Molecular mechanism of multiple dissimilatory nitrogen metabolisms by eukaryotic and prokaryotic cells

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(1) Molecular mechanisms of fungal denitrification system: The genes encoding copper-containing nitrite reductase (nirK) were isolated from several fungi including Fusarium oxysporum, in which fungal denitrifying activity was first found. The NirK protein previously isolated from F. oxysporum, was shown to be the product of the nirK gene. This is the first isolation from eukaryote of an orthologoue of the bacterial counterpart involved in denitrification. Phylogenetic analysis shows that these nirK genes and their homologue genes that have been recently found in many eukaryotic microorganisms are originated from the single ancestor, suggesting that they are derived from the protomitochondrion. It was shown that the nitrate reducing system of F. oxysporum comprises nitrate reductase (dNaR, possibly NarGHI, which is universally found in bacterial nitrate respiration) and ubiquinone-dependent formate dehydrogenase. Unlike the bacterial system the fungal dNaR system was shown to function s … More imultaneously with cytochrome oxidase under micro-aerobic conditions, which we termed hybrid respiration. These results have revealed general occurrence of fungal denitrification in nature.(2) Reaction mechanism of cytochrome P450nor: P450nor participates in fungal denitrification as nitric oxide (NO) reductase, performing an unusual electron transfer, direct transfer of 2 electrons from NADH to the hemeprotein. Structural and kinetic analyses such as determination of the crystal structure of P450nor in complex with an NAD analogue revealed-the detailed reaction mechanism of P450nor, including the mechanism of the prochiral hydride transfer from NADH to the ferric heme-NO complex and identification of the chemical entity of the key spectral intermediate. The results should have gave an marked impact, to many field such as bioinorganic chemistry and P450 biochemistry.(3) Ammonia fermentation and anaerobic growth of fungi: Involvement of the assimilatory nitrate reducing system in the fungal ammonia fermentation was confirmed by characterizing the uiaD or niiA-defective mutant of the fungus Aspergillus nidulans. The denitrifying fungus F. oxysporum was shown to perform hetero-lactic acid fermentation, which is coupled to ammonia fermentation. The results together with those of denitrification show that al least a portion of soil fungi are facultative with respect to aeration, not obligate aerobe as previously thought.(4) Other subjects: Codenitrification was shown to be a biological reaction but not chemical reaction, which was supported by the results of its saturation kinetics against codenitrification substrates, and inhibition by respiration inhibitors such as cyanide. Actinomycetes are thought to be obligate aerobes, By contrast, we provided evidence for occurrence of nitrate respiration in the actinomycetes Streptomyces antibioticus and Streptomyces coelicolor. Aerobic denitrifying bacteria were isolated in order to apply them to the control of green house-effect gas emission, and their properties were characterized. Less