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CysK2 from Mycobacterium tuberculosis is an O-phospho-L-serine-dependent S-sulfocysteine synthase

Steiner, Eva Maria LU orcid ; Bo¨th, Dominic ; Lo¨ssl, Philip ; Vilaplana, Francisco ; Schnell, Robert and Schneider, Gunter (2014) In Journal of Bacteriology 196(19). p.3410-3420
Abstract

Mycobacterium 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... (More)

Mycobacterium 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 kmax/Ks=(3.97×103)±619M-1 s-1, which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a kmax/Ks of (1.34×103)±48.2. Notably, CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates. The specificity constant kcat/Km 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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author
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publishing date
type
Contribution to journal
publication status
published
in
Journal of Bacteriology
volume
196
issue
19
pages
3410 - 3420
publisher
American Society for Microbiology
external identifiers
  • pmid:25022854
  • scopus:84907016411
ISSN
0021-9193
DOI
10.1128/JB.01851-14
language
English
LU publication?
no
additional info
Publisher Copyright: © 2014, American Society for Microbiology.
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cc5747e3-fc3f-4dda-9980-32a596f0e766
date added to LUP
2024-06-24 11:28:12
date last changed
2025-01-07 06:35:12
@article{cc5747e3-fc3f-4dda-9980-32a596f0e766,
  abstract     = {{<p>Mycobacterium 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<sub>max</sub>/K<sub>s</sub>=(3.97×10<sup>3</sup>)±619M<sup>-1</sup> s<sup>-1</sup>, which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a k<sub>max</sub>/K<sub>s</sub> of (1.34×10<sup>3</sup>)±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<sub>cat</sub>/K<sub>m</sub> 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.</p>}},
  author       = {{Steiner, Eva Maria and Bo¨th, Dominic and Lo¨ssl, Philip and Vilaplana, Francisco and Schnell, Robert and Schneider, Gunter}},
  issn         = {{0021-9193}},
  language     = {{eng}},
  number       = {{19}},
  pages        = {{3410--3420}},
  publisher    = {{American Society for Microbiology}},
  series       = {{Journal of Bacteriology}},
  title        = {{CysK2 from Mycobacterium tuberculosis is an O-phospho-L-serine-dependent S-sulfocysteine synthase}},
  url          = {{http://dx.doi.org/10.1128/JB.01851-14}},
  doi          = {{10.1128/JB.01851-14}},
  volume       = {{196}},
  year         = {{2014}},
}