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.
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 alpha-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.[1]References
- 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. Watanabe, S., Shimada, N., Tajima, K., Kodaki, T., Makino, K. J. Biol. Chem. (2006) [Pubmed]
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