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Site‐directed mutagenesis of a novel secondary alcohol dehydrogenase from M. luteus WIUJH20
Author(s) -
Shoger Nicholas,
Huang JenqKuen,
Wen Lisa
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.463.15
Subject(s) - dehydrogenase , alcohol dehydrogenase , stereochemistry , enzyme , biochemistry , substrate (aquarium) , chemistry , mutagenesis , amino acid , active site , protein primary structure , primary alcohol , mutant , peptide sequence , biology , gene , catalysis , ecology
A novel secondary alcohol dehydrogenase (2 o ‐ADH) has been characterized in this lab previously. This 2 o ‐ADH is categorized as secondary alcohol dehydrogenase based on its biochemical reactions, but it belongs to NAD + ‐dependent short‐chain L‐3‐hydroxyacyl‐CoA dehydrogenase (SCADH) based on amino acid sequence homology. In literatures, 2 o ‐ADH and SCADHs are distinguished in term of their primary structure and substrate specificities. SCADH has tight substrate specificity; it only catalyzes L‐3‐hydroxyacyl‐CoAs with various hydrocarbon chain‐length. In comparison the 2 o ‐ADH has low substrate specificity; it can catalyze hydroxy fatty acids with hydroxyl group in either D or L form on a number of carbon positions. However, the enzyme can not use hydroxyacyl‐CoA as a substrate. This 2 o ‐ADH is an excellent model system to study structure‐function relationship and determine what distinguishes their substrate specificity. We report here generation of single and double amino acid substitution mutants (D60S, A67K, and D60S + A67K) of 2 o ‐ADH in order to probe substrate functional group specificity. The research was supported in part by Western Illinois University Foundation.