Cloning and characterization of XXXX dehydrogenases from two pseudomonads and @entity62581 .

@entity38236 @entity59428 @entity14348 @entity15250 @entity26731 @entity3437 @entity8686 @entity41379 @entity505 @entity62581 @entity526
@entity38236 dehydrogenase has been cloned from @entity14348 , @entity26731 , and @entity62581 . The genes were identified by using a novel complementation assay employing an @entity526 mutant incapable of consuming @entity8686 as the sole @entity505 source but capable of growth on glucarate. A shotgun library of @entity3437 was screened in the mutant @entity526 by growing transformed cells on minimal medium containing @entity8686 . Colonies that survived were evaluated for @entity38236 dehydrogenase, which is capable of converting @entity8686 to @entity41379 . In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in @entity15250 and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in @entity526 were purified and characterized. For all three enzymes, the turnover number (k(cat)) with @entity8686 as a substrate was higher than that with @entity59428 ; however, the Michaelis constant (K(m)) for @entity59428 was lower than that for @entity8686 . The A. tumefaciens enzyme was found to have the highest rate constant (k(cat) = 1.9 x 10(2) s(-1) on @entity8686 ), which was more than twofold higher than those of both of the pseudomonad enzymes.
