The missing piece of the type II fatty acid synthase system from Mycobacterium tuberculosis

Abstract

The Mycobacterium tuberculosis fatty acid synthase type II (FAS-II) system has the unique property of producing unusually long-chain fatty acids involved in the biosynthesis of mycolic acids, key molecules of the tubercle bacillus. The enzyme(s) responsible for dehydration of (3R)-hydroxyacyl-ACP during the elongation cycles of the mycobacterial FAS-II remained unknown. This step is classically catalyzed by FabZ- and FabA-type enzymes in bacteria, but no such proteins are present in mycobacteria. Bioinformatic analyses and an essentiality study allowed the identification of a candidate protein cluster, Rv0635-Rv0636-Rv0637. Its expression in recombinant Escherichia coli strains leads to the formation of two heterodimers, Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC), which also occurs in Mycobacterium smegmatis, as shown by split-Trp assays. Both heterodimers exhibit the enzymatic properties expected for mycobacterial FAS-II dehydratases: a marked specificity for both long-chain (>or=C(12)) and ACP-linked substrates. Furthermore, they function as 3-hydroxyacyl dehydratases when coupled with MabA and InhA enzymes from the M. tuberculosis FAS-II system. HadAB and HadBC are the long-sought (3R)-hydroxyacyl-ACP dehydratases. The correlation between the substrate specificities of these enzymes, the organization of the orthologous gene cluster in different Corynebacterineae, and the structure of their mycolic acids suggests distinct roles for both heterodimers during the elongation process. This work describes bacterial monofunctional (3R)-hydroxyacyl-ACP dehydratases belonging to the hydratase 2 family. Their original structure and the fact that they are essential for M. tuberculosis survival make these enzymes very good candidates for the development of antimycobacterial drugs.

Fig. 1.

Protein purification. SDS/PAGE and gel filtration chromatograms. The cloned genes are mentioned. Lane a, total soluble proteins; lane b, Ni Sepharose fraction; lanes c and d, Superdex 75 fractions. Monomeric sizes of proteins: H-HadA, 18.3 kDa; HadB, 14.8 kDa; H-HadB, 15.9 kDa; HadC, 18.9 kDa.

Fig. 2.

Split-Trp growth assay in M. smegmatis. Recombinant M. smegmatis ΔhisA-expressing pairs of proteins fused to Ntrp and Ctrp were diluted 1- or 10-fold in water and spotted in parallel onto minimal medium and medium plus Trp. The pair of Ntrp-Esat6 and Cfp10-Ctrp was used as a positive control (the early secreted T cell antigens Esat6 and Cfp10 from Mtb form a tight, 1:1 complex). Negative controls and extra positive controls were performed (see SI Materials and Methods). Images were taken after a 3-week incubation.

Fig. 3.

Chain length specificity profiles of HadAB and HadBC heterodimers. Assays were performed at fixed concentrations of substrate and enzyme. (A and B) HadAB with 2.5 μM enoyl-CoA (A) or 25 μM enoyl-CoA (B). (C) HadBC with 25 μM enoyl-CoA. Data are means ± SD. The activities measured for both heterodimers in the presence of short chain substrates (C₄ and C₈) were not significantly above the control values (without enzyme). The large SDs obtained sometimes for long-chain substrates are due to solubility problems.

Fig. 4.

Comparison of the specific activities in the presence of octenoyl-CoA and octenoyl-ACP. Assays were performed at 2 μM substrate and fixed concentrations of enzymes HadAB (A) and HadBC (B). Data are means ± SD. The activities measured for both heterodimers in the presence of C8:1-CoA were not significantly above the control values (without enzyme).

Fig. 5.

Coupled assay of HadAB in the presence of MabA and InhA. Shown are the results of MALDI-TOF MS analyses of the reaction media containing 3-ketododecanoyl-CoA, NADPH, NADH, and MabA plus InhA (A), or plus 280 nM HadAB (B), or plus both HadAB and InhA (C). In A and B, the peaks at m/z 966, 988, 1,010, 1,032, and 1,054 stand for [M+H]⁺, [M+Na]⁺, [M-H+2Na]⁺, [M-2H+3Na]⁺, and [M-3H+4Na]⁺ ions of 3-hydroxydodecanoyl-CoA (product of MabA), respectively. In B, the minor peaks at m/z 948, 970, 992, 1,014, and 1,036 stand for [M+H]⁺ ion and the monosodium to tetrasodium adducts of the unsaturated species, dodecenoyl-CoA. In C, the peaks at m/z 950, 972, 994, 1,016, and 1,038 stand for [M+H]⁺ ion and the monosodium to tetrasodium adducts of the saturated species, dodecanoyl-CoA. Insets in A and C display the kinetics of the respective reactions followed at 340 nm (oxidation of InhA coenzyme, NADH). The three spectra correspond to the time points of 3 min of reaction.

Fig. 6.

Model of the roles of HadAB and HadBC heterodimers in the MA biosynthesis pathway. HadAB would take part, like KasA, in the early FA elongation cycles catalyzed by the FAS-II system, leading to the formation of the intermediate-size (C₂₂–C₄₂) meromycolic chains and consequently to medium-length MAs found in Rhodococcus and Nocardia. In mycobacteria, HadBC, like KasB, would elongate further the intermediate-size meromycolic chains to full-size molecules (C₅₂–C₆₄) during the late elongation cycles performed by FAS-II, resulting in the synthesis of eumycolic acids (C₇₄–C₉₀). For clarity, only some proteins of the FAS-II system are mentioned.