alpha-ketoglutaric semialdehyde dehydrogenase isozymes involved in metabolic pathways of D-glucarate, D-galactarate, and hydroxy-L-proline. Molecular and metabolic convergent evolution.
Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism in which alpha-ketoglutaric semialdehyde (alphaKGSA) dehydrogenase (KGSADH) is involved in the last step, the conversion of alphaKGSA to alpha-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 alphaKGSA 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.
Watanabe
Seiya
S
Faculty of Engineering, Kyoto University, Kyotodaigaku-katsura, Saikyo-ku, Kyoto 615-8530, Japan.
Yamada
Masaki
M
Ohtsu
Iwao
I
Makino
Keisuke
K
D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001089:Arabinose / Q000378:metabolism; D015806:Azospirillum brasilense / Q000378:metabolism; D019143:Evolution, Molecular; D005937:Glucaric Acid / Q000378:metabolism*; D006909:Hydroxyproline / Q000378:metabolism*; D007527:Isoenzymes; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D010802:Phylogeny; D013400:Sugar Acids / Q000378:metabolism*
D007527:Isoenzymes; D013400:Sugar Acids; D001089:Arabinose; C000090:galactaric acid; D000445:Aldehyde Oxidoreductases; C020070:2,5-dioxovalerate dehydrogenase; D005937:Glucaric Acid; D006909:Hydroxyproline