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

Abstract

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.

Keywords: Mycobacterium; dehydrogenase; mycolic acid; reductase; tuberculosis.

Figure 1

Proposed reaction catalysed by Rv2509. The putative substrate is trehalose mono‐premycolate and the product is trehalose monomycolate. The examples shown here is that of a α‐mycolic acid from Mycobacterium tuberculosis [Colour figure can be viewed at https://www.wileyonlinelibrary.com]

Figure 2

A structural alignment of the predicted Rv2509 structure from Mycobacterium tuberculosis, with annotated secondary structure elements (top) with the sequences of the Mycobacterial and Corynebacterial homologues highlighting the unique sequence motifs of the Rv2509 subfamily of SDRs. The GxxQNIG motif along with RR pair identifies a novel NADP(H) binding consensus, limited to Mycobacteriaceae (highlighted in yellow). GxxSGIG is the wider consensus seen in the more remotely related Corynebacteria. Similarly, the unique Rossmann core‐stabilisation signature CANAGT reported here is also restricted to the Mycobacteriaceae (highlighted in yellow) and reverts to the more common (C)NNAG(I/F) in the more remote homologues. Active site residues N‐G‐S‐Y‐K in alpha‐helix 4 and alpha‐helix 5 are preserved throughout the family; however, a unique additional tyrosine residue can be seen to be present in Mycobacteriaceae (highlighted in yellow). The C‐terminal extension is also uniquely conserved across the wider Corynebacterial species

Figure 3

Complementation of the M. smegmatis ΔMSMEG4722 mutant with the Corynebacterial mycolyl reductase. TLC analysis of ¹⁴C‐labelled mycolic acid methyl esters (MAMEs) isolated from different strains. α; α MAMEs, α′; α′ MAMEs, e; epoxy MAMEs, O; origin, D, degradation products derived from 3‐oxo‐mycolate precursors. Solvent system; petroleum ether:acetone (95:5, v:v)

Figure 4

Visualisation of the predicted structure of Mycobacterium tuberculosis Rv2509 monomer. Two different orientations are given; side‐view and top‐down view towards the coenzyme binding site. The protein is coloured like a rainbow from blue (N‐terminus) to red (C‐terminus) and the main secondary structure elements are labelled, as well as the predicted C‐terminal extension domain is circled in a red oval. The location of the key sequence and structural motifs discussed in the text is also provided

Figure 5

TLC analysis of ¹⁴C‐labelled MAMEs isolated from the strains of M. smegmatis ΔMSMEG4722 mutant transformed with the different deletion constructs of Rv2509. α; α MAMEs, α′; α′ MAMEs, e; epoxy MAMEs, O; origin, D, degradation products derived from 3‐oxo‐mycolate precursors. Solvent system; petroleum ether:acetone (95:5, v:v)

Figure 6

Conditional depletion of the mycolyl reductase in BCG::P_Tet‐BCG2529 leads to the loss of growth. (a) growth in different concentrations of ATc in 7H9 broth, (b) growth on 7H10 agar containing 5 µg/ml of ATc. Control strain; BCG transformed with pMV261::rev‐tetR‐RBS‐D

Figure 7

TLC analysis of ¹⁴C‐labelled MAMEs isolated from BCG::P_Tet‐BCG2529 strain. α; α MAMEs, keto; keto MAMEs, O; origin, D, degradation products derived from 3‐oxo‐mycolate precursors. Solvent system; petroleum ether:acetone (95:5, v:v)