Journal
ACS CATALYSIS
Volume 9, Issue 6, Pages 5480-5485Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.9b01464
Keywords
computational protein design; multistate design; multistate analysis; biocatalysis; enzyme engineering; aminotransferase; transaminase; amino acids
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Funding
- Ontario Ministry of Economic Development Innovation [ER14-10-139]
- Natural Sciences and Engineering Research Council of Canada [RGPIN-2016-04831]
- Canada Foundation for Innovation [26503]
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The engineering of multisubstrate enzyme specificity is highly desirable to foster the application of biocatalysis in industry. Here, we develop a multistate computational protein design methodology called multichemical state analysis (MCSA) that can optimize enzyme sequences on large structural ensembles for productive binding of multiple target substrates. Using MCSA, we redesigned E. coli branched-chain amino acid aminotransferase to accept both alpha-ketoglutarate and the non-native substrate L-histidine. Screening of a designed combinatorial library comprising 32 mutants for enhanced L-histidine transamination activity yielded four variants displaying up to similar to 200-fold improvements to k(cat)/K-M. MCSA opens the door to the design of broad-specificity biocatalysts and multisubstrate enzymes displaying tailored specificity.
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