Identification of the dehydratase component of the mycobacterial mycolic acid-synthesizing fatty acid synthase-II complex

Abstract

Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall and are essential for survival. While most components of the fatty acid synthase-II (FAS-II) enzymic machinery that synthesizes these long chain a-alkyl, b-hydroxy fatty acids have been identified, the gene encoding the b-hydroxyacyl-acyl carrier protein (ACP) dehydratase activity has remained elusive. Recent bioinformatics-based studies and drug inhibition experiments have identified the M. tuberculosis gene Rv0636 as a promising candidate for this role. Using a recently described, specialized transduction-based genetic tool we now demonstrate that MSMEG1341, the Mycobacterium smegmatis homologue of Rv0636, is an essential gene; null mutants of the gene could only be generated in a merodiploid strain which contained a second integrated acetamide-inducible copy of MSMEG1341. Growth of the conditional mutant in the absence of acetamide resulted in loss of mycolic acid biosynthesis and eventually loss of viability due to cell lysis. Null MSMEG1341 mutants could also be generated in a M. smegmatis strain containing an integrated copy of Rv0636, indicating that Rv0636 was the functional counterpart of MSMEG1341 in M. tuberculosis. Our results demonstrate that MSMEG1341 is an essential gene involved in mycolic acid biosynthesis and encodes the FAS-II b-hydroxyacyl-ACP dehydratase.

Phosphorylation of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA Regulates Mycolic Acid Biosynthesis

“…This was further supported by analysis of an additional round of mass spectrometry of MabA_T21A/T114A/T191A pre-treated with ATP and PknB, which failed to identify any additional phosphate groups (data not shown). Taken together, these results indicate that Thr 21 , Thr 114 , and Thr 191 are likely to play critical roles in the regulation of MabA activity. However, as shown in Fig.…”

“…Thr 21 is part of the conserved glycine-rich loop (TGXXXGXG) involved in cofactor phosphate binding (42), so a mutation at this position is very likely to alter cofactor binding activity. Also, Thr 21 lies in the N-terminal part of the buried ␤ sheet ␤1, making it difficult to explain how a kinase interacts and phosphorylates this particular site.…”

“…MabA belongs to the family of short-chain dehydrogenases/ reductases (SDR) (40), and its three-dimensional structure revealed a conserved Rossman-fold and the Ser-Tyr-Lys catalytic triad conserved among short-chain dehydrogenases/reductase members (41). To gain insight into the possible effect(s) of Thr 21 , Thr 114 , and Thr 191 phosphorylation on the enzymatic behavior of MabA, we determined the positions of all three residues with respect to the substrate-binding site and the catalytic residues, taking advantage of the available three-dimensional structure of the protein (41). Interestingly, Thr 191 lies close to the substrate-and cofactor-binding site, implying that it may potentially be involved in ␤-ketoacyl-ACP reductase activity (41).…”

“…The fact that phosphorylation was not completely abrogated in MabA_T191A indicated the presence of additional phosphorylation sites. Therefore, the MabA_T191A protein was subjected to another round of mass spectrometric analysis after in vitro phosphorylation with PknB, which allowed us to identify Thr 21 as an additional phosphorylation site (Table 1 and supplemental Fig. S2).…”

“…During the second step of the elongation cycle, the resulting ␤-ketoacyl-ACP product is reduced by MabA, the NADPH-dependent ␤-ketoacyl reductase of M. tuberculosis (19,20). The resulting ␤-hydroxyacyl-ACP is then dehydrated by a set of dehydratases, HadABC (21,22), and finally reduced by the enoyl-ACP reductase, InhA, the primary target of isoniazid (23). The succeeding steps of condensation of the elongating chain with malonyl-ACP units are performed by the ␤-ketoacyl-ACP synthases KasA and KasB (8,24,25), leading to very long-chain meromycolyl-ACPs (up to C 56 ), which are the direct precursors of mycolic acids.…”