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J. Biol. Chem., Vol. 281, Issue 5, 2612-2623, February 3, 2006

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Cloning, Expression, and Characterization of Bacterial L-Arabinose 1-Dehydrogenase Involved in an Alternative Pathway of L-Arabinose Metabolism^*

Seiya Watanabe^¶||, Tsutomu Kodak^||, and Keisuke Makino^¶||1

From the Faculty of Engineering, Kyoto University, Kyotodaigakukatsura, Saikyo-ku, Kyoto 615-8530, Institute of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto 611-0011, ^¶International Innovation Center, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto 606-8501,and^||CREST, Japan Science and Technology Agency, Gokasyo, Uji, Kyoto 611-0011, Japan

Azospirillum brasiliense converts L-arabinose to -ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46 [EC] ), catalyzing the conversion of L-arabinose to L-arabino--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.

Received for publication, June 14, 2005 , and in revised form, November 28, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB211983 [GenBank] .

^* This work was supported by the Center of Excellence program for the"Establishment of Centers of Excellence on Sustainable Energy System,"a grant-in-aid for scientific research, and grants for regional science and technology promotion from the Ministry of Education, Science, Sports, and Culture, Japan, and by CREST of the Japan Science and Technology Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 81-774-38-3517; Fax: 81-774-38-3524; E-mail: kmak{at}iae.kyoto-u.ac.jp.

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