N-terminal processing and amino acid sequence of two isoforms of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum.
Cytochrome P450nor (P450nor), a nitric oxide reductase involved in the denitrifying system of the fungus Fusarium oxysporum, revealed molecular multiplicity. Two isoforms of P450nor, termed P450norA (norA) and P450norB (norB), were isolated. They had distinct isoelectric points of 5.1 (norA) and 4.9 (norB). However, their catalytic, spectroscopic, and immunological properties were almost identical. Partial amino acid sequences, involving 263 amino acid residues of norA and 278 residues of norB among 404 and 402 residues, respectively, were determined. Corresponding sequences in the isoforms were identical, and all of the determined partial sequences of norA or norB coincided with the sequence deduced from the CYP 55 gene or its cDNA. The amino acid sequence determination ruled out the possibility that there is a redox center in P450nor derived from amino acid residues, e.g., quinonoid cofactors. The only difference between norA and norB was in their N-termini. The N-terminus of norA was a threonine residue, whereas that of norB was an N-acetylated alanyl residue and norB was shorter by 2 residues than norA. The results suggested that norA and norB may be the products of the same gene, but translated from different initiation codons. The hypothetical precursor of norA would have a presequence containing targeting and sorting signals for transportation to the intermembrane space of mitochondria. This is consistent with the results of a Western-blot analysis which showed that norA was recovered only in particulate fractions, whereas norB was in the soluble fraction. It is therefore likely that the intracellular localizations as well as the N-termini of norA and norB are different, owing to the differences in the translational initiation codons and co/post-translational processings.
Nakahara
K
K
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki.
Shoun
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification; D005670:Fusarium / Q000201:enzymology*; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D013347:Subcellular Fractions / Q000201:enzymology
D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C080610:nitric-oxide reductase (P450)