The mycolic acid reductase Rv2509 has distinct structural motifs and is essential for growth in slow‐growing mycobacteria

The final step in mycolic acid biosynthesis in Mycobacterium tuberculosis is catalysed by mycolyl reductase encoded by the Rv2509 gene. Sequence analysis and homology modelling indicate that Rv2509 belongs to the short‐chain fatty acid dehydrogenase/reductase (SDR) family, but with some distinct features that warrant its classification as belonging to a novel family of short‐chain dehydrogenases. In particular, the predicted structure revealed a unique α‐helical C‐terminal region which we demonstrated to be essential for Rv2509 function, though this region did not seem to play any role in protein stabilisation or oligomerisation. We also show that unlike the M. smegmatis homologue which was not essential for growth, Rv2509 was an essential gene in slow‐growing mycobacteria. A knockdown strain of the BCG2529 gene, the Rv2509 homologue in Mycobacterium bovis BCG, was unable to grow following the conditional depletion of BCG2529. This conditional depletion also led to a reduction of mature mycolic acid production and accumulation of intermediates derived from 3‐oxo‐mycolate precursors. Our studies demonstrate novel features of the mycolyl reductase Rv2509 and outline its role in mycobacterial growth, highlighting its potential as a new target for therapies. The Mycobacterium tuberculosis gene Rv2509 encodes a reductase enzyme involved in the last step of synthesis of mycolic acids, a key cell wall lipid. By generating a predicted model for the protein encoded by Rv2509, we show that the enzyme has distinct structural motifs including a distinct C‐terminal region, which we show by complementation studies to be vital for function. The gene is also essential for growth and viability underlining its potential as a new drug target.