Abstract:
:Optically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high-value chiral chemicals is often time-, money-, and resource-intensive; traditional semi-rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot-spot residues. In this study, through ligand-protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity toward rac-mandelonitrile (MAN). All the four selected residues had numerous collisions with rac-MAN. Five mutants significantly shifting stereoselectivity towards (S)-MAN were obtained from site-saturation mutagenesis, one of them, at position βPhe37, exhibiting efficient production of (S)-MAN with 96.8% eep , was isolated and further analyzed. The increased pulling force observed during SMD simulation was found to be in good coincidence with the formation of hydrogen bonds between (R)-MAN and residue βHis37. (R)-MAN had to break these barriers to enter the active site of nitrile hydratase and S selectivity was thus improved. The results indicated that combining steered molecular dynamics simulation with a traditional semi-rational design significantly reduced the select range of hot-spot residues for the evolution of NHase stereoselectivity, which could serve as an alternative for the modulation of enzyme stereoselectivity.
journal_name
Biotechnol Bioengjournal_title
Biotechnology and bioengineeringauthors
Cheng Z,Peplowski L,Cui W,Xia Y,Liu Z,Zhang J,Kobayashi M,Zhou Zdoi
10.1002/bit.26484subject
Has Abstractpub_date
2018-03-01 00:00:00pages
524-535issue
3eissn
0006-3592issn
1097-0290journal_volume
115pub_type
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