Identification of an Amidotransferase Involved in Mycobacterium tuberculosis Peptidoglycan Synthesis

Abstract

Peptidoglycan (PG) is a complex polymer providing shape and the strength to withstand osmotic pressure in bacteria. PG from Mycobacterium tuberculosis, Escherichia coli and bacilli is classified as A1γ and is composed of linear chains of N-acetyl glucosamine and N-acetyl muramic acid modified by a peptide side-chain (L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala) that may be cross-linked to the peptide side-chain of other glycan strand. However, there are differences in PG from these organisms; in E. coli PG is not amidated, whereas most bacilli and mycobacteria have one or more amidated amino acids per unit. Most of the enzymes associated with PG biosynthesis have been identified; exceptions include the enzyme(s) responsible for amidation of the peptides of PG. We identified Rv2201 as a potential amidotranferase involved in the synthesis of M. tuberculosis PG based on similarity to the Asn synthetase family of enzymes and the presence of orthologs in species known to have amidated PG. Rv2201 is reported to be essential in M. tuberculosis and orthologs to be associated with lysozyme and drug sensitivity in other Actinomycetales. We cloned and expressed Rv2201 in E. coli, and strains expressing recombinant Rv2201 synthesized modified lipid II with properties similar to amidated lipid II isolated from mycobacteria. Since our earlier studies indicated that lipid II is likely the substrate for amidation in mycobacteria we hypothesize that Rv2201 represents a novel amidotransferase involved in the biosynthesis of amidated PG in M. tuberculosis and may represent a novel drug target. Supported by NIAID grants AI18357 and AI49151