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 oxygena se (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 a cid residues corresponding to a molecular mass of 41.7 kDa, the same a s that previously estimated for MHPCO. MHPCO was expressed in Escheric hia coli and found to have the same properties as the native enzyme fr om Pseudomonas sp. MA-1. This study shows that MHPCO is a homotetramer ic protein with one flavin adenine dinucleotide bound per subunit. Seq uence comparison of the enzyme with other hydroxylases reveals regions that are conserved among aromatic flavoprotein hydroxylases.