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.