Vinyl ketone reduction by three distinct Gluconobacter oxydans 621H enzymes.
Three cytosolic NADPH-dependent flavin-associated proteins (Gox2107, Gox0502, and Gox2684) from Gluconobacter oxydans 621H were overproduced in Escherichia coli, and the recombinant enzymes were purified and characterized. Apparent native molecular masses of 65.2, 78.2, and 78.4 kDa were observed for Gox2107, Gox0502, and Gox2684, corresponding to a trimeric structure for Gox2107 and dimers for Gox0502 and Gox2684. Analysis of flavin content revealed Gox2107 was flavin adenine dinucleotide dependent, whereas Gox0502 and Gox2684 contained flavin mononucleotide. The enzymes were able to reduce vinyl ketones and quinones, reducing the olefinic bond of vinyl ketones as shown by (1)H nuclear magnetic resonance. Additionally, Gox0502 and Gox2684 stereospecifically reduced 5S-(+)-carvone to 2R,5S-dihydrocarvone. All enzymes displayed highest activities with 3-butene-2-one and 1,4-naphthoquinone. Gox0502 and Gox2684 displayed a broader substrate spectrum also reducing short-chain alpha-diketones, whereas Gox2107 was most catalytically efficient.
Schweiger
Paul
P
Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA.
Gross
Harald
H
Wesener
Shane
S
Deppenmeier
Uwe
U
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002074:Butanones / Q000378:metabolism; D003001:Cloning, Molecular; D000081005:Cyclohexane Monoterpenes; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D015870:Gene Expression; D020600:Gluconobacter oxydans / Q000201:enzymology*; D007659:Ketones / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D039821:Monoterpenes / Q000378:metabolism; D009285:Naphthoquinones / Q000378:metabolism; D011809:Quinones / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014753:Vinyl Compounds / Q000378:metabolism*
D001426:Bacterial Proteins; D002074:Butanones; D000081005:Cyclohexane Monoterpenes; D007659:Ketones; D039821:Monoterpenes; D009285:Naphthoquinones; D011809:Quinones; D011994:Recombinant Proteins; D014753:Vinyl Compounds; D005182:Flavin-Adenine Dinucleotide; C006923:carvone; D005486:Flavin Mononucleotide; C057920:3-buten-2-one; C035342:1,4-naphthoquinone