Marcella Aaron M, Culbertson Sannie J, Shogren-Knaak Michael A, Barb Adam W
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, 2437 Pammel Drive, Molecular Biology Building, rm 4210, Iowa State University, Ames, IA 50011, United States.
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, 2437 Pammel Drive, Molecular Biology Building, rm 4210, Iowa State University, Ames, IA 50011, United States.
J Mol Biol. 2017 Nov 24;429(23):3763-3775. doi: 10.1016/j.jmb.2017.10.015. Epub 2017 Oct 18.
The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05and 4.10Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a K=62±13nM, followed by the binding of two more equivalents of holo-ACPP with K=1.2±0.2μM. Cooperativity was not observed for apo-ACPP which bound with K=2.4±0.1μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.
大肠杆菌全（酰基载体蛋白）合成酶（ACPS）在脂肪酸生物合成的早期步骤中催化辅酶A依赖性的脱辅基ACPP激活，以生成全（酰基载体蛋白）（全ACPP）。大肠杆菌ACPS与人类脂肪酸合酶有足够的差异，这为开发新型靶向ACPS的抗生素提供了依据。通过X射线晶体学分别解析到2.05Å和4.10Å的未结合配体和结合全ACPP形式的大肠杆菌ACPS模型显示，ACPS结合了三个产物全ACPP分子以形成3:3六聚体。溶液核磁共振波谱实验使用化学位移扰动并通过拟合剩余偶极耦合来确定全ACPP与ACPS的相对取向，从而验证了全ACPP上的ACPS结合界面。该结合界面的组织方式是使带正电荷的ACPS残基与全ACPP的磷酸泛酰巯基乙胺部分之间形成接触，这表明产物中包含比酶：底物复合物中预期更多的稳定相互作用。实际上，全ACPP与酶的结合亲和力高于底物脱辅基ACPP，并且具有负协同性。第一个全ACPP分子以K = 62±13 nM的解离常数结合，随后另外两个全ACPP分子以K = 1.2±0.2μM的解离常数结合。脱辅基ACPP的结合未观察到协同性，其解离常数为K = 2.4±0.1μM。细胞中强烈的产物结合和高水平的全ACPP表明ACPS在脂肪酸生物合成中具有潜在的调节作用。
