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Substrate-induced conformational changes in the essential peripheral membrane-associated mannosyltransferase PimA from mycobacteria: implications for catalysis.

ABSTRACT: Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), which are key components of the mycobacterial cell envelope. PimA is the paradigm of a large family of peripheral membrane-binding GTs for which the molecular mechanism of substrate/membrane recognition and catalysis is still unknown. Strong evidence is provided showing that PimA undergoes significant conformational changes upon substrate binding. Specifically, the binding of the donor GDP-Man triggered an important interdomain rearrangement that stabilized the enzyme and generated the binding site for the acceptor substrate, phosphatidyl-myo-inositol (PI). The interaction of PimA with the beta-phosphate of GDP-Man was essential for this conformational change to occur. In contrast, binding of PI had the opposite effect, inducing the formation of a more relaxed complex with PimA. Interestingly, GDP-Man stabilized and PI destabilized PimA by a similar enthalpic amount, suggesting that they formed or disrupted an equivalent number of interactions within the PimA complexes. Furthermore, molecular docking and site-directed mutagenesis experiments provided novel insights into the architecture of the myo-inositol 1-phosphate binding site and the involvement of an essential amphiphatic alpha-helix in membrane binding. Altogether, our experimental data support a model wherein the flexibility and conformational transitions confer the adaptability of PimA to the donor and acceptor substrates, which seems to be of importance during catalysis. The proposed mechanism has implications for the comprehension of the peripheral membrane-binding GTs at the molecular level.

SUBMITTER: Guerin ME

PROVIDER: S-EPMC2755885 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Substrate-induced conformational changes in the essential peripheral membrane-associated mannosyltransferase PimA from mycobacteria: implications for catalysis.

Guerin Marcelo E ME Schaeffer Francis F Chaffotte Alain A Gest Petra P Giganti David D Korduláková Jana J van der Woerd Mark M Jackson Mary M Alzari Pedro M PM

The Journal of biological chemistry 20090611 32

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), which are key components of the mycobacterial cell envelope. PimA is the paradigm of a large family of peripheral membrane-binding GTs for which the molecular mechanism of substrate/membrane recognition and catalysis is still unknown. Strong evidence is provided showing that PimA undergoes significant conformational changes u ...[more]

PMID: 19520856

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Project description:Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.

Dataset Information

Substrate-induced conformational changes in the essential peripheral membrane-associated mannosyltransferase PimA from mycobacteria: implications for catalysis.

Publications

Substrate-induced conformational changes in the essential peripheral membrane-associated mannosyltransferase PimA from mycobacteria: implications for catalysis.

Similar Datasets

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