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1.
Int J Mol Sci ; 20(20)2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31640223

RESUMO

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.


Assuntos
Bactérias/enzimologia , Cisteína Sintase/química , Cisteína Sintase/metabolismo , Serina O-Acetiltransferase/química , Serina O-Acetiltransferase/metabolismo , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cisteína Sintase/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , Mapas de Interação de Proteínas , Espalhamento a Baixo Ângulo , Serina O-Acetiltransferase/genética , Difração de Raios X
2.
Microb Pathog ; 131: 218-226, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974158

RESUMO

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.


Assuntos
Produtos Biológicos/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Serina O-Acetiltransferase/efeitos dos fármacos , Serina O-Acetiltransferase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biofilmes/efeitos dos fármacos , Domínio Catalítico , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Cinética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Serina O-Acetiltransferase/genética
3.
Appl Microbiol Biotechnol ; 103(6): 2609-2619, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30729285

RESUMO

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.


Assuntos
Corynebacterium glutamicum/metabolismo , Cisteína/biossíntese , Engenharia Metabólica , Proteínas de Bactérias/genética , Clonagem Molecular , Corynebacterium glutamicum/genética , Cistationina gama-Liase/genética , L-Lactato Desidrogenase/genética , Mutação , Serina O-Acetiltransferase/genética , Serina O-Acetiltransferase/metabolismo
4.
Prep Biochem Biotechnol ; 49(4): 368-374, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30734630

RESUMO

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.


Assuntos
Clonagem Molecular/métodos , Serina O-Acetiltransferase/biossíntese , Streptococcus pneumoniae/enzimologia , Sequência de Bases , Códon , Detergentes/química , Escherichia coli/genética , Octoxinol/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Serina O-Acetiltransferase/química , Serina O-Acetiltransferase/genética , Serina O-Acetiltransferase/isolamento & purificação
5.
Appl Microbiol Biotechnol ; 103(3): 1325-1338, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30564850

RESUMO

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.


Assuntos
Reatores Biológicos/microbiologia , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Cisteína/biossíntese , Engenharia Metabólica/métodos , Cistationina gama-Liase/genética , Cisteína Sintase/biossíntese , Fermentação/genética , Fermentação/fisiologia , Deleção de Genes , Serina O-Acetiltransferase/genética
6.
Sci Rep ; 8(1): 2377, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29402922

RESUMO

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.


Assuntos
Proteínas Mitocondriais/metabolismo , Saccharomycetales/enzimologia , Serina O-Acetiltransferase/metabolismo , Enxofre/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Deleção de Genes , Teste de Complementação Genética , Proteínas Mitocondriais/genética , Sinais Direcionadores de Proteínas , Transporte Proteico , Serina/análogos & derivados , Serina/metabolismo , Serina O-Acetiltransferase/genética
7.
Sci Rep ; 8(1): 1776, 2018 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-29379050

RESUMO

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.


Assuntos
Cisteína/biossíntese , Cisteína/metabolismo , Clonagem Molecular , Cisteína Sintase/metabolismo , Escherichia coli/metabolismo , Metionina/metabolismo , Serina/metabolismo , Serina O-Acetiltransferase/metabolismo , Enxofre/metabolismo
8.
Int J Biol Macromol ; 111: 1010-1018, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29366889

RESUMO

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.


Assuntos
Cisteína/biossíntese , Disenteria Bacilar/enzimologia , Serina O-Acetiltransferase/química , Shigella flexneri/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Cisteína/genética , Farmacorresistência Bacteriana/genética , Estabilidade Enzimática , Escherichia coli/genética , Humanos , Simulação de Dinâmica Molecular , Mutação , Estrutura Quaternária de Proteína , Serina O-Acetiltransferase/genética , Shigella flexneri/genética , Shigella flexneri/patogenicidade
9.
Plant Biotechnol J ; 16(5): 1057-1067, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29044890

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Galinhas/crescimento & desenvolvimento , Metionina/metabolismo , Serina O-Acetiltransferase/genética , Esterol O-Aciltransferase/metabolismo , Zea mays/genética , Zeína/metabolismo , Animais , Proteínas de Arabidopsis/genética , Expressão Gênica , Valor Nutritivo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Serina O-Acetiltransferase/metabolismo , Esterol O-Aciltransferase/genética , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Zeína/química
10.
Sci Rep ; 7(1): 15649, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-29142277

RESUMO

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.


Assuntos
Cisteína Sintase/genética , Cisteína/biossíntese , Entamoeba histolytica/genética , Serina O-Acetiltransferase/genética , Sequência de Aminoácidos/genética , Vias Biossintéticas/genética , Cisteína Sintase/biossíntese , Entamoeba histolytica/metabolismo , Perfilação da Expressão Gênica , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Serina O-Acetiltransferase/biossíntese , Especificidade por Substrato
11.
Sci Rep ; 7(1): 8817, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28821763

RESUMO

Contact-dependent growth inhibition (CDI) is a wide-spread mechanism of inter-bacterial competition. CDI+ bacteria deliver CdiA-CT toxins into neighboring bacteria and produce specific immunity proteins that protect against self-intoxication. The CdiA-CT toxin from uropathogenic Escherichia coli 536 is a latent tRNase that is only active when bound to the cysteine biosynthetic enzyme CysK. Remarkably, the CysK:CdiA-CT binding interaction mimics the 'cysteine synthase' complex of CysK:CysE. The C-terminal tails of CysE and CdiA-CT each insert into the CysK active-site cleft to anchor the respective complexes. The dissociation constant for CysK:CdiA-CT (K d ~ 11 nM) is comparable to that of the E. coli cysteine synthase complex (K d ~ 6 nM), and both complexes bind through a two-step mechanism with a slow isomerization phase after the initial encounter. However, the second-order rate constant for CysK:CdiA-CT binding is two orders of magnitude slower than that of the cysteine synthase complex, suggesting that CysE should outcompete the toxin for CysK occupancy. However, we find that CdiA-CT can effectively displace CysE from pre-formed cysteine synthase complexes, enabling toxin activation even in the presence of excess competing CysE. This adventitious binding, coupled with the very slow rate of CysK:CdiA-CT dissociation, ensures robust nuclease activity in target bacteria.


Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Cisteína Sintase/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Cisteína Sintase/química , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Serina O-Acetiltransferase/química , Serina O-Acetiltransferase/metabolismo
12.
Biochemistry ; 56(37): 5011-5025, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28805060

RESUMO

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.


Assuntos
Alanina/análogos & derivados , Proteínas de Bactérias/antagonistas & inibidores , Cisteína Sintase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Haemophilus influenzae/enzimologia , Modelos Moleculares , Salmonella enterica/metabolismo , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Alanina/farmacologia , Regulação Alostérica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ligação Competitiva , Domínio Catalítico , Cristalografia por Raios X , Cisteína Sintase/química , Cisteína Sintase/genética , Cisteína Sintase/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Haemophilus influenzae/metabolismo , Cinética , Ligantes , Conformação Molecular , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Salmonella enterica/enzimologia , Serina/química , Serina/metabolismo , Serina O-Acetiltransferase/química , Serina O-Acetiltransferase/genética , Serina O-Acetiltransferase/metabolismo , Serina O-Acetiltransferase/farmacologia
13.
Mol Biotechnol ; 59(7): 294-304, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28585118

RESUMO

Intergenic regions of divergent gene pairs show bidirectional promoter activity but whether regulatory sequences for gene expression in opposite directions are shared is not established. In this study, promoters of divergently arranged gene pair At4g35640-At4g35650 (SERAT3;2-IDH-III) of Arabidopsis thaliana were analyzed to identify overlapping regulatory regions. Both genes showed the highest expression in flower buds and flowers. 5' RACE experiments extended the intergenic region from 161 bp shown in TAIR annotation to 512 bp. GUS analysis of transgenic A. thaliana plants carrying the 691 bp fragment (512 bp intergenic region plus 5' UTR of both the genes) linked to uidA gene revealed that SERAT3;2 promoter drives gene expression in the tapetum, whereas IDH-III promoter functions specifically in microspores/pollen. Serial 5' deletion of the 691 bp fragment showed SERAT3;2 promoter extends up to -355 position, whereas IDH-III promoter encompasses the 512 bp intergenic region. In transgenics, uidA transcript levels were lower than native SERAT3;2 and IDH-III transcripts indicating presence of additional cis regulatory elements beyond the 691 bp fragment. The present study demonstrated for the first time occurrence of a nested promoter in plants and identified a novel bidirectional promoter capable of driving gene expression in tapetum and microspores/pollen.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Isocitrato Desidrogenase/genética , Regiões Promotoras Genéticas , Serina O-Acetiltransferase/genética , Regiões 5' não Traduzidas , Arabidopsis/genética , Flores/genética , Flores/crescimento & desenvolvimento , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sítio de Iniciação de Transcrição
14.
J Bacteriol ; 199(16)2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28559296

RESUMO

Serratia marcescens causes health care-associated opportunistic infections that can be difficult to treat due to a high incidence of antibiotic resistance. One of the many secreted proteins of S. marcescens is the PhlA phospholipase enzyme. Genes involved in the production and secretion of PhlA were identified by screening a transposon insertion library for phospholipase-deficient mutants on phosphatidylcholine-containing medium. Mutations were identified in four genes (cyaA, crp, fliJ, and fliP) that are involved in the flagellum-dependent PhlA secretion pathway. An additional phospholipase-deficient isolate harbored a transposon insertion in the cysE gene encoding a predicted serine O-acetyltransferase required for cysteine biosynthesis. The cysE requirement for extracellular phospholipase activity was confirmed using a fluorogenic phospholipase substrate. Phospholipase activity was restored to the cysE mutant by the addition of exogenous l-cysteine or O-acetylserine to the culture medium and by genetic complementation. Additionally, phlA transcript levels were decreased 6-fold in bacteria lacking cysE and were restored with added cysteine, indicating a role for cysteine-dependent transcriptional regulation of S. marcescens phospholipase activity. S. marcescenscysE mutants also exhibited a defect in swarming motility that was correlated with reduced levels of flhD and fliA flagellar regulator gene transcription. Together, these findings suggest a model in which cysteine is required for the regulation of both extracellular phospholipase activity and surface motility in S. marcescensIMPORTANCESerratia marcescens is known to secrete multiple extracellular enzymes, but PhlA is unusual in that this protein is thought to be exported by the flagellar transport apparatus. In this study, we demonstrate that both extracellular phospholipase activity and flagellar function are dependent on the cysteine biosynthesis pathway. Furthermore, a disruption of cysteine biosynthesis results in decreased phlA and flagellar gene transcription, which can be restored by supplying bacteria with exogenous cysteine. These results identify a previously unrecognized role for CysE and cysteine in the secretion of S. marcescens phospholipase and in bacterial motility.


Assuntos
Cisteína/biossíntese , Fosfolipases/metabolismo , Serina O-Acetiltransferase/metabolismo , Serratia marcescens/enzimologia , Serratia marcescens/metabolismo , Meios de Cultura/química , Cisteína/metabolismo , Elementos de DNA Transponíveis , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Teste de Complementação Genética , Locomoção , Mutagênese Insercional , Fosfolipases/genética , Serina/análogos & derivados , Serina/metabolismo , Serina O-Acetiltransferase/genética , Serratia marcescens/genética , Serratia marcescens/fisiologia
15.
Biochemistry ; 56(18): 2385-2399, 2017 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-28414426

RESUMO

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


Assuntos
Cisteína Sintase/química , Cisteína/química , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/enzimologia , Serina O-Acetiltransferase/química , Sítios de Ligação , Clonagem Molecular , Cisteína/biossíntese , Cisteína Sintase/genética , Cisteína Sintase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/genética , Serina O-Acetiltransferase/genética , Serina O-Acetiltransferase/metabolismo , Especificidade por Substrato
16.
FEBS Lett ; 591(9): 1212-1224, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28337759

RESUMO

In bacteria and plants, serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase-A sulfhydrylase (CysK) collaborate to synthesize l-Cys from l-Ser. CysE and CysK bind one another with high affinity to form the cysteine synthase complex (CSC). We demonstrate that bacterial CysE is activated when bound to CysK. CysE activation results from the release of substrate inhibition, with the Ki for l-Ser increasing from 4 mm for free CysE to 16 mm for the CSC. Feedback inhibition of CysE by l-Cys is also relieved in the bacterial CSC. These findings suggest that the CysE active site is allosterically altered by CysK to alleviate substrate and feedback inhibition in the context of the CSC.


Assuntos
Cisteína Sintase/metabolismo , Cisteína/metabolismo , Proteínas de Escherichia coli/metabolismo , Serina O-Acetiltransferase/metabolismo , Regulação Alostérica , Biocatálise , Domínio Catalítico , Ativação Enzimática , Retroalimentação Fisiológica , Cinética , Ligação Proteica , Espectrometria de Fluorescência , Especificidade por Substrato
17.
Mol Med Rep ; 15(3): 1343-1347, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28138713

RESUMO

Serine acetyltransferase (CysE) belongs to the hexapeptide acetyltransferase family and is involved in the biosynthesis of L­cysteine in microorganisms. Mycobacterium tuberculosis CysE is regarded as a potential target for anti­tuberculosis (TB) drugs; however, the structure and active sites of M. tuberculosis CysE remain unknown. The present study aimed to predict the secondary structure and to construct a 3D model for M. tuberculosis CysE using bioinformatics analysis. To determine the essential amino acids that are associated with CysE enzymatic activity, amino acid sequences from several microorganisms were compared, and a consensus sequence was identified. Subsequently, site­directed mutagenesis was used to generate mutant M. tuberculosis CysE proteins. Enzyme assays demonstrated that D67A, H82A and H117A mutants abolished ~75% activity of M. tuberculosis CysE. Prediction of the protein structure and identification of the active amino acids for M. tuberculosis CysE is essential for designing inhibitors, which may aid the discovery of effective anti­TB drugs.


Assuntos
Aminoácidos/química , Domínio Catalítico , Modelos Moleculares , Mycobacterium tuberculosis , Conformação Proteica , Serina O-Acetiltransferase/química , Sequência de Aminoácidos , Catálise , Mutagênese Sítio-Dirigida , Mutação , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Estrutura Secundária de Proteína , Serina O-Acetiltransferase/genética , Serina O-Acetiltransferase/metabolismo , Relação Estrutura-Atividade
18.
Biochem J ; 474(7): 1221-1239, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28126739

RESUMO

Cysteine biosynthesis takes place via a two-step pathway in bacteria, fungi, plants and protozoan parasites, but not in humans, and hence, the machinery of cysteine biosynthesis is an opportune target for therapeutics. The decameric cysteine synthase complex (CSC) is formed when the C-terminal tail of serine acetyltransferase (SAT) binds in the active site of O-acetylserine sulfydrylase (OASS), playing a role in the regulation of this pathway. Here, we show that OASS from Brucella abortus (BaOASS) does not interact with its cognate SAT C-terminal tail. Crystal structures of native BaOASS showed that residues Gln96 and Tyr125 occupy the active-site pocket and interfere with the entry of the SAT C-terminal tail. The BaOASS (Q96A-Y125A) mutant showed relatively strong binding (Kd = 32.4 µM) to BaSAT C-terminal peptides in comparison with native BaOASS. The mutant structure looks similar except that the active-site pocket has enough space to bind the SAT C-terminal end. Surface plasmon resonance results showed a relatively strong (7.3 µM Kd) interaction between BaSAT and the BaOASS (Q96A-Y125A), but no interaction with native BaOASS. Taken together, our observations suggest that the CSC does not form in B. abortus.


Assuntos
Proteínas de Bactérias/química , Brucella abortus/química , Cisteína Sintase/química , Cisteína/biossíntese , Serina O-Acetiltransferase/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Brucella abortus/enzimologia , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Cisteína Sintase/genética , Cisteína Sintase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina O-Acetiltransferase/genética , Serina O-Acetiltransferase/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
19.
Biochimie ; 131: 29-44, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27638321

RESUMO

Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a Km of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania.


Assuntos
Cisteína Sintase/metabolismo , Leishmania donovani/enzimologia , Proteínas de Protozoários/metabolismo , Serina O-Acetiltransferase/metabolismo , Vias Biossintéticas , Clonagem Molecular , Cisteína/biossíntese , Cisteína Sintase/genética , Concentração de Íons de Hidrogênio , Immunoblotting , Leishmania donovani/genética , Leishmania donovani/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Microscopia de Fluorescência , Ligação Proteica , Proteínas de Protozoários/genética , Serina O-Acetiltransferase/genética , Espectrofotometria , Regulação para Cima
20.
Curr Microbiol ; 73(4): 527-33, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27376536

RESUMO

We have previously demonstrated that in Acidithiobacillus ferrooxidans, resistance to the highly toxic divalent cation Cd(2+) is mediated in part by the sulfur assimilation pathway (SAP) and enhanced intracellular concentrations of cysteine and glutathione(GSH) (Zheng et al., Extremophiles 19:429-436, 2015). In this paper, we investigate the interplay between Zn(2+) and Pb(2+) resistances, SAP gene expression, and thiol-containing metabolite levels. Cells grown in the presence of 300 mM Zn(2+) had enhanced activities of the following enzymes: adenosylphosphosulphate reductase (APR, 40-fold), serine acetyltransferase (SAT, 180-fold), and O-acetylserine (thiol) lyase (OAS-TL, 230-fold). We investigated the concentrations of mRNA transcripts of the genes encoding these enzymes in cells grown in the presence of 600 mM Zn(2+): transcripts for 4 SAP genes-ATPS(ATP sulphurylase), APR, SiR(sulfite reductase), SAT, and OAS-TL-each showed a more than three-fold increase in concentration. At the metabolite level, concentrations of intracellular cysteine and glutathione (GSH) were nearly doubled. When cells were grown in the presence of 10 mM Pb(2+), SAP gene transcript concentrations, cysteine, and GSH concentrations were all decreased, as were SAP enzyme activities. These results suggested that Zn(2+) induced SAP pathway gene transcription, while Pb(2+) inhibited SAP gene expression and enzyme activities compared to the pathway in most organisms. Because of the detoxification function of thiol pool, the results also suggested that the high resistance of A. ferrooxidans to Zn(2+) may also be due to regulation of GSH and the cysteine synthesis pathway.


Assuntos
Acidithiobacillus/enzimologia , Proteínas de Bactérias/metabolismo , Cisteína Sintase/metabolismo , Chumbo/metabolismo , Oxirredutases/metabolismo , Serina O-Acetiltransferase/metabolismo , Enxofre/metabolismo , Zinco/metabolismo , Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Proteínas de Bactérias/genética , Cisteína/metabolismo , Cisteína Sintase/genética , Regulação Bacteriana da Expressão Gênica , Glutationa/metabolismo , Oxirredutases/genética , Serina O-Acetiltransferase/genética
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