Bonnett Shilah A, Whicher Jonathan R, Papireddy Kancharla, Florova Galina, Smith Janet L, Reynolds Kevin A
Department of Chemistry, Portland State University, Portland, OR. 97201.
Chemical Biology Graduate Program, University of Michigan, Ann Arbor, Michigan 48109.
Chem Biol. 2013 Jun 20;20(6):772-83. doi: 10.1016/j.chembiol.2013.04.014.
The formation of an activated cis-3-cyclohexylpropenoic acid by Plm1, the first extension module of the phoslactomycin polyketide synthase, is proposed to occur through an L-3-hydroxyacyl-intermediate as a result of ketoreduction by an A-type ketoreductase (KR). Here, we demonstrate that the KR domain of Plm1 (PlmKR1) catalyzes the formation of an L-3-hydroxyacyl product. The crystal structure of PlmKR1 revealed a well-ordered active site with a nearby Trp residue characteristic of A-type KRs. Structural comparison of PlmKR1 with B-type KRs that produce D-3-hydroxyacyl intermediates revealed significant differences. The active site of cofactor-bound A-type KRs is in a catalysis-ready state, whereas cofactor-bound B-type KRs are in a precatalytic state. Furthermore, the closed lid loop in substrate-bound A-type KRs restricts active site access from all but one direction, which is proposed to control the stereochemistry of ketoreduction.
磷霉素聚酮合酶的首个延伸模块Plm1催化生成顺式-3-环己基丙烯酸，据推测这是通过一种A型酮还原酶（KR）进行酮还原，以L-3-羟基酰基中间体为媒介而发生的。在此，我们证明Plm1的KR结构域（PlmKR1）催化生成L-3-羟基酰基产物。PlmKR1的晶体结构显示其活性位点排列有序，附近有一个A型KR特有的色氨酸残基。将PlmKR1与生成D-3-羟基酰基中间体的B型KR进行结构比较，发现了显著差异。结合辅因子的A型KR的活性位点处于催化就绪状态，而结合辅因子的B型KR处于预催化状态。此外，底物结合型A型KR中闭合的盖子环限制了除一个方向外从其他所有方向进入活性位点，这被认为可控制酮还原的立体化学。
