Gene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase

Abstract

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.

Figure 1

Location of restriction enzyme EagI and BssHII sites in the probe sequence. The EagI site is located 66 nucleotides and BssHII site is located 192 nucleotides from the 5′ end. The mark indicates the length representing 75 nucleotides.

Figure 2

DNA mapping of pBluMH. (A) Molecular mass markers (lanes 1 and 7). pBluMH digested with PstI (lane 2), EagI (lane 3), BssHII (lane 4), EagI + PstI (lane 5), and BssHII + PstI (lane 6). (B) Southern hybridization of the gel in A. pBluMH digested with BssHII + PstI (lane 1), EagI + PstI (lane 2), BssHII (lane 3), EagI (lane 4), PstI (lane 5), and DNA molecular mass markers (lane 6).

Figure 3

Construction of pPC1 expression plasmid. (A) Plasmid pBluMH. The thin line indicates the plasmid region derived from pBluescript vector, the empty box indicates the probe area. Number in parenthesis indicates the length of DNA (in kb) with reference to the PstI site near T3 promoter as the origin point. MHPCO gene (B) was isolated from pBluMH by PCR and ligated to pET-11a (C), resulting in the expression plasmid pPC1 (D). The mark indicates the length representing 1,000 nucleotides.

Figure 4

Nucleotide sequence of MHPCO gene, including upstream and downstream flanking regions and the predicted MHPCO sequence.

Figure 5

Alignment of MHPCO with other aromatic flavoprotein hydroxylases. Black areas indicate amino acid residues conserved in all six hydroxylases, and the gray boxes indicate amino acid residues conserved in two or more hydroxylases. (A) Conserved region A: residues 18–34 in MHPCO or 9–21 in PHBH. (B) Conserved region B: residues 154–157 in MHPCO, residues 157–160 in PHBH. (C) Conserved region C: residues 281–295 in MHPCO or residues 279–293 in PHBH. The underlined sequence is the conserved region C1, and the nonunderlined sequence is the conserved region C2.

Figure 6

The postulated model of binding of NADPH molecule in PHBH. FAD is indicated as the molecule on the left (line drawing), and NADPH is indicated as the molecule on the right (ball and stick drawing). The PHBH molecule is represented as gray ribbon. Residues 157–160 and 163 are indicated as the black loop on the left. Residues 287–289 and 293 are indicated as the black loop on the right.