Hereditary
methemoglobinemia associated with
nicotinamide adenine dinucleotide-
cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary
methemoglobinemia. However, the underlying molecular mechanisms of canine and feline
methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary
methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at
codon 194 in exon 7, replacing an
isoleucine residue with
leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant
enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant
enzyme exhibited decreased heat stability and increased susceptibility to
trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant
enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant
enzyme properties with normal enzymatic activity would be associated with hereditary
methemoglobinemia in the Pomeranian dog family.