title: An in-frame deletion of codon 298 of the NADH-cytochrome b5 reductase gene results in hereditary methemoglobinemia type II (generalized type). A functional implication for the role of the COOH-terminal region of the enzyme.
abstract: The nucleotide sequence was determined for the gene of NADH-cytochrome b5 reductase of a patient of type II hereditary methemoglobinemia found in Yokohama, Japan. An in-frame deletion of 3 base pairs corresponding to codon 298 (TTC) was identified in the patient. The patient was homozygous for the mutation as shown by hybridization experiments using allele-specific oligonucleotides. The mutation causes deletion of Phe-298, which is the third to the COOH-terminal residue, indicating that in this mutant enzyme the sequence of this region has changed from -Cys-Phe-Val-Phe-COOH to -Cys-Val-Phe-COOH. The mutant enzyme, whose Phe-298 was deleted (F298 delta), was prepared by means of a bacterial expression system and site-directed mutagenesis. The kcat/Km value (NADH) of the enzyme was 5.7 s-1 M-1, which corresponds to 0.4% of that of the wild type. Moreover, the enzyme was much less thermostable than the wild type. To examine further the role of the COOH-terminal portion of the enzyme, various mutant enzymes were also prepared and characterized. The enzymatic properties of F298L, F300L, and F298L/F300L were essentially the same as that of the wild type. The kinetic properties of F298A, and F300A were not greatly affected, but the stability of the enzymes was somewhat impaired. Since Val-299 is naturally Ala in steer enzyme, no specific residues in the carboxyl-terminal region (298-300) are essential to the enzyme function. The instability of the F298/F300A double mutant indicates that the hydrophobicity of the carboxyl-terminal region of the enzyme might be important to maintain the conformation of the enzyme. high impairment of the activity of the F298 delta, F298stop, and F300stop mutants might be caused by the loss of the residue(s) in the carboxyl-terminal portion. These results indicate that the hydrophobicity, but not the specific amino acid residues, of the carboxyl-terminal portion of the enzyme is important for the stability of the enzyme.
authors.affiliation: Department of Biochemistry, Oita Medical University, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Shirabe
authors.forename: Y
authors.initials: Y
authors.lastname: Fujimoto
authors.forename: T
authors.initials: T
authors.lastname: Yubisui
authors.forename: M
authors.initials: M
authors.lastname: Takeshita
mesh_terms: D000293:Adolescent; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D003062:Codon; D003579:Cytochrome Reductases / Q000235:genetics* / Q000378:metabolism; D042966:Cytochrome-B(5) Reductase; D004795:Enzyme Stability; D006358:Hot Temperature; D006801:Humans; D008297:Male; D008708:Methemoglobinemia / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D017384:Sequence Deletion
chemical_list: D003062:Codon; D009838:Oligodeoxyribonucleotides; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase