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

摘要

The biosynthesis of cysteine is a crucial metabolic pathway supplying a building block for de novo protein synthesis but also a reduced thiol as a component of the oxidative defense mechanisms that appear particularly vital in the dormant state of Mycobacterium tuberculosis. We here show that the cysteine synthase CysM is, in contrast to previous annotations, an O-phosphoserine-specific cysteine synthase. CysM belongs to the fold type II pyridoxal 5′-phosphate-dependent enzymes, as revealed by the crystal structure determined at 2.1-Å resolution. A model of O-phosphoserine bound to the enzyme suggests a hydrogen bonding interaction of the side chain of Arg220 with the phosphate group as a key feature in substrate selectivity. Replacement of this residue results in a significant loss of specificity for O-phosphoserine. Notably, reactions with sulfur donors are not affected by the amino acid replacement. The specificity of CysM toward O-phosphoserine together with the previously established novel mode of sulfur delivery via thiocarboxylated CysO (Burns, K. E., Baumgart, S., Dorrestein, P. C., Zhai, H., McLafferty, F. W., and Begley, T. P. (2005) J. Am. Chem. Soc. 127, 11602–11603) provide strong evidence for an O-phosphoserine-based cysteine biosynthesis pathway in M. tuberculosis that is independent of both O-acetylserine and the sulfate reduction pathway. The existence of an alternative biosynthetic pathway to cysteine in this pathogen has implications for the design strategy aimed at inhibition of this metabolic route. The biosynthesis of cysteine is a crucial metabolic pathway supplying a building block for de novo protein synthesis but also a reduced thiol as a component of the oxidative defense mechanisms that appear particularly vital in the dormant state of Mycobacterium tuberculosis. We here show that the cysteine synthase CysM is, in contrast to previous annotations, an O-phosphoserine-specific cysteine synthase. CysM belongs to the fold type II pyridoxal 5′-phosphate-dependent enzymes, as revealed by the crystal structure determined at 2.1-Å resolution. A model of O-phosphoserine bound to the enzyme suggests a hydrogen bonding interaction of the side chain of Arg220 with the phosphate group as a key feature in substrate selectivity. Replacement of this residue results in a significant loss of specificity for O-phosphoserine. Notably, reactions with sulfur donors are not affected by the amino acid replacement. The specificity of CysM toward O-phosphoserine together with the previously established novel mode of sulfur delivery via thiocarboxylated CysO (Burns, K. E., Baumgart, S., Dorrestein, P. C., Zhai, H., McLafferty, F. W., and Begley, T. P. (2005) J. Am. Chem. Soc. 127, 11602–11603) provide strong evidence for an O-phosphoserine-based cysteine biosynthesis pathway in M. tuberculosis that is independent of both O-acetylserine and the sulfate reduction pathway. The existence of an alternative biosynthetic pathway to cysteine in this pathogen has implications for the design strategy aimed at inhibition of this metabolic route. In the dormant phase Mycobacterium tuberculosis is surviving within granulomas in the lungs of infected individuals where the environment is characterized by hypoxia, nutrient starvation, and oxidative stress (1Wayne L.G. Eur. J. Clin. Microbiol. Infect. Dis. 1994; 13: 908-914Crossref PubMed Scopus (319) Google Scholar, 2Zhang Y. Front. Biosci. 2004; 9: 1136-1156Crossref PubMed Scopus (160) Google Scholar, 3Fenton M.J. Vermeulen M.W. Infect. Immun. 1996; 64: 683-690Crossref PubMed Google Scholar). The latter involves nitrogen monoxide and its spontaneously formed derivatives (reactive nitrogen intermediates), which are used by phagocytic cells to kill internalized bacteria (4MacMicking J.D. North R.J. LaCourse R. Mudgett J.S. Shah S.K. Nathan C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5243-5248Crossref PubMed Scopus (911) Google Scholar). The prime targets of reactive nitrogen intermediate