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

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.

Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants

“…In some cases, researchers use the M01 variant without the G415S mutation yet still name it M01; for this reason, we specify this variant's name and identity in the database. (25, 163−165, 168, 175, 184, 185, 187, 343, 348, 366), 354 (19,352,353), 359 (341), 363 (352,353), 366 (166), 370 (338), 377 (165,175,379), 383 (338,339,368), 390 (189), 392 (189), 393 (189,358), 399 (203), 401 (5,167,176,177,184,186,187,193,341,343,344,380), 403 (187,203,339), 405 (9, 358), 407 (368,381,382), 408 (338−340), 410 (25), 413 (339), 415 (345,(349)(350)(351)354), 417 (340), 425 (165,175,379), 434 (357−359, 361, 368), 435 (361), 437 (...…”

“…All positions of the P450 BM3 heme domain that have been mutated and show improvement toward small alkyl hydroxylation are shown (yellow spheres). Mutated positions in the heme domain (residues 1–471) and in the reductase domain (472–1049) are listed, with refs: 4 (), 26 ( , , , , ), 47 ( , , , , , , , , , , , , , , , , , , , , , − ), 51 ( , , , , , , , , , , ), 52 ( , , , − ), 64 ( , ), 70 (), 72 (), 74 ( , , , , , , , , , , , , − , , − ), 75 ( , , , , , , ), 78 ( , , , − , − …”

“…It has been the topic of extensive research over the past three decades and numerous laboratories have worked on evolving this enzyme to generate novel enzymatic properties and features. As a result, P450 BM3 and its variants have been shown to catalyze a wide variety of reactions, including hydroxylation, − epoxidation, − carbene transfer, − and nitrene transfer, − with a diverse range of substrates. The advances reported by the group of Frances Arnold on the evolution of P450 BM3 toward novel biocatalytic reactions, which were prominently featured in her 2018 Nobel-winning research, are among the most transformative.…”

“…), 47(28,221,226,331,429,430,448,449,481), 51(28,221,331,449,481), 69 (24), 72(24,226), 74(19,28,223,331,429,430), 75(19,226,309), 78(24,226,309,366,424,462), 81 (462), 82(226,462,482), 87(19,21,22,24,28,221,223,226,227,309,331,332,429,430,445,448,449,…”

Non-Ewald methods for evaluating the electrostatic interactions of charge systems: similarity and difference

“…ZMM is implemented in myPresto (Mashimo et al 2013), omega-gene (Kasahara et al 2016), and GROMACS (Sakuraba and Fukuda 2018). Its applications to biomolecular systems are found in literature (Kamiya et al 2013;Kasahara et al 2014;Nishikawa et al 2014;Iida et al 2016;Kamiya et al 2016;Nishigami et al 2016;Bekker et al 2017;Kasahara et al 2017;Kasahara et al 2018b;Hayami et al 2019;Higo et al 2020;Hayami et al 2021;Ikebe et al 2021;Higo et al 2022).…”