Evans Simon E, Williams Christopher, Arthur Christopher J, Burston Steven G, Simpson Thomas J, Crosby John, Crump Matthew P
School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS (UK), Fax: (+44) 117-9298611.
Chembiochem. 2008 Oct 13;9(15):2424-32. doi: 10.1002/cbic.200800180.
The actinorhodin (act) synthase acyl carrier protein (ACP) from Streptomyces coelicolor plays a central role in polyketide biosynthesis. Polyketide intermediates are bound to the free sulfhydryl group of a phosphopantetheine arm that is covalently linked to a conserved serine residue in the holo form of the ACP. The solution NMR structures of both the apo and holo forms of the ACP are reported, which represents the first high resolution comparison of these two forms of an ACP. Ensembles of twenty apo and holo structures were calculated and yielded atomic root mean square deviations of well-ordered backbone atoms to the average coordinates of 0.37 and 0.42 A, respectively. Three restraints defining the protein to the phosphopantetheine interface were identified. Comparison of the apo and holo forms revealed previously undetected conformational changes. Helix III moved towards helix II (contraction of the ACP), and Leu43 on helix II subtly switched from being solvent exposed to forming intramolecular interactions with the newly added phosphopantetheine side chain. Tryptophan fluorescence and S. coelicolor fatty acid synthase (FAS) holo-synthase (ACPS) assays indicated that apo-ACP has a twofold higher affinity (K(d) of 1.1 muM) than holo-ACP (K(d) of 2.1 muM) for ACPS. Site-directed mutagenesis of Leu43 and Asp62 revealed that both mutations affect binding, but have differential affects on modification by ACPS. Leu43 mutations in particular strongly modulate binding affinity for ACPS. Comparison of apo- and holo-ACP structures with known models of the Bacillus subtilis FAS ACP-holo-acyl carrier protein synthase (ACPS) complex suggests that conformational modulation of helix II and III between apo- and holo-ACP could play a role in dissociation of the ACP-ACPS complex.
来自天蓝色链霉菌的放线紫红素（act）合酶酰基载体蛋白（ACP）在聚酮化合物生物合成中起核心作用。聚酮化合物中间体与磷酸泛酰巯基乙胺臂的游离巯基结合，该臂共价连接到ACP全酶形式中一个保守的丝氨酸残基上。本文报道了ACP的脱辅基形式和全酶形式的溶液核磁共振结构，这是这两种形式的ACP的首次高分辨率比较。计算了20个脱辅基和全酶结构的集合，得到有序主链原子相对于平均坐标的原子均方根偏差分别为0.37 Å和0.42 Å。确定了三个将蛋白质与磷酸泛酰巯基乙胺界面连接起来的限制条件。脱辅基形式和全酶形式的比较揭示了以前未检测到的构象变化。螺旋III向螺旋II移动（ACP收缩），螺旋II上的Leu43从暴露于溶剂中微妙地转变为与新添加的磷酸泛酰巯基乙胺侧链形成分子内相互作用。色氨酸荧光和天蓝色链霉菌脂肪酸合酶（FAS）全酶合酶（ACPS）分析表明，脱辅基-ACP对ACPS的亲和力（K(d)为1.1 μM）是全酶-ACP（K(d)为2.1 μM）的两倍。Leu43和Asp62的定点诱变表明，这两个突变都影响结合，但对ACPS的修饰有不同影响。特别是Leu43突变强烈调节对ACPS的结合亲和力。将脱辅基-ACP和全酶-ACP结构与枯草芽孢杆菌FAS ACP-全酶酰基载体蛋白合酶（ACPS）复合物的已知模型进行比较表明，脱辅基-ACP和全酶-ACP之间螺旋II和III的构象调节可能在ACP-ACPS复合物的解离中起作用。
