Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism.
Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.
Watanabe
Seiya
S
Faculty of Engineering, Kyoto University, Kyotodaigakukatsura, Saikyo-ku, Kyoto 615-8530.
Kodaki
Tsutomu
T
Kodak
Tsutomu
T
Makino
Keisuke
K
D000595:Amino Acid Sequence; D001089:Arabinose / Q000378:metabolism* / Q000494:pharmacology; D015806:Azospirillum brasilense / Q000201:enzymology; D001426:Bacterial Proteins; D002237:Carbohydrate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D001089:Arabinose; D002237:Carbohydrate Dehydrogenases; C033519:L-arabinose dehydrogenase; D005947:Glucose