MYCOTHIOL BIOSYNTHESIS AND METABOLISM - CELLULAR-LEVELS OF POTENTIAL INTERMEDIATES IN THE BIOSYNTHESIS AND DEGRADATION OF MYCOTHIOL IN MYCOBACTERIUM-SMEGMATIS

Mycothiol (MSH; -L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside (Ac Cys-GlcN-Ins)) is a novel thiol produced at millimolar levels by mycob acteria and other actinomycetes that do not make glutathione. We devel oped 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 d ry weight) of AcCys (similar to 30), Cys-GlcN-Ins (similar to 8), and GlcN-Ins (similar to 100) but not Cys-GlcN (<3) or AcCys-GlcN (<80) du ring exponential growth in Middlebrook 7H9 medium. The level of GlcN-I ns declined 10-fold in stationary phase and similar to 5-fold in 7H9 m edium lacking glucose. Incubation in 10 mM AcCys produced 50- and 1000 -fold increases in cellular Cys and AcCys levels, respectively, a 10-f old decrease in GlcN-Ins and a transient 3-fold increase in Cys-GlcN-I ns. These results exclude Cys-GlcN and AcCys-GlcN as intermediates in MSH biosynthesis and implicate GlcN-Ins and Cys-GlcN-Ins as key interm ediates. Assay of GlcN-Ins/ATP-dependent ligase activity with Cys and AcCys as substrates revealed that Cys was at least an order of magnitu de better substrate. Based on the cellular measurements, MSH biosynthe sis involves assembly of GlcN-Ins, ligation with Cys to produce Cys-Gl cN-Ins, and acetylation of the latter to produce MSH.