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Exploring the Conversion of a d ‐Sialic Acid Aldolase into a l ‐KDO Aldolase
Author(s) -
RomeroRivera Adrian,
IglesiasFernández Javier,
Osuna Sílvia
Publication year - 2018
Publication title -
european journal of organic chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.825
H-Index - 155
eISSN - 1099-0690
pISSN - 1434-193X
DOI - 10.1002/ejoc.201800103
Subject(s) - aldolase a , chemistry , enzyme , directed evolution , active site , sialic acid , biochemistry , stereochemistry , rational design , fructose bisphosphate aldolase , substrate (aquarium) , mutant , gene , genetics , biology , ecology
Directed evolution involves iterative rounds of genetic diversification followed by selection of variants with improved enzyme properties. A common trend in these studies is the introduction of multiple scattered mutations remote from the active site. Rationalizing how distal mutations influence the conformational states or ensemble of conformations formed by enzymes, and its relation to enzymatic catalysis is highly appealing for the improvement of current rational enzyme design strategies. Directed evolution was applied to convert d ‐sialic acid aldolase into an efficient l ‐3‐deoxy‐ manno ‐2‐octulosonic acid ( l ‐KDO) aldolase. In this study, we computationally evaluate the conformational dynamics of both enzymes to shed light on the specificity of the evolved variant. We further demonstrate the role of distal mutations on the modulation of enzyme conformational dynamics and its relation to substrate accessibility and selectivity. Mutations markedly altered the active site shape and substrate access tunnels in the evolved l ‐KDO aldolase, thus affecting the enzyme specificity.