Mofid Mohammad Reza, Finking Robert, Essen Lars Oliver, Marahiel Mohamed A
Fachbereich Chemie/Biochemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany.
Biochemistry. 2004 Apr 13;43(14):4128-36. doi: 10.1021/bi036013h.
The activation of apo-peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases (NRPSs), apo-acyl carrier proteins (ACPs) of polyketide synthases (PKSs), and fatty acid synthases (FASs) to their active holo form is accomplished with dedicated 4'-phosphopantetheinyl transferases (PPTases). They catalyze the transfer of the essential prosthetic group 4'-phosphopantetheine (4'-Ppant) from coenzyme A (CoA) to a highly conserved serine residue in all PCPs and ACPs. PPTases, based on sequence and substrate specifity, have been classified into three types: bacterial holo-acyl carrier protein synthase (AcpS), fatty acid synthase of eukaryotes (FAS2) and Sfp, a PPTase of secondary metabolism. The recently solved crystal structures of AcpS and Sfp-type PPTases with CoA revealed a common alpha + beta-fold with a beta(1)alpha(3)beta(2) motif and similarities in CoA binding and polymerization mode. However, it was not possible to discern neither the PCP binding region of Sfp nor the priming reaction mechanism from the Sfp-CoA cocrystal. In this work, we provide a model for the reaction mechanism based on mutational analysis of Sfp that suggests a reaction mechanism in which the highly conserved E151 deprotonates the hydroxyl group of the invariant serine of PCP. That, in turn, acts as a nucleophile to attack the beta-phosphate of CoA. The Sfp mutants K112, E117, and K120 further revealed that the loop region between beta4 and alpha5 (residues T111-S124) in Sfp is the PCP binding region. Also, residues T44, K75, S89, H90, D107, E109, E151, and K155 that have been shown in the Sfp-CoA cocrystal structure to coordinate CoA are now all confirmed by mutational and biochemical analysis.
非核糖体肽合成酶（NRPS）的载脂蛋白肽载体蛋白（PCP）、聚酮合酶（PKS）的载脂蛋白酰基载体蛋白（ACP）以及脂肪酸合成酶（FAS）激活为其活性全酶形式是由专门的4'-磷酸泛酰巯基乙胺基转移酶（PPTase）完成的。它们催化将必需的辅基4'-磷酸泛酰巯基乙胺（4'-Ppant）从辅酶A（CoA）转移至所有PCP和ACP中一个高度保守的丝氨酸残基上。基于序列和底物特异性，PPTase已被分为三种类型：细菌全酶酰基载体蛋白合成酶（AcpS）、真核生物脂肪酸合成酶（FAS2）以及参与次生代谢的PPTase Sfp。最近解析的AcpS和Sfp型PPTase与CoA的晶体结构显示出具有β(1)α(3)β(2)基序的常见α + β折叠结构，并且在CoA结合和聚合模式上具有相似性。然而，从Sfp-CoA共晶体中既无法辨别Sfp的PCP结合区域，也无法确定引发反应机制。在这项工作中，我们基于Sfp的突变分析提供了一种反应机制模型，该模型表明一种反应机制，即高度保守的E151使PCP不变丝氨酸的羟基去质子化。反过来，该羟基作为亲核试剂攻击CoA的β-磷酸基团。Sfp突变体K112、E117和K120进一步表明，Sfp中β4和α5之间的环区域（残基T111 - S124）是PCP结合区域。此外，在Sfp-CoA共晶体结构中显示与CoA配位的残基T44、K75、S89、H90、D107、E109、E151和K155现在都通过突变和生化分析得到了证实。
