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Chembiochem. 2019 Jan 2. doi: 10.1002/cbic.201800799. [Epub ahead of print]

Hidden Specificities in Enzyme Catalysis: Structural Basis of Substrate Structure-Selectivity Relationship of a Ketoreductase.

Author information

1
Albert-Ludwigs-Universität Freiburg, GERMANY.
2
University of Cambridge, UNITED KINGDOM.
3
Albert-Ludwigs-Universität Freiburg, Institut für Pharmazeutische Wissenschaften, Albertstr. 25, 79104, Freiburg, GERMANY.

Abstract

Enzymes often convert both physiological and non-physiological substrates with high stereoselectivity - yet, for some enzymes, opposite product chirality is observed. A possible explanation is the existence of hidden specificities becoming apparent when non-physiological substrates confer different substrate-enzyme interactions than the physiological substrate. To test this hypothesis, a series of α-methylated β-keto esters were converted with Tyl-KR1, a ketoreductase from polyketide synthesis in Streptomyces fradiae. The conversions of six substrates with different physicochemical properties exhibited enantioselectivities ranging from 84% ee for R,R to 84% ee for S,S, yet high and uniform diastereoselectivity (anti, dr >9:1). The exchange of a single atom, namely an oxygen ester instead of a thioester, led to almost complete loss of enantioselectivity (<5% ee). An additional S,S-selective binding mode as a hidden specificity in Tyl-KR1 has been identified through molecular modeling and site-directed mutagenesis.

KEYWORDS:

Molecular modeling; Vibrational Circular Dichroism; diastereoselectivity; enantioselectivity; enzyme catalysis

PMID:
30600894
DOI:
10.1002/cbic.201800799

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