Ppm1, a novel polyprenol monophosphomannose synthase from Mycobacterium tuberculosis

Abstract

Dolichol monophosphomannose (DPM) is an ever-present donor of mannose (Man) in various eukaryotic glycosylation processes. Intriguingly, the related polyprenol monophosphomannose (PPM) is involved in the biosynthesis of lipomannan and lipoarabinomanan, key bacterial factors termed modulins that are found in mycobacteria. Based on similarities to known DPM synthases, we have identified and characterized the PPM synthase of Mycobacterium tuberculosis, now termed Mt-Ppm1. In the present study, we demonstrate that Mt-Ppm1 possesses an unusual two-domain architecture, by which the second domain is sufficient for PPM synthesis. However, when overexpressed separately in mycobacteria, domain 1 of Mt-Ppm1 appears to increase the synthesis of PPM. Interestingly, other mycobacteria such as M. smegmatis, M. avium and M. leprae produce two distinct proteins, which are similar to the two domains found in Mt-Ppm1. Using an in vitro assay, we also demonstrate that Mt-Ppm1 transfers Man from GDP-Man to a structurally diverse range of lipid monophosphate acceptors. The identification of the PPM synthase as a key enzyme in lipoarabinomannan biosynthesis now provides an attractive candidate for gene disruption to generate mutants for subsequent immunological studies. PPM synthase can also be exploited as a target for specific inhibitors of M. tuberculosis.

Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold ‘em, knowing when to fold ‘em

“…In fact, there are two Lnt homologues in S. coelicolor, SCO1014 and SCO1336. SCO1014 also shows homology to a domain associated with mycobacterial polyprenol monophosphomannose (PPM) synthases [42]. Notably M. tuberculosis Ppm is a two-domain protein (Rv2051c) in which the Nterminus is similar to Lnt and the C-terminus has PPM synthase activity [42].…”

“…SCO1014 also shows homology to a domain associated with mycobacterial polyprenol monophosphomannose (PPM) synthases [42]. Notably M. tuberculosis Ppm is a two-domain protein (Rv2051c) in which the Nterminus is similar to Lnt and the C-terminus has PPM synthase activity [42]. PPM is an alkali-stable sugar donor used in the formation of the cell envelope glycolipids lipomannan (LM) and lipoarabinomannan (LAM) in M. tuberculosis [42].…”

“…Notably M. tuberculosis Ppm is a two-domain protein (Rv2051c) in which the Nterminus is similar to Lnt and the C-terminus has PPM synthase activity [42]. PPM is an alkali-stable sugar donor used in the formation of the cell envelope glycolipids lipomannan (LM) and lipoarabinomannan (LAM) in M. tuberculosis [42]. The function of these mycobacterial Lnt homologues might instead relate to their belonging to the CN hydrolase enzyme superfamily rather than their being directly orthologous to E. coli Lnt.…”

Novel prenyl-linked benzophenone substrate analogues of mycobacterial mannosyltransferases

“…Sequences corresponding to the C-terminal domain of the Rv2051c gene product (Mt-ppm/D2) required for cloning into pUC8 were obtained by PCR using Vent polymerase (New England Biolabs, Beverly, MA, U.S.A.) as described previously [22]. Liquid cultures of Escherichia coli (pUC8-Mt-ppm1/D2) were grown in LB broth at 37 • C with 100 µg/ml ampicillin to an A 600 0.4 and induced for 4 h with 1 mM isopropyl β-D-thiogalactoside [22].…”

“…Besra et al [17] demonstrated that Ac 1 PIM 2 (according to the nomenclature of Kordulakova et al [21]) is specifically extended by an addition of Manp residues from the alkali-stable sugar donor C 50 /C 40 /C 35 -PPM (where PPM stands for polyprenol monophosphomannose) to form higher PIMs and further to a 'linear' α(1 → 6)-LM via a PPM-dependent α(1 → 6)mannosyltransferase. The generation of PPM in M. tuberculosis H37Rv results from the transfer of Manp from GDP-Manp to polyprenyl phosphates catalysed by the C-terminal domain(s) of a PPM synthase (Mt-Ppm1), now termed as Mt-Ppm1/D2 [22]. However, the identity of the mycobacterial PPM-dependent α(1 → 6)mannosyltransferase is unknown.…”