Molecular cloning, expression, and properties of an unusual aldo-keto reductase family enzyme, pyridoxal 4-dehydrogenase, that catalyzes irreversible oxidation of pyridoxal.
Microbacterium luteolum YK-1 has pyridoxine degradation pathway I. We have cloned the structural gene for the second step enzyme, pyridoxal 4-dehydrogenase. The gene consists of 1,026-bp nucleotides and encodes 342 amino acids. The enzyme was overexpressed under cold shock conditions with a coexpression system and chaperonin GroEL/ES. The recombinant enzyme showed the same properties as the M. luteolum enzyme. The primary sequence of the enzyme was 54% identical with that of d-threo-aldose 1-dehydrogenase from Agrobacterium tumefaciens, a probable aldo-keto reductase (AKR). Upon multiple alignment with enzymes belonging to the 14 AKR families so far reported, pyridoxal 4-dehydrogenase was found to form a new AKR superfamily (AKR15) together with A. tumefaciens d-threo-aldose 1-dehydrogenase and Pseudomonas sp. l-fucose dehydrogenase. These enzymes belong to a distinct branch from the two main ones found in the phylogenic tree of AKR proteins. The enzymes on the new branch are characterized by their inability to reduce the corresponding lactones, which are produced from pyridoxal or sugars. Furthermore, pyridoxal 4-dehydrogenase prefers NAD(+) to NADP(+) as a cofactor, although AKRs generally show higher affinities for the latter.
Yokochi
Nana
N
Department of Bioresources Science, Faculty of Agriculture and Research Institute of Molecular Genetics, Kochi University, Monobe-Otsu 200, Nankoku, Kochi 783-8502, Japan.
Yoshikane
Yu
Y
Trongpanich
Yanee
Y
Ohnishi
Kouhei
K
Yagi
Toshiharu
T
D000192:Actinomycetales / Q000201:enzymology*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010802:Phylogeny; D011730:Pyridoxal / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D011730:Pyridoxal; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase