Seiya Watanabe, Tsutomu Kodak, K. Makino
Feb 3, 2006
Citations
6
Influential Citations
73
Citations
Quality indicators
Journal
Journal of Biological Chemistry
Abstract
Azospirillum brasiliense converts l-arabinose to α-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-γ-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.