Enzyme modification using mutation site prediction method for enhancing the regioselectivity of substrate reaction sites

Jinzen Ikebe,Aya Komori,Tomoshi Kameda,Kaito Kobayashi,Munenori Suzuki

doi:10.1038/s41598-021-98433-7

Jinzen Ikebe, Aya Komori + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-98433-7

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Abstract

Enzymes with low regioselectivity of substrate reaction sites may produce multiple products from a single substrate. When a target product is produced industrially using these enzymes, the production of non-target products (byproducts) causes adverse effects such as increased processing costs for purification and the amount of raw material. Thus it is required the development of modified enzymes to reduce the amount of byproducts’ production. In this paper, we report a method called mutation site prediction for enhancing the regioselectivity of substrate reaction sites (MSPER). MSPER takes conformational data for docking poses of an enzyme and a substrate as input and automatically generates a ranked list of mutation sites to destabilize docking poses for byproducts while maintaining those for target products in silico. We applied MSPER to the enzyme cytochrome P450 CYP102A1 (BM3) and the two substrates to enhance the regioselectivity for four target products with different reaction sites. The 13 of the total 14 top-ranked mutation sites predicted by MSPER for the four target products succeeded in selectively enhancing the regioselectivity up to 6.4-fold. The results indicate that MSPER can distinguish differences of substrate structures and the reaction sites, and can accurately predict mutation sites to enhance regioselectivity without selection by directed evolution screening.

Highlights

Enzymes with low regioselectivity of substrate reaction sites may produce multiple products from a single substrate
MSPER significantly reduced the number of mutants tested by reducing the number of candidate substitution residues from the 455 residues that make up the enzyme to just a few residues
We describe the definition of the docking poses used in this work and the details of MSPER below

Summary

Introduction

Enzymes with low regioselectivity of substrate reaction sites may produce multiple products from a single substrate. When a target product is produced industrially using these enzymes, the production of non-target products (byproducts) causes adverse effects such as increased processing costs for purification and the amount of raw material It is required the development of modified enzymes to reduce the amount of byproducts’ production. In terms of rational design for enhancing regioselectivity, it is interesting that Seifert et al have developed an effective mutant that selectively generates the target product from a less regioselective cytochrome P450 CYP102A1 (BM3)[19] They proposed amino acid substitutions to narrow down the space of the enzyme active site and controlled the regioselectivity so that only the smallest methyl group in the substrate could access the active site. We would like to develop a method that allows us to freely control reaction sites of substrates and automatically predict mutation sites to enhance regioselectivity

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Conclusion