α-Ketoglutaric Semialdehyde Dehydrogenase Isozymes Involved in Metabolic Pathways of D-Glucarate, D-Galactarate, and Hydroxy-L-proline MOLECULAR AND METABOLIC CONVERGENT EVOLUTION

Abstract Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism in which α-ketoglutaric semialdehyde (αKGSA) dehydrogenase (KGSADH) is involved in the last step, the conversion of αKGSA to α-ketoglutarate. In the preceding studies, we identified a set of metabolic genes of the l-arabinose pathway including the KGSADH gene (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623; Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888; Watanabe, S., Shimada, N., Tajima, K., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 33521-33536). Here, we describe that A. brasilense possesses two different KGSADH isozymes from l-arabinose-related enzyme (KGSADH-I); that is, d-glucarate/d-galactarate-inducible KGSADH-II and hydroxy-l-proline-inducible KGSADH-III. They were purified homogeneously from A. brasilense cells grown on d-galactarate or hydroxy-l-proline, respectively. When compared with KGSADH-I, amino acid sequences of KGSADH-II and KGSADH-III were significantly similar but not totally identical. Physiological characterization using recombinant enzymes revealed that KGSADH-II and KGSADH-III showed similar high substrate specificity for αKGSA and different coenzyme specificity; that is, NAD+-dependent KGSADH-II and NADP+-dependent KGSADH-III. In the phylogenetic tree of the aldehyde dehydrogenase (ALDH) superfamily, KGSADH-II and KGSADH-III were poorly related to the known ALDH subclasses including KGSADH-I. On the other hand, ALDH-like ycbD protein involved in d-glucarate/d-galactarate operon from Bacillus subtilis is closely related to the methylmalonyl semialdehyde dehydrogenase subclass but not A. brasilense KGSADH isozymes. To estimate the correct function, the corresponding gene was expressed, purified, and characterized. Kinetic analysis revealed the physiological role as NADP+-dependent KGSADH. We conclude that three different types of KGSADH appeared in the bacterial evolutional stage convergently. Furthermore, even the same pathway such as l-arabinose and d-glucarate/d-galactarate metabolism also evolved by the independent involvement of KGSADH.