Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneum

Abstract

Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase. Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor‐producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family. TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial‐targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought.

Cloning and Enhanced Expression of the Cytochrome P450nor Gene (nicA;CYP55A5) Encoding Nitric Oxide Reductase fromAspergillus oryzae

“…There are two types of P450nor enzyme as to specificity against the electron donor. 11,12) The first is the NADH type, such as Fnor and Cnor1, that reduce nitric oxide employing only NADH. The second is the NAD(P)H type, such as Cnor2 and Tnor, that can utilize both NADH and NADPH.…”

“…5) agreed with the characters of other P450nors. 12) Two peaks at 414 and 390 (weak) nm along with minor peaks at 535, 568, and 643 nm indicated that the bound heme in the resting state was in an oxidized form with a mixture of high-spin and low-spin states. The dithionitereduced form exhibited two peaks at 412 nm and 541 nm, and its carbon monoxide-ligated form yielded a spectral species with absorption peaks at 446 and 548 nm as the characteristic form of P450.…”

“…7,9,[11][12][13][14] These are P450norA and P450norB from F. oxysporum (Fnor), 14) P450nor1 and P450nor2 from Cylindrocarpon tonkinense (Cnor1 and Cnor2), 7,11) and P450nor from Trichosporon cutaneum (Tnor). 12) The Fnor species has been analyzed extensively both physicochemically and kinetically, and the structure has been determined by x-ray crystallography. 10) These P450nor isoforms are specific for electron donors NADH and NADPH.…”

“…Fnor and Cnor1 are specific to NADH, 7,11,15) whereas Cnor2 and Tnor can utilize both. 12) In F. oxysporum, it has been reported that expression of the P450nor-encoding gene is induced in the presence of substrates as nitrate or nitrite for y To whom correspondence should be addressed. Tel: +81-75-623-2104; Fax: +81-75-623-2455; E-mail: kaya@gekkeikan.co.jp Abbreviations: P450, cytochrome P450; NO, nitric oxide; P450nor, cytochrome P450nor; Anor, a P450nor of Aspergillus oryzae; Fnor, a P450nor of Fusarium oxysporum; Cnor1 and Cnor2, P450nor1 and P450nor2 isozymes from Cylindrocarpon tonkinense; Tnor, a P450nor from Trichosporon cutaneum; GUS, -glucuronidase; glaB, glucoamylase-encoding gene; melO, tyrosinase-encoding gene denitrification in low concentrations of oxygen.…”

Response to Hypoxia, Reduction of Electron Acceptors, and Subsequent Survival by Filamentous Fungi

“…The genes encoding Nor purified from F. oxysporum, 10,11) Cylindrocarpon tonkinence, 16) and Trichosporon cutaneum, 17) have been isolated. Kaya et al also cloned and produced recombinant Nor from A. oryzae.…”