A novel alpha-ketoglutaric semialdehyde dehydrogenase: evolutionary insight into an alternative pathway of bacterial L-arabinose metabolism.

Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism, which is different from the known bacterial and fungal pathways. In a previous paper (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623), we identified and characterized l-arabinose 1-dehydrogenase, which catalyzes the first reaction step in this pathway, and we cloned the corresponding gene. Here we focused on the fifth enzyme, alpha-ketoglutaric semialdehyde (alphaKGSA) dehydrogenase, catalyzing the conversion of alphaKGSA to alpha-ketoglutarate. alphaKGSA dehydrogenase was purified tentatively as a NAD(+)-preferring aldehyde dehydrogenase (ALDH) with high activity for glutaraldehyde. The gene encoding this enzyme was cloned and shown to be located on the genome of A. brasilense separately from a gene cluster containing the l-arabinose 1-dehydrogenase gene, in contrast with Burkholderia thailandensis in which both genes are located in the same gene cluster. Higher catalytic efficiency of ALDH was found with alphaKGSA and succinic semialdehyde among the tested aldehyde substrates. In zymogram staining analysis with the cell-free extract, a single active band was found at the same position as the purified enzyme. Furthermore, a disruptant of the gene did not grow on l-arabinose. These results indicated that this ALDH gene was the only gene of the NAD(+)-preferring alphaKGSA dehydrogenase in A. brasilense. In the phylogenetic tree of the ALDH family, alphaKGSA dehydrogenase from A. brasilense falls into the succinic semialdehyde dehydrogenase (SSALDH) subfamily. Several putative alphaKGSA dehydrogenases from other bacteria belong to a different ALDH subfamily from SSALDH, suggesting strongly that their substrate specificities for alphaKGSA are acquired independently during the evolutionary stage. This is the first evidence of unique "convergent evolution" in the ALDH family.