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.
Watanabe
Seiya
S
Faculty of Engineering, Kyoto University, Kyotodaigaku-katsura, Saikyo-ku, Kyoto 615-8530, Japan.
Shimada
Naoko
N
Tajima
Kunihiko
K
Kodaki
Tsutomu
T
Makino
Keisuke
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001089:Arabinose / Q000378:metabolism*; D015806:Azospirillum brasilense / Q000201:enzymology / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D019143:Evolution, Molecular; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007783:Lactones / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D009876:Operon / Q000235:genetics; D010802:Phylogeny; D016415:Sequence Alignment; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D007783:Lactones; D013455:Sulfur; D001089:Arabinose; D007501:Iron; D006836:Hydro-Lyases