Advancements in inhibitors of crucial enzymes in the cysteine biosynthetic pathway: Serine acetyltransferase and O-acetylserine sulfhydrylase.

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.

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.

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.

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.

Key amino acid residues in homoserine-acetyltransferase from M. tuberculosis give insight into the evolution of MetX family of enzymes - HAT, SAT and HST.

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.

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.

ß-Cyanoalanine Synthase Regulates the Accumulation of ß-ODAP via Interaction with Serine Acetyltransferase in Lathyrus sativus.

ß-N-Oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), found in Lathyrus sativus at first, causes a neurological disease, lathyrism, when over ingested in an unbalanced diet. Our previous research suggested that ß-ODAP biosynthesis is related to sulfur metabolism. In this study, ß-cyanoalanine synthase (ß-CAS) was confirmed to be responsible for ß-ODAP biosynthesis via in vitro enzymatic analysis. LsCAS was found to be pyridoxal phosphate (PLP)-dependent via spectroscopic analysis and dual functional via enzymatic activity analysis. Generation of a M135T/M235S/S239T triple mutant of LsCAS, which are the key sites to control the ratio of CAS/cysteine synthase (CS) activity, switches reaction chemistry to that of a CS. LsCAS interactions were further screened and verified via Y2H, BiFC and pull-down assay. It was suggested that LsSAT2 interacts and forms a cysteine regulatory complex (CRC) with LsCAS in mitochondria, which improves LsSAT while reduces LsCAS activities to affect ß-ODAP content positively. These results provide new insights into the molecular regulation of ß-ODAP content in L. sativus.

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.