Premium
Cloning of a putative L‐3‐hydroxyacyl CoA dehydrogenase from micrococcus luteus
Author(s) -
Takkalapalli Vamshi,
Huang Jenq-Kuen,
Wen Lisa
Publication year - 2012
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.26.1_supplement.584.5
Subject(s) - micrococcus luteus , biochemistry , biology , dehydrogenase , gene , amino acid , micrococcus , microbiology and biotechnology , peptide sequence , enzyme , bacteria , genetics , escherichia coli
A novel secondary alcohol dehydrogenase (2‐ADH) was previously purified from Micrococcus luteus WIUJH20 and it N‐terminal amino acid sequence determined. This 2‐ADH gene has been assigned to NAD + dependant L‐3‐hydroxyacyl CoA dehydrogenase (an enzyme in fatty acid β‐oxidation) due to its high sequence homology (96%). The 2‐ADH can oxidize many hydroxyl fatty acids to keto fatty acids with hydroxyl groups in either D‐or L‐ form on a number of different carbon positions. Therefore this 2‐ADH has low substrate specificity unlike other L‐3‐hydroxyacyl CoA dehydrogenases that requires hydroxyl group in L‐form and on carbon 3. These two genes are excellent candidates for studying the structure‐function relationship that distinguishes their substrate specificities. The 2‐ADH gene has been cloned previously and the putative residues responsible for substrate specificity studied by site‐directed mutagenesis. In the present study, we report the cloning of a putative L‐3‐hydroxyacyl CoA dehydrogenase from micrococcus luteus WIUJH20. The gene has been amplified by PCR using the micrococcus luteus genomic DNA as template. The candidate gene was cloned into pET28a expression vector and transformed to JM109 host cell. The target protein will be expressed in BL21(DE3)pLysS host cells and the substrate specificity will be studied and compared to that of the 2‐ADH. Supported by Western Illinois University.