The genus Mycobacterium contains over one hundred species, many of which are true or opportunistic pathogens causing significant disease in humans. Tuberculosis (TB), one of the world’s leading infectious killers, is caused by the mycobacterial species Mycobacterium tuberculosis (Mtb). Multidrug-resistant tuberculosis (MDR-TB) has become increasingly difficult to treat, often requiring second-line drug treatments ranging from 9 to 20 months. The current catastrophic cost of containing the TB epidemic highlights the necessity to investigate possible targets for new antimicrobial drugs against Mtb.

Bacterial cell walls contain a protective layer of peptidoglycan (PG) that gives the cells structure and support. Most PG layers in bacterial cell walls are cross linked via a 4→3 crosslink that is catalyzed by enzymes known as D,D-transpeptidases (commonly known as penicillin binding proteins, PBPs). These enzymes are typically targeted by common antibiotics such as β-lactams like penicillins. However, mycobacterial species contain unique 3→3 PG cross links, and the nonhomologous L,D transpeptidases (Ldts) are the enzymes that catalyze these 3→3 crosslinks in the PG layer of mycobacteria. Mycobacteria have up to six Ldt enzymes, but the reason for this apparent redundancy is not well understood, in part because putative Ldt substrates are notoriously challenging to access.

Toward understanding the physiological roles of mycobacterial Ldts in order to design more effective drugs targeting their function, we will use PG biosynthetic enzymes to produce putative Ldt substrates in vitro. PG biosynthesis begins with the activated sugar nucleotide UDP-GlcNAc. MurA is a UDP-GlcNAc enolpyruvyl transferase and is the first enzyme in the PG biosynthetic pathway. Literature indicates that the S368P MurA variant is more active than the wild-type version of the enzyme. Furthermore, the protein CwlM, when phosphorylated by the serine/threonine protein kinase PknB, allosterically activates MurA more than 30-fold. Thus, MurA, together with PknB and CwlM, generate suitable amounts of product required for the subsequent enzymatic synthesis of Ldt substrates.

Here, we present the work our lab has undertaken toward accessing active MurA for synthesizing putative mycobacterial L,D-transpeptidase substrates. Our research is being conducted in an effort to understand the apparent redundancy of mycobacterial Ldt enzymes and to search for opportunities for antimicrobial development.

Colby MaGill

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