Structural characterization of the gene and corresponding cDNA for the cytochrome P450rm from Rhodotorula minuta which catalyzes formation of isobutene and 4-hydroxylation of benzoate.
Cytochrome P450rm was previously isolated from the basidiomycete yeast Rhodotorula minuta as a bifunctional enzyme with isobutene-forming and benzoate 4-hydroxylase activities. We cloned the gene and corresponding cDNA for P450rm in order to characterize the enzyme in the context of fungal phylogeny and physiology. From the cDNA sequence, P450rm was deduced to have 527 amino acids with a calculated molecular weight of 59136. P450rm shared 48% amino acid sequence identity with CYP53A1 from Aspergillus niger, indicating that the gene belongs to a novel subfamily of CYP53, CYP53B. However, the organization of the P450rm gene, which has eight exons and seven introns, differed completely to that of CYP53A1. Northern analysis demonstrated that the level of P450rm mRNA expression increased when L-phenylalanine was used as sole carbon source. These results suggest that P450rm has been well conserved during the evolution of fungi as a benzoate 4-hydroxylase in the dissimilation pathway starting from L-phenylalanine
Fujii
T
T
Department of Applied Microbial Technology, Kumamoto Institute of Technology, Japan.
Nakamura
K
K
Shibuya
K
K
Tanase
S
S
Gotoh
O
O
Ogawa
T
T
Fukuda
H
H
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000235:genetics; D050565:Benzoate 4-Monooxygenase; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D005091:Exons; D005800:Genes, Fungal; D007438:Introns; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000378:metabolism; D010957:Plasmids; D012331:RNA, Fungal / Q000032:analysis; D012248:Rhodotorula / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D004271:DNA, Fungal; D012331:RNA, Fungal; D010649:Phenylalanine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050565:Benzoate 4-Monooxygenase