The low-molecular-weight protein tyrosine phosphatase (LMWPTPase) belongs to a distinctive class of phosphotyrosine phosphatases widely distributed among prokaryotes and eukaryotes. We report here the crystal structure of LMWPTPase of microbial origin, the first of its kind from Mycobacterium tuberculosis. The structure was determined to be two crystal forms at 1.9-and 2.5-Å resolutions. These structural forms are compared with those of the LMWPTPases of eukaryotes. Though the overall structure resembles that of the eukaryotic LMWPTPases, there are significant changes around the active site and the protein tyrosine phosphatase (PTP) loop. The variable loop forming the wall of the crevice leading to the active site is conformationally unchanged from that of mammalian LMWPTPase; however, differences are observed in the residues involved, suggesting that they have a role in influencing different substrate specificities. The single amino acid substitution (Leu12Thr [underlined below]) in the consensus sequence of the PTP loop, CTGNI CRS, has a major role in the stabilization of the PTP loop, unlike what occurs in mammalian LMWPTPases. A chloride ion and a glycerol molecule were modeled in the active site where the chloride ion interacts in a manner similar to that of phosphate with the main chain nitrogens of the PTP loop. This structural study, in addition to identifying specific mycobacterial features, may also form the basis for exploring the mechanism of the substrate specificities of bacterial LMWPTPases.

No abstract

No abstract

BackgroundThe Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppAMTB.Methodology/Principal FindingsOppAMTB was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppAMTB confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1β, IL-6 and TNF-α was also compromised after inactivation of oppD.ConclusionsTaken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis.

No abstract