All four Mycobacterium tuberculosis glnA genes encode glutamine synthetase activities but only GlnA1 is abundantly expressed and essential for bacterial homeostasis

Abstract

Glutamine synthetases (GS) are ubiquitous enzymes that play a central role in every cell's nitrogen metabolism. We have investigated the expression and activity of all four genomic Mycobacterium tuberculosis GS - GlnA1, GlnA2, GlnA3 and GlnA4 - and four enzymes regulating GS activity and/or nitrogen and glutamate metabolism - adenylyl transferase (GlnE), gamma-glutamylcysteine synthase (GshA), UDP-N-acetylmuramoylalanine-D-glutamate ligase (MurD) and glutamate racemase (MurI). All eight genes are located in multigene operons except for glnA1, and all are transcribed in M. tuberculosis; however, some are not translated or translated at such low levels that the enzymes escape detection. Of the four GS, only GlnA1 can be detected. Each of the eight genes, as well as the glnA1-glnE-glnA2 cluster, was expressed separately in Mycobacterium smegmatis, and its gene product was characterized and assayed for enzymatic activity by analysing the reaction products. In M. smegmatis, all four recombinant-overexpressed GS are multimeric enzymes exhibiting GS activity. Whereas GlnA1, GlnA3 and GlnA4 catalyse the synthesis of L-glutamine, GlnA2 catalyses the synthesis of D-glutamine and D-isoglutamine. The generation of mutants in M. tuberculosis of the four glnA genes, murD and murI demonstrated that all of these genes except glnA1 are nonessential for in vitro growth. L-methionine-S,R-sulphoximine (MSO), previously demonstrated to inhibit M. tuberculosis growth in vitro and in vivo, strongly inhibited all four GS enzymes; hence, the design of MSO analogues with an improved therapeutic to toxic ratio remains a promising strategy for the development of novel anti-M. tuberculosis drugs.