The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACP Pks13 ). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACP Pks13 ) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce ␣-alkyl -ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening.
BackgroundShort-chain dehydrogenases/reductases (SDRs) form one of the largest and oldest NAD(P)(H) dependent oxidoreductase families. Despite a conserved ‘Rossmann-fold’ structure, members of the SDR superfamily exhibit low sequence similarities, which constituted a bottleneck in terms of identification. Recent classification methods, relying on hidden-Markov models (HMMs), improved identification and enabled the construction of a nomenclature. However, functional annotations of plant SDRs remain scarce.ResultsWide-scale analyses were performed on ten plant genomes. The combination of hidden Markov model (HMM) based analyses and similarity searches led to the construction of an exhaustive inventory of plant SDR. With 68 to 315 members found in each analysed genome, the inventory confirmed the over-representation of SDRs in plants compared to animals, fungi and prokaryotes. The plant SDRs were first classified into three major types — ‘classical’, ‘extended’ and ‘divergent’ — but a minority (10% of the predicted SDRs) could not be classified into these general types (‘unknown’ or ‘atypical’ types). In a second step, we could categorize the vast majority of land plant SDRs into a set of 49 families. Out of these 49 families, 35 appeared early during evolution since they are commonly found through all the Green Lineage. Yet, some SDR families — tropinone reductase-like proteins (SDR65C), ‘ABA2-like’-NAD dehydrogenase (SDR110C), ‘salutaridine/menthone-reductase-like’ proteins (SDR114C), ‘dihydroflavonol 4-reductase’-like proteins (SDR108E) and ‘isoflavone-reductase-like’ (SDR460A) proteins — have undergone significant functional diversification within vascular plants since they diverged from Bryophytes. Interestingly, these diversified families are either involved in the secondary metabolism routes (terpenoids, alkaloids, phenolics) or participate in developmental processes (hormone biosynthesis or catabolism, flower development), in opposition to SDR families involved in primary metabolism which are poorly diversified.ConclusionThe application of HMMs to plant genomes enabled us to identify 49 families that encompass all Angiosperms (‘higher plants’) SDRs, each family being sufficiently conserved to enable simpler analyses based only on overall sequence similarity. The multiplicity of SDRs in plant kingdom is mainly explained by the diversification of large families involved in different secondary metabolism pathways, suggesting that the chemical diversification that accompanied the emergence of vascular plants acted as a driving force for SDR evolution.
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