Structures of chloramphenicol acetyltransferase III and Escherichia coli β-ketoacylsynthase III co-crystallized with partially hydrolysed acetyl-oxa(dethia)CoA.
Structures of chloramphenicol acetyltransferase III and Escherichia coli β-ketoacylsynthase III co-crystallized with partially hydrolysed acetyl-oxa(dethia)CoA.
复制标题
DOI：
10.1107/s2053230x23001206
复制
发表时间：
2023-03-01
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
需要乙酰辅酶A的稳定类似物来支持乙酰转移酶的结构 - 功能研究。在此，报道了两种酶在一种乙酰辅酶A类似物存在下的结构，该类似物中硫酯被酯所取代。
乙酰辅酶A（acetyl - CoA）是一种活性代谢物，在结晶时间范围内会在许多酶的活性位点发生非生产性水解。为了阐明导致催化的酶 - 乙酰辅酶A相互作用，需要乙酰辅酶A底物类似物。一种可用于结构研究的可能类似物是乙酰 - 氧杂（去硫）辅酶A（AcOCoA），其中辅酶A的硫酯S原子被O原子取代。在此，呈现了在部分水解的AcOCoA和相应亲核试剂存在下生长的晶体中氯霉素乙酰转移酶III（CATIII）和大肠杆菌酮酰基合酶III（FabH）的结构。基于这些结构，AcOCoA在酶之间的行为不同，FabH与AcOCoA反应，而CATIII无反应。CATIII的结构揭示了对催化机制的深入了解，三聚体的一个活性位点对于AcOCoA和氯霉素具有相对清晰的电子密度，而其他活性位点对于AcOCoA具有较弱的密度。一种FabH结构包含水解的AcOCoA产物氧杂（去硫）辅酶A（OCoA），而另一种FabH结构包含带有OCoA的酰基 - 酶中间体。总之，这些结构为使用AcOCoA进行不同亲核试剂的酶结构 - 功能研究提供了初步见解。
Stable analogs of acetyl-CoA are needed to support structure–function studies of acetyltransferase enzymes. Here, the structures of two enzymes in the presence of an acetyl-CoA analog in which the thioester is replaced by an ester are reported. Acetyl coenzyme A (acetyl-CoA) is a reactive metabolite that nonproductively hydrolyzes in a number of enzyme active sites in the crystallization time frame. In order to elucidate the enzyme–acetyl-CoA interactions leading to catalysis, acetyl-CoA substrate analogs are needed. One possible analog for use in structural studies is acetyl-oxa(dethia)CoA (AcOCoA), in which the thioester S atom of CoA is replaced by an O atom. Here, structures of chloramphenicol acetyltransferase III (CATIII) and Escherichia coli ketoacylsynthase III (FabH) from crystals grown in the presence of partially hydrolyzed AcOCoA and the respective nucleophile are presented. Based on the structures, the behavior of AcOCoA differs between the enzymes, with FabH reacting with AcOCoA and CATIII being unreactive. The structure of CATIII reveals insight into the catalytic mechanism, with one active site of the trimer having relatively clear electron density for AcOCoA and chloramphenicol and the other active sites having weaker density for AcOCoA. One FabH structure contains a hydrolyzed AcOCoA product oxa(dethia)CoA (OCoA), while the other FabH structure contains an acyl-enzyme intermediate with OCoA. Together, these structures provide preliminary insight into the use of AcOCoA for enzyme structure–function studies with different nucleophiles.