coelicolor FabH with the acetyl-CoA-specific E. coli FabH (YL1/ecFabH mutant) results in a dramatic shift to a fatty acid profile of predominantly straight-chain fatty acids (Li et al., 2005). As predicted, FabH was able to use malonyl-RedQ in place of malonyl-FabC. Under saturating malonyl-RedQ VX-765 order conditions, FabH was able to use either acetyl-CoA or isobutyryl-CoA (Table 1). The Km values for each of these were comparable to those observed using

malonyl-FabC, and again there was almost a 40-fold higher catalytic efficiency (kcat/Km) for isobutyryl-CoA compared to acetyl-CoA. However, for both acyl-CoA substrates, the reaction rate kcat was at least 20-fold less using malonyl-RedQ vs. malonyl-FabC (Fig. 2). At fixed isobutyryl-CoA and acetyl-CoA concentrations and variable malonyl-RedQ or malonyl-FabC IDH inhibitor cancer concentrations, similar sets of observations were made. Greater catalytic efficiency was seen with isobutyryl-CoA relative to acetyl-CoA, and for each acyl-CoA substrate, the apparent reaction rate was much faster using malonyl-FabC than with malonyl-RedQ.

This set of analyses also demonstrated that the apparent Km for malonyl-FabC (4.53 μM) and malonyl-RedQ (7.80 μM) was comparable. Thus, the difference in overall catalytic efficiency of FabH using malonyl-ACP substrates arises predominantly from differences in apparent catalytic rates rather than Km values. The ability of FabH to utilize malonyl-RedQ and to have a preference for isobutyryl-CoA Thalidomide is consistent with a) genetic data which suggest that FabH can initiate prodiginine biosynthesis in SJM1, the S. coelicolor redP deletion mutant, and b) the observation of a significant

increase in branched-chain alkyl prodiginines in the SJM1 mutant relative to the wild-type S. coelicolor (Mo et al., 2005). A final observation from these analyses is that the maximal kinetic efficiency of FabH (kcat/Km of 9.84 μM−1 min−1 using isobutyryl-CoA and malonyl-FabC) is 66-fold higher than that of RedP (kcat/Km of 0.147 μM−1 min−1 using acetyl-CoA and malonyl-RedQ). This difference might arise from the ability of FabH to utilize isobutyryl-CoA (the enzymes have comparable efficiencies using acetyl-CoA), or because FabH is a primary metabolic enzyme. Initial characterization of many FabH enzymes, including those from streptomycetes, was carried out with a commercially available E. coli ACP (Han et al., 1998; Choi et al., 2000a, b; Khandekar et al., 2001). Subsequent work has revealed that these enzymes have ACP specificity. Improved catalytic activity and in some cases apparent changes in acyl group specificity can be observed when assays are performed using malonyl-ACP generated from the cognate ACP (Florova et al., 2002; Brown et al., 2005).