title: Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis complex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria.
abstract: Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.
authors.affiliation: Tuberculosis Research Group, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone KT15 3NB, United Kingdom. p.wheeler@vla.defra.gsi.gov.uk
authors.forename: Paul R
authors.initials: PR
authors.lastname: Wheeler
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Coldham
authors.forename: Lisa
authors.initials: L
authors.lastname: Keating
authors.forename: Stephen V
authors.initials: SV
authors.lastname: Gordon
authors.forename: Esen E
authors.initials: EE
authors.lastname: Wooff
authors.forename: Tanya
authors.initials: T
authors.lastname: Parish
authors.forename: R Glyn
authors.initials: RG
authors.lastname: Hewinson
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D001692:Biological Transport; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003542:Cystathionine gamma-Lyase / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004187:Disaccharides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D016678:Genome; D016680:Genome, Bacterial; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D006020:Glycopeptides; D006714:Homoserine / Q000737:chemistry; D007294:Inositol; D007477:Ions; D007700:Kinetics; D008715:Methionine / Q000737:chemistry*; D008956:Models, Chemical; D009154:Mutation; D009163:Mycobacterium bovis / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011720:Pyrazoles / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013455:Sulfur / Q000737:chemistry*; D013997:Time Factors; D014461:Ultracentrifugation; D014466:Ultraviolet Rays
chemical_list: D000480:Alkynes; D000596:Amino Acids; D004187:Disaccharides; D006020:Glycopeptides; D007477:Ions; D011720:Pyrazoles; D013438:Sulfhydryl Compounds; C089265:mycothiol; D007294:Inositol; C009055:propargylglycine; D006714:Homoserine; D013455:Sulfur; D008715:Methionine; C022696:O-succinylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases; D003542:Cystathionine gamma-Lyase; D003545:Cysteine; D005998:Glycine