Open AccessGene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
Author(s) -
Pimchai Chaiyen,
David P. Ballou,
Vincent Massey
Publication year - 1997
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.94.14.7233
Subject(s) - biochemistry , molecular cloning , oxygenase , flavoprotein , peptide sequence , microbiology and biotechnology , escherichia coli , biology , gene , nucleic acid sequence , enzyme , molecular mass , cloning (programming) , oligonucleotide , chemistry , computer science , programming language
The gene encoding 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO; EC1.14.12.4 ) was cloned by using an oligonucleotide probe corresponding to the N terminus of the enzyme to screen a DNA library ofPseudomonas 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 inEscherichia coli and found to have the same properties as the native enzyme fromPseudomonas 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.