Gene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.
The gene encoding 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO; EC 1.14.12.4) was cloned by using an oligonucleotide probe corresponding to the N terminus of the enzyme to screen a DNA library of Pseudomonas sp. MA-1. The gene encodes for a protein of 379 amino acid residues corresponding to a molecular mass of 41.7 kDa, the same as that previously estimated for MHPCO. MHPCO was expressed in Escherichia coli and found to have the same properties as the native enzyme from Pseudomonas sp. MA-1. This study shows that MHPCO is a homotetrameric protein with one flavin adenine dinucleotide bound per subunit. Sequence comparison of the enzyme with other hydroxylases reveals regions that are conserved among aromatic flavoprotein hydroxylases.
Chaiyen
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Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606, USA.
Ballou
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DP
Massey
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011549:Pseudomonas; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D006899:Mixed Function Oxygenases; C019483:methylhydroxypyridine-carboxylate dioxygenase