35 ± 2.76 μM and for malonyl-RedQ 6.73 ± 0.31 μM). However, no detectable activities were observed with any other pairing (limit of detection

was < 1% of activity observed with acetyl-CoA and malonyl-RedQ), demonstrating that neither isobutyryl-CoA nor malonyl-FabC are substrates for RedP. These observations demonstrate a clear substrate preference for RedP and provide biochemical evidence to support the role of RedP catalyzing the first step in the biosynthesis of the undecylpyrrole component of undecylprodiginine. The specificity for acetyl-CoA plays a key role in controlling the formation of a straight-chain dodecanoyl-ACP and thus the formation of acetate-derived alkyl prodiginines in S. coelicolor. The RedP specificity for malonyl-RedQ demonstrates that the process to generate acetate-derived alkyl prodiginines via a dodecanoyl-ACP (Fig. 1) occurs H 89 price Ivacaftor using a dedicated ACP. We have recently demonstrated that RedJ is a thioesterase that can catalyze the hydrolysis of dodecanoyl-RedQ to provide dodecanoic acid (Whicher et al., 2011),

and genetic evidence has shown that it is converted to undecylpyrrole by the actions of RedL and RedK (Mo et al., 2008). RedJ has been demonstrated to have much greater activity with longer-chain acyl substrates (up to C10 in length) and to efficiently discriminate between acyl-RedQ substrates and other acyl-ACPs. This ACP selectivity is thus observed at both the first (RedP) and the last step (RedJ) in the formation of dodecanoic acid for prodiginine biosynthesis and presumably plays a key role in keeping this process and the fatty acid biosynthetic process separate. An 80% decrease in prodiginine production upon deletion of redP in S. coelicolor (SJM1) indicates that RedP is an important enzyme for prodiginine biosynthesis, but not essential (Mo et al., 2005). A significant restoration of prodiginine biosynthesis is observed in SJM1 with plasmid-based expression of FabH, indicating that FabH can function in place of RedP. The specificity of RedP and RedJ for malonyl-RedQ would predict that in order to support prodiginine biosynthesis, FabH should be able

to utilize malonyl-RedQ as well as malonyl-FabC. The streptomycetes FabH was initially assayed using the E. coli Thiamine-diphosphate kinase ACP to generate the malonyl-ACP. The cognate ACP from streptomycetes (FabC) was not used in these assays. Isobutyryl-CoA was observed to have a threefold slower Vmax than acetyl-CoA, and a lower Km (Han et al., 1998). In this study, we sought to extend these analyses to include both the cognate ACP (malonyl-FabC) and malonyl-RedQ. As shown in Table 1, a lower Km (1.74 μM) for isobutyryl-CoA than acetyl-CoA (8.36 μM) was also observed using malonyl-FabC. However, in this case, the overall reaction rate (kcat) was 10 times faster for isobutyryl-CoA in comparison with acetyl-CoA (Table 1 and Fig. 2). The FabH is approximately 50-fold more efficient using isobutyryl-CoA vs.