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

Abstract

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 α-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. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.