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| Proposed pathway for the early steps of PIM biosynthesis in mycobacteria. (a) Chemical structure of PIM 2/6 and their acylated forms Ac 1/2 PIM 2 and Ac 1/2 PIM 6. (b) The two pathways originally proposed for the biosynthesis of Ac 1 PIM 2 in mycobacteria are shown: (i) PI is mannosylated to form PIM 1. PIM 1 is then mannosylated to PIM 2 , which is acylated to form Ac 1 PIM 2 ; (ii) PIM 1 is first acylated to Ac 1 PIM 1 and then mannosylated to Ac 1 PIM 2. Our experimental evidence indicates that although both pathways might co-exist in mycobacteria, the sequence of events PI-PIM 1-PIM 2-Ac 1 PIM 2 is favoured. As an important part of the literature concerning PIMs studies refers to the nomenclature based on the Mtb H37Rv sequences, we also include the Rv numbers to identify the proteins.
Source publication
The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs)
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... crystal structure of PatA from M. smegmatis was solved using single-wavelength anomalous dispersion with a K 2 PtCl 4 derivative at 2.06 Å resolution in C 2 space group (PatA-C16-1). Two other crystal forms were obtained in P 2 1 (PatA-C16-2) and P 4 2 2 1 2 (PatA-C16-3) space groups, and the corresponding crystal structures solved at 2.90 and 2.43 Å resolution, respectively, by using molecular replace- ment methods (see Methods and Supplementary Information for details; Supplementary Fig. 1; Supplementary Tables 1 and 2). The high quality of the electron density maps allowed the trace of residues 41 to 302 (PatA-C16-1), 48 to 295 (PatA-C16-2) and 48 to 303 (PatA-C16-3; Supplementary Fig. 2). ...
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... Interestingly, docking calculations put the myo-inositol and Manp moieties of PIM 1 in an equivalent position to that observed for PIM 2 , leaving free space in the pocket, a fact that might account for the acceptor substrate specificity of PatA ( Supplementary Fig. 10). To experimentally validate the proposed model, we designed three single-point substitution, E149A, R164A and H284A, predicted to impact the PIM 2 interaction with PatA ( Fig. 6c; Supplementary Fig. 8). ...
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... structure of CmGPAT is composed of two domains: (i) a helical domain comprising the first 78 residues of the protein displays a four-helix bundle architecture of unknown function, and (ii) an a/b domain, consisting of a nine-stranded continuous b-sheet surrounded by 11 a-helices (Fig. 7a). As depicted in Fig. 7a, the central b-sheet of CmGPAT superimposes well with that observed in PatA, with the exception of its outermost strands b 1 , b 2 and b 9 , which are missing in PatA (see also Supplementary Fig. 11). ...
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... region completely covered the narrowest and most hydrophobic of the three tunnels, tunnel-2 (Fig. 7b,c), which was proposed to be involved in fatty-acid recognition 38 . Strikingly, the acyl-binding pocket identified in the crystal structures of the palmitoyl-PatA and PatA-S-C16CoA complexes superimposed very well with tunnel-2 of CmGPAT ( Supplementary Fig. 11). On the basis of the experimental location of the S-C16CoA in PatA, a palmitoyl- CoA molecule was fitted into tunnel-2 and subjected to energy minimization ( Fig. 7d,e; Supplementary Fig. 11; see Methods for details Interestingly, the CoA-binding site observed in PatA is also conserved in CmGPAT. ...
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... the acyl-binding pocket identified in the crystal structures of the palmitoyl-PatA and PatA-S-C16CoA complexes superimposed very well with tunnel-2 of CmGPAT ( Supplementary Fig. 11). On the basis of the experimental location of the S-C16CoA in PatA, a palmitoyl- CoA molecule was fitted into tunnel-2 and subjected to energy minimization ( Fig. 7d,e; Supplementary Fig. 11; see Methods for details Interestingly, the CoA-binding site observed in PatA is also conserved in CmGPAT. Specifically, the acyl donor is located in the region corresponding to the entrance of the main groove (Fig. 7c,e). The two a-helices a 9 and a 10 , flanking the entrance of ...
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... is worth noting that when the individual palmitate molecules observed in the three crystal structures of palmitoyl-PatA were superimposed, they showed important structural flexibility at the carboxylate region ( Supplementary Fig. 3c), suggesting that the carboxylate group is not in a catalytically competent position in the crystal structures. Moreover, the palmitate is not a substrate neither an inhibitor of the reaction catalysed by PatA ( Supplementary Fig. 12). Our binary complexes correspond most likely to the hydrolysis reaction product of palmitoyl- CoA, thus one of the oxygen atoms found in the palmitate that interacts with H126 most likely come from a water molecule activated by H126. ...
