Department of Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, D-91058 Erlangen, Germany.
Phytochemistry. 2012 May;77:53-9. doi: 10.1016/j.phytochem.2012.01.022. Epub 2012 Feb 20.
Progesterone 5β-reductases (P5βR; EC 1.3.99.6) encoded by Vein Patterning 1 (VEP1) genes are capable of reducing the CC double-bond of a variety of enones enantioselectively. Sequence and activity data of orthologous P5βRs were used to define a set of residues possibly responsible for the large differences in enzyme activity seen between rAtSt5βR and rDlP5βR, recombinant forms of P5βRs from Arabidopsis thaliana and Digitalis lanata, respectively. Tyrosine-156, asparagine-205 and serine-248 were identified as hot spots in the rDlP5βR responsible for its low catalytic efficiency. These positions were individually substituted for amino acids found in the strong rAtSt5βR in the corresponding sites. Kinetic constants were determined for rDlP5βR and its mutants as well as for rAtSt5βR using progesterone and 2-cyclohexen-1-one as substrates. Enzyme mutants in which asparagine-205 was substituted for methionine or alanine showed considerably lower km and higher K(cat)/k(m) values than the wild-type DlP5βR, approaching the catalytic efficiency of strong P5βRs. The introduced mutations not only lead to an improved capability to reduce progesterone but also to altered substrate preference. Our findings provided structural insights into the differences seen among the natural P5βRs with regard to their substrate preferences and catalytic efficiencies.
类固酮 5β-还原酶（P5βR；EC 1.3.99.6）由静脉模式基因 1（VEP1）编码，能够对各种烯酮的 CC 双键进行立体选择性还原。为了定义一组可能导致 rAtSt5βR 和 rDlP5βR 之间酶活性差异的残基，我们使用了同源 P5βR 的序列和活性数据，rAtSt5βR 和 rDlP5βR 分别是拟南芥和毛地黄的 P5βR 的重组形式。鉴定出酪氨酸-156、天冬酰胺-205 和丝氨酸-248 是导致 rDlP5βR 催化效率低的热点。这些位置分别被相应位置上在强 rAtSt5βR 中发现的氨基酸取代。使用孕酮和 2-环己烯-1-酮作为底物，测定了 rDlP5βR 及其突变体以及 rAtSt5βR 的动力学常数。将天冬酰胺-205 突变为蛋氨酸或丙氨酸的酶突变体的 km 和 K(cat)/k(m) 值明显低于野生型 DlP5βR，接近强 P5βR 的催化效率。引入的突变不仅提高了还原孕酮的能力，而且改变了底物偏好。我们的研究结果为自然 P5βR 之间在底物偏好和催化效率方面的差异提供了结构上的见解。
