title: CysK2 from Mycobacterium tuberculosis is an O-phospho-L-serine-dependent S-sulfocysteine synthase.
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 pyridoxal phosphate-dependent enzyme family. The apparent second-order rate of the first half-reaction with OPS was determined as kmax/Ks = (3.97 × 10(3)) ± 619 M(-1) s(-1), which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a kmax/Ks 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 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.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Eva Maria
authors.initials: EM
authors.lastname: Steiner
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Dominic
authors.initials: D
authors.lastname: Böth
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Philip
authors.initials: P
authors.lastname: Lössl
authors.affiliation: Division of Glycoscience, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Francisco
authors.initials: F
authors.lastname: Vilaplana
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Robert
authors.initials: R
authors.lastname: Schnell
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden gunter.schneider@ki.se.
authors.forename: Gunter
authors.initials: G
authors.lastname: Schneider
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D012694:Serine; D008190:Lyases; C022706:S-sulfocysteine synthase