Lobo S, Florova G, Reynolds K A
Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23219, USA.
Biochemistry. 2001 Oct 2;40(39):11955-64. doi: 10.1021/bi011325a.
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.
在植物和细菌的II型可解离脂肪酸合成酶中启动脂肪酸生物合成的3-酮酰基-ACP合酶III（KASIII）已被证明具有乙酰辅酶A：酰基载体蛋白（ACP）转酰基酶（ACT）活性。有几条证据表明ACT活性可能与KASIII以外的蛋白质有关。我们使用天蓝色链霉菌的粗提物，从KASIII中分离出了另一种具有ACT活性的蛋白质，并随后通过五步色谱法将其纯化了85倍。凝胶过滤显示，这种45 kDa的蛋白质的天然酶分子量为185±35 kDa，与同四聚体结构一致。相应的基因（fadA）被克隆并测序，结果表明它编码的蛋白质与II型硫解酶具有氨基酸序列同源性。fadA在大肠杆菌中表达，通过金属螯合色谱法纯化得到的重组FadA酶显示同时具有ACT和硫解酶活性。动力学研究表明，在ACT测定中，FadA对二碳乙酰辅酶A底物具有底物特异性（K(m) 8.7±1.4 microM），但能够使用来自II型脂肪酸和聚酮化合物合成酶的ACP（天蓝色链霉菌FabC ACP，K(m) 10.7±1.4 microM；大肠杆菌FabC ACP，K(m) 8.8±2 microM；FrenN ACP，K(m) 44±12 microM）。在硫解酶测定中，动力学分析显示乙酰乙酰辅酶A（K(m) 9.8±0.8 microM）和辅酶A（K(m) 10.9±1.8 microM）结合的K(m)值相似。FadA的Cys92Ser突变体对乙酰乙酰辅酶A和辅酶A的K(m)值几乎没有变化，但硫解酶活性的k(cat)下降了99%以上。Cys92Ser突变体未观察到可检测到的ACT活性，这表明这两种活性都与FadA有关，并且可能涉及形成相同的共价乙酰-S-半胱氨酸酶中间体。以前在硫解酶中未观察到与ACP的ACT活性，就天蓝色链霉菌FadA而言，其ACT活性明显低于FadA的硫解酶活性（k(cat) 3 min(-1)）低于硫解酶活性（k(cat) 2170 min(-1)）。FadA的ACT活性与KAS活性相当，并且明显高于报道的链霉菌KASIII的ACT活性。
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