Cysteine Synthase (CysM) of Mycobacterium tuberculosis Is an O-Phosphoserine Sulfhydrylase

Ågren, Daniel; Schnell, R.; Oehlmann, Wulf; Singh, Mahavir; Schneider, Günter

doi:10.1074/jbc.m804877200

Cited by 54 publications

(37 citation statements)

References 43 publications

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“…These spectral changes are consistent with the formation of the enzyme-aminoacrylate intermediate (Fig. 2) commonly observed in this class of PLP-dependent enzymes (12,13,27). None of the other tested compounds, in particular not OAS, induced significant absorbance shifts indicative of a specific reaction with CysK2 (Fig.…”

Section: Amino Acids Such As O-acetyl-l-serine (Oas) O-phospho-l-sersupporting

confidence: 67%

“…The reaction of CysK2 with OPS and CysO-SH was carried out as described previously (13). However, we could not detect any CysO-cysteine adduct by ESI-MS,…”

Section: Amino Acids Such As O-acetyl-l-serine (Oas) O-phospho-l-sermentioning

confidence: 99%

“…Production and purification of M. tuberculosis CysK1 (CysK1 Mtb ), CysO, and CysM (CysM Mtb ) followed protocols described previously (12,13).…”

Section: Methodsmentioning

confidence: 99%

“…Amino acid sequence comparisons (Fig. 1B) suggested that R243 of CysK2 might be equivalent to the position of R220 in CysM Mtb , a residue that is crucial for OPS specificity in this enzyme (13). R243 in CysK2 was therefore replaced by alanine using site-directed mutagenesis.…”

Section: Figmentioning

confidence: 99%

“…The classical pathway is based on the serine acetyltransferase CysE and the cysteine synthase CysK1, whereby acetylation of L-serine catalyzed by CysE results in O-acetyl-L-serine (OAS) (11), which is converted to Lcysteine by CysK1 using hydrogen sulfide as the sulfur donor (12). The alternative pathway based on CysM uses O-phospho-L-serine (OPS) as the acceptor substrate (13,14) and thiocarboxylated CysO as a sulfur donor (15,16) to produce cysteine. Protection of the unstable enzyme-aminoacrylate intermediate by CysM from small oxidants such as H 2 O 2 secures cysteine biosynthesis under oxidative stress and likely supports the long-term intracellular survival within the host macrophages (17).…”

mentioning

confidence: 99%

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CysK2 from Mycobacterium tuberculosis Is an O -Phospho- l -Serine-Dependent S -Sulfocysteine Synthase

et al. 2014

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bMycobacterium tuberculosis is dependent on cysteine biosynthesis, and reduced sulfur compounds such as mycothiol synthesized from cysteine serve in first-line defense mechanisms against oxidative stress imposed by macrophages. Two biosynthetic routes to L-cysteine, each with its own specific cysteine synthase (CysK1 and CysM), have been described in M. tuberculosis, but the function of a third putative sulfhydrylase in this pathogen, CysK2, has remained elusive. We present biochemical and biophysical evidence that CysK2 is an S-sulfocysteine synthase, utilizing O-phosphoserine (OPS) and thiosulfate as substrates. The enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal phosphate-dependent enzyme family. The apparent second-order rate of the first half-reaction with OPS was determined as k max /K s ‫؍‬ (3.97 ؋ 10 3 ) ؎ 619 M ؊1 s ؊1 , which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a k max /K s of (1.34 ؋ 10 3 ) ؎ 48.2. Notably, CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates. The specificity constant k cat /K m for thiosulfate is 40-fold higher than for sulfide, suggesting an annotation as S-sulfocysteine synthase. Mycobacterial CysK2 thus provides a third metabolic route to cysteine, either directly using sulfide as donor or indirectly via S-sulfocysteine. Hypothetically, S-sulfocysteine could also act as a signaling molecule triggering additional responses in redox defense in the pathogen upon exposure to reactive oxygen species during dormancy.

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Section: Amino Acids Such As O-acetyl-l-serine (Oas) O-phospho-l-sersupporting

confidence: 67%

“…The reaction of CysK2 with OPS and CysO-SH was carried out as described previously (13). However, we could not detect any CysO-cysteine adduct by ESI-MS,…”

Section: Amino Acids Such As O-acetyl-l-serine (Oas) O-phospho-l-sermentioning

confidence: 99%

“…Production and purification of M. tuberculosis CysK1 (CysK1 Mtb ), CysO, and CysM (CysM Mtb ) followed protocols described previously (12,13).…”

Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

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CysK2 from Mycobacterium tuberculosis Is an O -Phospho- l -Serine-Dependent S -Sulfocysteine Synthase

et al. 2014

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Amino Acid Biosynthesis

Parker

Pratt

2010

Amino Acids, Peptides and Proteins in Organic Chemistry

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The ribosomal synthesis of proteins utilizes a family of 20 a-amino acids that are universally coded by the translation machinery; in addition, two further a-amino acids, selenocysteine and pyrrolysine, are now believed to be incorporated into proteins via ribosomal synthesis in some organisms. More than 300 other amino acid residues have been identified in proteins, but most are of restricted distribution and produced via post-translational modification of the ubiquitous protein amino acids [1]. The ribosomally encoded a-amino acids described here ultimately derive from a-keto acids by a process corresponding to reductive amination. The most important biosynthetic distinction relates to whether appropriate carbon skeletons are pre-existing in basic metabolism or whether they have to be synthesized de novo and this division underpins the structure of this chapter.There are a small number of a-keto acids ubiquitously found in core metabolism, notably pyruvate (and a related 3-phosphoglycerate derivative from glycolysis), together with two components of the tricarboxylic acid cycle (TCA), oxaloacetate and a-ketoglutarate (a-KG). These building blocks ultimately provide the carbon skeletons for unbranched a-amino acids of three, four, and five carbons, respectively. a-Amino acids with shorter (glycine) or longer (lysine and pyrrolysine) straight chains are made by alternative pathways depending on the available raw materials. The strategic challenge for the biosynthesis of most straight-chain amino acids centers around two issues: how is the a-amino function introduced into the carbon skeleton and what functional group manipulations are required to generate the diversity of side-chain functionality required for the protein function?The core family of straight-chain amino acids does not provide all the functionality required for proteins. a-Amino acids with branched side-chains are used for two purposes; the primary need is related to protein structural issues. Proteins fold into well-defined three-dimensional shapes by virtue of their amphipathic nature: a significant fraction of the amino acid side-chains are of low polarity and the hydrophobic effect drives the formation of ordered structures in which these side-chains are buried away from water. In contrast to the straight-chain amino

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Computational Insights into the Mechanism of Inhibition of OASS‐A by a Small Molecule Inhibitor

Bruno

Amori

Costantino

2013

Molecular Informatics

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O-Acetylserine sulfhydrylase (isoform A, OASS-A) is a PLP-dependent enzyme involved in the last step of cysteine biosynthesis in many pathogens. Many microorganisms use cysteine as the main building block for sulfur-containing antioxidants, and cysteine depletion in several pathogens resulted in a reduced antibiotic resistance, thus leading to the identification of OASS as novel suitable molecular targets to overcome antimicrobial resistances. The precise molecular mechanism of OASS-A inhibition by small peptides or by small molecule inhibitors is still unclear. To shed more lights on the structural basis underlying the inhibition mechanism for OASS, we engaged ourselves in studying the dynamic properties of this enzyme. In this paper, we describe a computational study involving unbiased MD simulations of OASS-A from Haemophilus influenzae (HiOASS) in its inhibitor free, PLP-bound form, and in complex with a pentapeptide inhibitor and with UPAR40, a small molecule which we have recently reported as a potent OASS-A inhibitors. We proposed that UPAR40 inhibits HiOASS-A through the stabilization of a closed conformation. Moreover, preliminary docking studies and sequence analysis allow us to speculate about the non-specificity of UPAR40 toward a particular OASS enzyme species or isoforms.

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Cysteine Synthase (CysM) of Mycobacterium tuberculosis Is an O-Phosphoserine Sulfhydrylase

Cited by 54 publications

References 43 publications

CysK2 from Mycobacterium tuberculosis Is an O -Phospho- l -Serine-Dependent S -Sulfocysteine Synthase

CysK2 from Mycobacterium tuberculosis Is an O -Phospho- l -Serine-Dependent S -Sulfocysteine Synthase

Amino Acid Biosynthesis

Computational Insights into the Mechanism of Inhibition of OASS‐A by a Small Molecule Inhibitor

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