The C‐terminal of CysM from <i>Mycobacterium tuberculosis</i> protects the aminoacrylate intermediate and is involved in sulfur donor selectivity

Ågren, Daniel; Schnell, R.; Schneider, Günter

doi:10.1016/j.febslet.2008.12.019

Cited by 22 publications

(14 citation statements)

References 41 publications

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“…This change occurred within 20 min, and the resulting spectrum corresponds with that of the enzyme-bound PLP. Compared with the results from similar experiments using O-acetylserine sulfhydrylases (36), the lifetime of the ␣-aminoacrylate intermediate in DcsD is obviously shorter than that in normal O-acetylserine sulfhydrylases. This may be another reason for the high K m value of DcsD toward L-OAS.…”

Section: In Vitro D-cs Synthesiscontrasting

confidence: 63%

Establishment of anIn Vitrod-Cycloserine-Synthesizing System by UsingO-Ureido-l-Serine Synthase andd-Cycloserine Synthetase Found in the Biosynthetic Pathway

Uda

Matoba

Kumagai

et al. 2013

Antimicrob Agents Chemother

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We have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic, D-cycloserine. The gene cluster is composed of 10 open reading frames, designated dcsA to dcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization of O-ureidoserine. DcsD is similar to O-acetylserine sulfhydrylase, which generates L-cysteine using O-acetyl-L-serine with sulfide, and therefore, DcsD may be a synthase to generate O-ureido-L-serine using O-acetyl-L-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase converting O-ureido-D-serine into D-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed in Escherichia coli and purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substrates O-acetyl-L-serine and hydroxyurea, synthesis of D-cycloserine was successfully attained. These in vitro studies yield the conclusion that DcsD and DcsG are necessary for the syntheses of O-ureido-L-serine and D-cycloserine, respectively. DcsD was also able to catalyze the synthesis of L-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclic D-amino acid analogs, such as D-homocysteine thiolactone.

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Section: In Vitro D-cs Synthesiscontrasting

confidence: 63%

Establishment of anIn Vitrod-Cycloserine-Synthesizing System by UsingO-Ureido-l-Serine Synthase andd-Cycloserine Synthetase Found in the Biosynthetic Pathway

Uda

Matoba

Kumagai

et al. 2013

Antimicrob Agents Chemother

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“…Based on E. coli, three enzymes were identified that may have cysteine desulfhydrase activity in S. enterica: CysK, CysM, and STM1557 (34% identical to E. coli MalY) (27). These PLP-dependent enzymes generate pyruvate, hydrogen sulfide, and ammonia from cysteine, and it was plausible that these reactions would proceed through a 2AA intermediate (29,46,47). Deletions of cysK, cysM, and stm1557 were generated by gene replacement (39), and strains that contained each mutation alone or in combination with a ridA mutation were constructed.…”

Section: Resultsmentioning

confidence: 99%

Endogenous Synthesis of 2-Aminoacrylate Contributes to Cysteine Sensitivity in Salmonella enterica

et al. 2014

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RidA, the archetype member of the widely conserved RidA/YER057c/UK114 family of proteins, prevents reactive enamine/imine intermediates from accumulating in Salmonella enterica by catalyzing their hydrolysis to stable keto acid products. In the absence of RidA, endogenous 2-aminoacrylate persists in the cellular environment long enough to damage a growing list of essential metabolic enzymes. Prior studies have focused on the dehydration of serine by the pyridoxal 5=-phosphate (PLP)-dependent serine/threonine dehydratases, IlvA and TdcB, as sources of endogenous 2-aminoacrylate. The current study describes an additional source of endogenous 2-aminoacrylate derived from cysteine. The results of in vivo analysis show that the cysteine sensitivity of a ridA strain is contingent upon CdsH, the predominant cysteine desulfhydrase in S. enterica. The impact of cysteine on 2-aminoacrylate accumulation is shown to be unaffected by the presence of serine/threonine dehydratases, revealing another mechanism of endogenous 2-aminoacrylate production. Experiments in vitro suggest that 2-aminoacrylate is released from CdsH following cysteine desulfhydration, resulting in an unbound aminoacrylate substrate for RidA. This work expands our understanding of the role played by RidA in preventing enamine stress resulting from multiple normal metabolic processes.

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“…Similar collapsing of the active site upon PLP-α-aminoacrylate intermediate formation is observed in structures determined for homologues CysK1 and CysM from M. tuberculosis . 25,56 Loss of the phosphate group upon formation of the stable PLP-α-aminoacrylate intermediate from OPS precluded direct structural identification of residues involved in OPS specificity. However, the active site of SbnA-AA revealed three residues: Arg132, Tyr152, and Ser185 are in close proximity to the β-carbon of the α-aminoacrylate intermediate (Figure 3B), and all three residues were shown to be involved in formation of the intermediate.…”

Section: Discussionmentioning

confidence: 99%

Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis

et al. 2016

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Staphylococcus aureus assembles the siderophore, staphyloferrin B, from l-2,3-diaminopropionic acid (l-Dap), α-ketoglutarate, and citrate. Recently, SbnA and SbnB were shown to produce l-Dap and α-ketoglutarate from O-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5′-phosphate (PLP)-dependent enzyme with homology to O-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of O-acetyl-l-serine (OAS), and l-glutamate serves as a nitrogen donor instead of a sulfide. In this work, we examined how SbnA dictates substrate specificity for OPS and l-glutamate using a combination of X-ray crystallography, enzyme kinetics, and site-directed mutagenesis. Analysis of SbnA crystals incubated with OPS revealed the structure of the PLP-α-aminoacrylate intermediate. Formation of the intermediate induced closure of the active site pocket by narrowing the channel leading to the active site and forming a second substrate binding pocket that likely binds l-glutamate. Three active site residues were identified: Arg132, Tyr152, Ser185 that were essential for OPS recognition and turnover. The Y152F/S185G SbnA double mutant was completely inactive, and its crystal structure revealed that the mutations induced a closed form of the enzyme in the absence of the α-aminoacrylate intermediate. Lastly, l-cysteine was shown to be a competitive inhibitor of SbnA by forming a nonproductive external aldimine with the PLP cofactor. These results suggest a regulatory link between siderophore and l-cysteine biosynthesis, revealing a potential mechanism to reduce iron uptake under oxidative stress.

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The C‐terminal of CysM from Mycobacterium tuberculosis protects the aminoacrylate intermediate and is involved in sulfur donor selectivity

Cited by 22 publications

References 41 publications

Establishment of anIn Vitrod-Cycloserine-Synthesizing System by UsingO-Ureido-l-Serine Synthase andd-Cycloserine Synthetase Found in the Biosynthetic Pathway

Establishment of anIn Vitrod-Cycloserine-Synthesizing System by UsingO-Ureido-l-Serine Synthase andd-Cycloserine Synthetase Found in the Biosynthetic Pathway

Endogenous Synthesis of 2-Aminoacrylate Contributes to Cysteine Sensitivity in Salmonella enterica

Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis

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