Mycothiol biosynthesis and metabolism. Cellular levels of potential intermediates in the biosynthesis and degradation of mycothiol in mycobacterium smegmatis.

Mycothiol (MSH; 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D- glucop yranoside (AcCys-GlcN-Ins)) is a novel thiol produced at millimolar levels by mycobacteria and other actinomycetes that do not make glutathione. We developed methods to determine the major components of MSH (AcCys, Cys-GlcN, AcCys-GlcN, Cys-GlcN-Ins, GlcN-Ins) in cell extracts. Mycobacterium smegmatis was shown to produce measurable levels (nmol/g of residual dry weight) of AcCys ( approximately 30), Cys-GlcN-Ins (approximately 8), and GlcN-Ins (approximately 100) but not Cys-GlcN (<3) or AcCys-GlcN (<80) during exponential growth in Middlebrook 7H9 medium. The level of GlcN-Ins declined 10-fold in stationary phase and approximately 5-fold in 7H9 medium lacking glucose. Incubation in 10 mM AcCys produced 50- and 1000-fold increases in cellular Cys and AcCys levels, respectively, a 10-fold decrease in GlcN-Ins and a transient 3-fold increase in Cys-GlcN-Ins. These results exclude Cys-GlcN and AcCys-GlcN as intermediates in MSH biosynthesis and implicate GlcN-Ins and Cys-GlcN-Ins as key intermediates. Assay of GlcN-Ins/ATP-dependent ligase activity with Cys and AcCys as substrates revealed that Cys was at least an order of magnitude better substrate. Based on the cellular measurements, MSH biosynthesis involves assembly of GlcN-Ins, ligation with Cys to produce Cys-GlcN-Ins, and acetylation of the latter to produce MSH.