Vein Patterning 1-encoded progesterone 5尾-reductase: Activity-guided improvement of catalytic efficiency

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• 作者：Peter Bauer^a ; Kristin Rudolph^a ; Frieder Mü ; ller-Uri^a ; Wolfgang Kreis^a ; ^b ; ^{wkreis@biologie.uni-erlangen.de}
• 关键词：P5尾R1/2 ; progesterone 5尾-reductase 1/2 ; PRO ; progesterone ; CH ; 2-cyclohexen-1-one ; SDR ; short-chain dehydrogenase/reductase ; SDM ; site-directed mutagenesis
• 刊名：Phytochemistry
• 出版年：2012
• 期刊代码：85_00319422
• 类别：ch
• 出版时间：May, 2012
• 卷：77
• 期：Complete
• 页码：53-59
• 文件大小：789 K

摘要

Progesterone 5尾-reductases (P5尾R; 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 P5尾Rs were used to define a set of residues possibly responsible for the large differences in enzyme activity seen between rAtSt5尾R and rDlP5尾R, recombinant forms of P5尾Rs from Arabidopsis thaliana and Digitalis lanata, respectively. Tyrosine-156, asparagine-205 and serine-248 were identified as hot spots in the rDlP5尾R responsible for its low catalytic efficiency. These positions were individually substituted for amino acids found in the strong rAtSt5尾R in the corresponding sites. Kinetic constants were determined for rDlP5尾R and its mutants as well as for rAtSt5尾R 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 DlP5尾R, approaching the catalytic efficiency of strong P5尾Rs. 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 P5尾Rs with regard to their substrate preferences and catalytic efficiencies.