Tuning enzymatic properties by protein engineering toward catalytic tetrad of carbonyl reductase. Issue 12 (3rd September 2021)

• Record Type: Journal Article
• Authors: Cheng, Feng; Zhai, Qiu‐Yao; Gao, Xiao‐Fan; Liu, Hua‐Tao; Qiu, Shuai; Wang, Ya‐Jun; Zheng, Yu‐Guo
• Is Part Of: Biotechnology and bioengineering. Volume 118:Issue 12(2021)
• Page Start: 4643
• Page End: 4654
• Publication Date: 2021-09-03
• Subjects: biocatalysis -- carbonyl reductase -- catalytic tetrad -- protein engineering; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27925
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: Enzyme engineering toward catalytic‐tetrad residues usually results in activity loss. Unexpectedly, we found that a directed evolution campaign yielded a beneficial residue A100 in Km CR (a carbonyl reductase from Kluyveromyces marxianus ZJB14056), which is a residue of catalytic tetrad and conserved according to multiple sequence alignment. Inspired by this finding, we performed saturation mutagenesis on all the four residues of catalytic tetrad of Km CR. A number of variants with improved enzymatic activities were obtained. Among them, the variant Km CR_A100S exhibited increased catalytic efficiency ( k cat /K M = 47.3 s −1 ·mM −1 ), improved stereoselectivity (from moderate selectivity ( de P = 66.7%) to strict ( S ) ‐ selectivity ( de P > 99.5%)), and extended substrate scope, compared to those of Km CR_WT. In silico analysis showed that a relay system was rebuilt in Km CR via the beneficial residue S100. Furthermore, comparison of 11 protein engineering campaigns indicated that the beneficial position is easily overlooked due to the long distance (>10 Å) from ketone substrates. Since CRs share similar catalytic mechanism, the knowledge gained from this study has universal significance to CR engineering. Abstract : Enzyme engineering toward catalytic‐tetrad residues usually results in activity loss. Unexpectedly, engineering of the catalytic tetrad of Km CR (a carbonyl reductase from Kluyveromyces marxianus ) yielded a variant Km CR_A100S that exhibited increased catalytic efficiency, improved stereoselectivity, and extended substrate scope. Since CRs share similar catalytic mechanism, the gained knowledge has universal significance to CR engineering.