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 ␥-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine ␥-synthase (CGS), but demonstration of a ␥-elimination reaction with cystathionine as well as the ␥-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.

Monocyte Differentiation, Activation, and Mycobacterial Killing Are Linked to Transsulfuration-dependent Redox Metabolism

“…Interestingly, unlike the host, Mycobacterium does not accumulate cysteine to detectable levels. Excess cysteine is apparently eliminated by an active cysteine desulfhydrase, suggesting that cysteine may be toxic (29). The metabolic interplay between the host and the sulfur needs of pathogen is therefore likely to play an important role in modulating the outcome of their interaction.…”

“…Mycobacteria obtain sulfur either via uptake of sulfate (which is assimilated into cysteine or homocysteine (Fig. 1) or methionine, with the latter being the preferred sulfur source in the host (29). Methionine is subsequently converted to cysteine via the transsulfuration pathway.…”

Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria

“…14 C]cysteine was converted to pyruvate by Cys desulfhydrase (15,156), recently identified as Rv3025c in M. tuberculosis (131). Pyruvate was metabolized through the Krebs cycle, with the loss of label as carbon dioxide.…”

Mycothiol Import by Mycobacterium smegmatis and Function as a Resource for Metabolic Precursors and Energy Production

“…Cysteine desulfhydrase activity was assayed as described for extracts of M. tuberculosis by Wheeler et al (39). The activity was determined in dialyzed M. smegmatis mc 2 155 extracts by the analysis of pyruvate as the 1,2-diamino-4,5-dimethoxybenzene (DDB; Invitrogen) derivative.…”

“…Wheeler et al (39) have measured a cysteine desulfhydrase activity in extracts of Mycobacterium bovis and M. tuberculosis that generates pyruvate as the product. Since conversion of Cys to pyruvate was a plausible step in conversion of Cys to CO 2 , we assayed the dialyzed extract of the Tn1 mutant for cysteine desulfhydrase activity.…”