A Streptomyces collinus Thiolase with Novel Acetyl-CoA:Acyl Carrier Protein Transacylase Activity^,

Abstract

Acetyl-CoA:acyl carrier protein (ACP) transacylase (ACT) activity has been demonstrated for the 3-ketoacyl-ACP synthase III (KASIII) which initiates fatty acid biosynthesis in the type II dissociable fatty acid synthases of plants and bacteria. Several lines of evidence have indicated the possibility of ACT activity being associated with proteins other than KASIII. Using a crude extract of Streptomyces collinus, we have resolved from KASIII an additional 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 homotetrameric structure for the native enzyme. The corresponding gene (fadA) was cloned and sequenced and shown to encode a protein with amino acid sequence homology to type II thiolases. The fadA was expressed in Escherichia coli, and the resulting recombinant FadA enzyme purified by metal chelate chromatography was shown to have both ACT and thiolase activities. Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for a two-carbon acetyl-CoA substrate (K(m) 8.7 +/- 1.4 microM) but was able to use ACPs from both type II fatty acid and polyketide synthases (Streptomyces glaucescens FabC ACP, K(m) 10.7 +/- 1.4 microM; E. coli FabC ACP, K(m) 8.8 +/- 2 microM; FrenN ACP, K(m) 44 +/- 12 microM). In the thiolase assay kinetic analyses revealed similar K(m) values for binding of substrates acetoacetyl-CoA (K(m) 9.8 +/- 0.8 microM) and CoA (K(m) 10.9 +/- 1.8 microM). A Cys92Ser mutant of FadA possessed virtually unchanged K(m) values for acetoacetyl-CoA and CoA but had a greater than 99% decrease in k(cat) for the thiolase activity. No detectable ACT activity was observed for the Cys92Ser mutant, demonstrating that both activities are associated with FadA and likely involve formation of the same covalent acetyl-S-Cys enzyme intermediate. An ACT activity with ACP has not previously 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 FadA (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 streptomycete KASIII.

Alteration of the Fatty Acid Profile of Streptomyces coelicolor by Replacement of the Initiation Enzyme 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

“…The E. coli and S. glaucescens ACPs were obtained as described previously (12). Malonyl-CoA and CoASH were obtained from Sigma Chemical Co. (St. Louis, MO).…”

1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

“…For saFabH assays, MACP was prepared from Streptomyces glaucescens ACP (FabC). FabC was used in these studies because it was readily generated in our laboratory (42) and because a reliable commercial source for E. coli ACP was no longer available. We observed a K m for MACP (FabC) of 2.4 Ϯ 0.5 M in a saFabH-coupled assay, which is indistinguishable from the 1.76 Ϯ 0.4 M value previously reported for the assay with purified MACP made from E. coli ACP (27).…”

Characterization of FabG and FabI of the Streptomyces coelicolor Dissociated Fatty Acid Synthase

“…FabC, RedQ, and E. coli ACP were sourced as described previously. [1,30,31] Preparation of malonyl-ACPs and crotonoyl-ACPs was carried out as described previously. [14] S. coelicolor and E. coli fabG: The genes encoding SCO1815, SCO1345, and SCO1346 (fabG) were amplified from the appropriate S. coelicolor cosmids by PCR by using the primers 5'-CATATG AGCCGC TCGGTT CTC-3' and 5'-GGATCC TCAGTG ACCCAT TCCCAG TCC-3' (SCO1815); 5'-CATATG ACTGAA CTGCCC GAGCCC TCC-3' and 5'-GGATCC TCAGGC CACGCC GGCGTT C-3' (SCO1345); and 5'-CATATG TCCACC ACTGAG CAGCG-3' and 5'-GGATCC CTAGTC GAGCGG TCCGCC-3' (SCO1346).…”