxidase, protecting and optimizing photosynthesis by reducing its toxic byproducts (e.g., H2O2). Reduction of 2CPA then inactivates its peroxidase activity, suppressing the peroxide detoxification machinery, whereas the activation of SAT1 promotes thiol synthesis and builds up reduction capacity, which in turn triggers the retrograde regulation of defense gene expressions against abiotic stress. Thus, we conclude that CYP20-3 is a unique metabolic hub conveying resource allocations between plant growth and defense responses (trade-offs), ultimately balancing optimal growth phonotype.

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.

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.

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.

Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes.

Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.

Overexpression of serine acetyltransferase in maize leaves increases seed-specific methionine-rich zeins.

Maize kernels do not contain enough of the essential sulphur-amino acid methionine (Met) to serve as a complete diet for animals, even though maize has the genetic capacity to store Met in kernels. Prior studies indicated that the availability of the sulphur (S)-amino acids may limit their incorporation into seed storage proteins. Serine acetyltransferase (SAT) is a key control point for S-assimilation leading to Cys and Met biosynthesis, and SAT overexpression is known to enhance S-assimilation without negative impact on plant growth. Therefore, we overexpressed Arabidopsis thaliana AtSAT1 in maize under control of the leaf bundle sheath cell-specific rbcS1 promoter to determine the impact on seed storage protein expression. The transgenic events exhibited up to 12-fold higher SAT activity without negative impact on growth. S-assimilation was increased in the leaves of SAT overexpressing plants, followed by higher levels of storage protein mRNA and storage proteins, particularly the 10-kDa δ-zein, during endosperm development. This zein is known to impact the level of Met stored in kernels. The elite event with the highest expression of AtSAT1 showed 1.40-fold increase in kernel Met. When fed to chickens, transgenic AtSAT1 kernels significantly increased growth rate compared with the parent maize line. The result demonstrates the efficacy of increasing maize nutritional value by SAT overexpression without apparent yield loss. Maternal overexpression of SAT in vegetative tissues was necessary for high-Met zein accumulation. Moreover, SAT overcomes the shortage of S-amino acids that limits the expression and accumulation of high-Met zeins during kernel development.

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.