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J. Biol. Chem., Vol. 281, Issue 44, 33521-33536, November 3, 2006

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Identification and Characterization of L-Arabonate Dehydratase, L-2-Keto-3-deoxyarabonate Dehydratase, and L-Arabinolactonase Involved in an Alternative Pathway of L-Arabinose Metabolism

NOVEL EVOLUTIONARY INSIGHT INTO SUGAR METABOLISM^*

Seiya Watanabe^¶, Naoko Shimada, Kunihiko Tajima^||, Tsutomu Kodaki^¶, and Keisuke Makino^¶1

From the Faculty of Engineering, Kyoto University, Kyotodaigaku-katsura, Saikyo-ku, Kyoto 615-8530, Japan, the Institute of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto 611-0011, Japan, the ^||Department of Biomolecular Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan, and ^¶CREST, JST (Japan Science and Technology Agency), Gokasyo, Uji, Kyoto 611-0011, Japan

Azospirillum brasiliense possesses an alternative pathway of L-arabinose metabolism, different from the known bacterial and fungal pathways. In the preceding articles, we identified and characterized L-arabinose-1-dehydrogenase and -ketoglutaric semialdehyde dehydrogenase, which catalyzes the first and final reaction steps in this pathway, respectively (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623 and Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888). We here report the remaining three enzymes, L-arabonate dehydratase, L-2-keto-3-deoxyarabonate (L-KDA) dehydratase, and L-arabinolactonase. N-terminal amino acid sequences of L-arabonate dehydratase and L-KDA dehydratase purified from A. brasiliense cells corresponded to those of AraC and AraD genes, which form a single transcriptional unit together with the L-arabinose-1-dehydrogenase gene. Furthermore, the L-arabinolactonase gene (AraB) was also identified as a component of the gene cluster. Genetic characterization of the alternative L-arabinose pathway suggested a significant evolutional relationship with the known sugar metabolic pathways, including the Entner-Doudoroff (ED) pathway and the several modified versions. L-Arabonate dehydratase belongs to the ILVD/EDD family and spectrophotometric and electron paramagnetic resonance analysis revealed it to contain a [4Fe-4S]²⁺ cluster. Site-directed mutagenesis identified three cysteine ligands essential for cluster coordination. L-KDA dehydratase was sequentially similar to DHDPS/NAL family proteins. D-2-Keto-3-deoxygluconate aldolase, a member of the DHDPS/NAL family, catalyzes the equivalent reaction to L-KDA aldolase involved in another alternative L-arabinose pathway, probably associating a unique evolutional event between the two alternative L-arabinose pathways by mutation(s) of a common ancestral enzyme. Site-directed mutagenesis revealed a unique catalytic amino acid residue in L-KDA dehydratase, which may be a candidate for such a natural mutation.

Received for publication, July 14, 2006 , and in revised form, August 31, 2006.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB241136 [GenBank] .

^* This work was supported by the Center of Excellence (COE) program for the "Establishment of COE on Sustainable Energy System", a Grant-in-aid for Scientific Research, and Grants for Regional Science and Technology Promotion "Kyoto Nanotechnology Cluster" project from the Ministry of Education, Science, Sports and Culture, Japan. This work was also supported by CREST and "Research for Promoting Technological Seeds" of the Japan Science and Technology Corporation. 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: Inst. of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto 611-0011 Japan. Tel.: 81-774-38-3517; Fax: 81-774-38-3524; E-mail: kmak{at}iae.kyoto-u.ac.jp.

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