A Streptomyces collinus thiolase with novel Acetyl-CoA : Acyl carrier protein transacylase activity

Acetyl-CoA:acyl carrier protein (ACP) transacylase (ACT) activity has been demonstrated for the 3-ketoacyl-ACP synthase III (KASIII) which initiates f atty acid biosynthesis in the type II dissociable fatty acid synthases of p lants and bacteria. Several lines of evidence have indicated the possibilit y of ACT activity being associated with proteins other than KASIII. Using a crude extract of Streptomyces collinus, we have resolved from KASIII an ad ditional protein with ACT activity and subsequently purified it 85-fold in five chromatographic steps. The 45 kDa protein was shown by gel filtration to have a molecular mass of 185 +/- 35 kDa, consistent with a homotetrameri c structure for the native enzyme. The corresponding gene (fadA) was cloned and sequenced and shown to encode a protein with amino acid sequence homol ogy to type II thiolases. The fadA was expressed in Escherichia coli, and t he resulting recombinant FadA enzyme purified by metal chelate chromatograp hy was shown to have both ACT and thiolase activities. Kinetic studies reve aled that in an ACT assay FadA had a substrate specificity for a two-carbon acetyl-CoA substrate (K-m 8.7 +/- 1.4 muM) but was able to use ACPs from b oth type II fatty acid and polyketide synthases (Streptomyces glaucescens F abC ACP, K-m 10.7 +/- 1.4 muM; E. coli FabC ACP, K-m 8.8 +/- 2 muM; FrenN A CP, K-m 44 +/- 12 muM). In the thiolase assay kinetic analyses revealed sim ilar K-m values for binding of substrates acetoacetyl-CoA (K-m 9.8 +/- 0.8 muM) and CoA (K-m 10.9 +/- 1.8 muM). A Cys92Ser mutant of FadA possesed vir tually unchanged K-m values for acetoacetyl-CoA and CoA but had a greater t han 99% decrease in k(cat) for the thiolase activity. No detectable ACT act ivity was observed for the Cys92Ser mutant, demonstrating that both activit ies are associated with FadA and likely involve formation of the same coval ent acetyl-S-Cys enzyme intermediate. An ACT activity with ACP has not prev iously been observed for thiolases and in the case of the S. collinus FadA is significantly lower (k(cat) 3 min(-1)) than the thiolase activity of Fad A (k(cat) 2170 min(-1)). The ACT activity of FadA is comparable to the KAS activity and significantly higher than the ACT activity, reported for a str eptomycete KASIII.