Nitric Oxide Reductase Cytochrome P-450 Gene, CYP 55, of the Fungus Fusarium oxysporum Containing a Potential Binding-Site for FNR, the Transcription Factor Involved in the Regulation of Anaerobic Growth of Escherichia coli1

Abstract

A unique cytochrome P-450 (P-450) is involved in fungal denitrification, acting as a nitric oxide reductase. The phylogenetic classification of the P-450 (P-450nor) into the group of bacterial P-450s along with its involvement in the anaerobic process are of evolutional interest. The corresponding gene, CYP 55, of the fungus Fusarium oxysporum was isolated and sequenced. The P-450nor gene contained 7 introns that consisted of 49 to 55 bp. The presence of the introns suggested that horizontal transfer of the gene from bacteria to the fungus, if it has occurred, was an early event in evolution. Besides a TATA box, several inverted repeats were found in the 5'-upstream flanking region. One inverted repeat exhibited the same sequence as the binding site of FNR (fumarate and nitrate reduction), a DNA-binding, O2 (dioxygen)-sensor protein that positively regulates expressions of many hypoxic genes in Escherichia coli and other bacteria. The result suggested that the expression of P450nor is regulated in response to oxygen tension by an FNR-like system. Northern blot analyses showed that both nitrate and nitrite are the actual inducers of P-450nor and that its expression is predominantly regulated at the transcriptional level. These results raise the interesting possibility that the expression of the fungal denitrification system is regulated, as in the case of bacterial nitrate respiration, by a set of mechanisms, i.e., a combination of an FNR-like system and a system responding to nitrate/nitrite.

Oxygen requirement for denitrification by the fungus Fusarium oxysporum

“…This might arise from the same phenomenon as that observed with the bacterial denitrifying systems, that O 2 represses their expression and inhibits their denitrifying enzymes. This assumption is supported by our findings from F. oxysporum, showing that transcription of the gene for P450nor is strictly repressed under highly oxic conditions (Tomura et al 1994) and that Nir activity is inhibited by O 2 (Shoun and Tanimoto 1991).…”

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

“…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. 16) Aspergillus oryzae is used in the Japanese fermentation industries of sake, soy sauce, and miso manufacturing because this fungus can produce vast amounts and many kinds of hydrolytic enzymes, such as -amylases, glucoamylases, and proteases. Recently, an EST library of A. oryzae has been constructed in various culture conditions such as submerged, solid-state, nutrient, and starved cultures.…”

A Possible Role of NADPH-Dependent Cytochrome P450nor Isozyme in Glycolysis under Denitrifying Conditions

“…In spite of isolation of P450nor genes from several fungi, 7,9,26,27) existence of isozymes of P450nor that are encoded by distinct genes has been found to date only in C. tonkinense. Presence of only P450nor1, like two isoforms of P450nor in F. oxysporum, would be su‹cient for carrying out its putative physiological roles in detoxiˆcation and electron sink in both mitochondria and cytosol.…”

“…Since the discovery of fungal denitriˆcation, 3) we have been clarifying the fungal denitrifying system and have shown that the denitrifying fungi have constructed a unique nitraterespiration system during evolution that is distinct from the bacterial denitrifying system or the nondenitrifying nitrate respiration (ammoniˆcation) system. [4][5][6][7][8][9][10][11][12][13][14][15][16][17] The most unusual feature of the fungal system is the involvement of cytochrome P450nor (P450nor) that acts as nitric oxide (NO) reductase. 6) Fusarium oxysporum was shown to contain a unique couple of nitrate reductase (Nar) and formate dehydrogenase (Fdh) that should act as the major energy-producing process in the denitrifying system of the fungal mitochondrion.…”