90NISR RESEARCH GRANT
2002 Young Investigator Research Grant

Analysis of the Novel Nitrate Dissimilatory Mechanisms byFilamentous Fungi
Naoki TAKAYA
Institute of Applied Biochemistry, University of Tsukuba
Filamentous fungi are important in Japanese fermentation industries and their roles in the fermentation processes
have been extensively studied. Most of them grow wellunder the aerobic conditions and thus they have been longthought to be aerobic organisms, however, a variety of filamentous fungi were shown to employ the anaerobic metabolic mechanisms, nitrate (NO3) respiration and ammonia(NH3) fermentation. These metabolic mechanisms had been
exclusively described in facultative and restricted anaerobic
bacteria, respectively, thus these findings are surprising andimplicate that the filamentous fungi might adapt intochanges of environmental oxygen (O2) tension by using
these complicated mechanisms for growth. Until now, studies of Dr. Shoun and his colleagues have revealed the components involved in the mechanisms. The fungus Fusarium
oxysporum respires with NO3 under the O2 limited conditions by the sequential reaction of NO3 reductase, nitrite(NO2) reductase, and nitric oxide (NO) reductase, by which
NO3 is reduced to nitrous oxide (N2O) (NO3 respiration).
These enzymes are located in mitochondrion and are considered to be physiologically significant for producing ATPfor growth through respiratory electron transferring chain.
The fungal NH3 fermentation is occurred under moreanoxic conditions than NO3 respiration. It was originallyfound in F. oxysporum. This process consists of NO3 reduction to NH4 coupled with the catabolic oxidation ofelectron donors (ethanol) to acetate and substrate-level
phosphorylation that supports growth under anaerobic conditions. This process resembles ammonification by theobligatory anaerobe Clostridium, and is termed NH3 fermentation. It was also shown that 15 of 17 tested fungal
strains ferment NH3 under anaerobic conditions, suggestingthat this activity is widely distributed among fungi. In themechanism, cytosolic NAD(P)H-dependent NO3 and NO2
reductases, which are similar to assimilating NO3 and NO2
reductases, are responsible for substrate level phosphorylation.
This study describes properties of the genetically manipulated and the naturally occurred mutants of the fungi thathave lesions in the possible genes involved in the fungal
anaerobic metabolisms. The data presented here give a conclusive evidence of the components and physiological significance of these fungal metabolic mechanisms.
1. Physiological function of NO reduction and oxidation by F. oxysporum

Although NO3 respiration (denitrification) is importantfor microorganisms to grow under anoxic conditions, it
could often produce NO, which is hazardous for cellularcomponents and their activity since NO is a free radical
concerning to various biological functions. Most microorganisms have an NO degrading enzyme flavohemoglobin(fhb), to survive against the damaging effects of exogenousNO. This study demonstrated that the fungus F. oxysporum
produced NO under the NO3 respiratory condition and induced production of two hemoproteins, fhb and cytochrome
P450nor (P450nor). Both proteins have been known to metabolize NO to less hazardous forms, N2O and NO3, respectively. Detailed investigation of the mutants defectivein either protein showed that they had a redundant function
for supporting cell growth against highly toxic NO stress.
In contrast, only P450nor rescued mitochondrial damages
such as inhibition of respiration and breakage of cellulariron-sulfur proteins caused by NO, indicating that fhb andP450nor could have different targets dependent with thetargets for eliminating NO stress. They must constitute a
dual heme NO degrading system that is specific for filamentous fungi since both the hemoproteins are unique formicroorganisms and P450nor is only seen in filamentous
fungi. This observation is also significant on P450 biologysince P450nor is the first example that might make evolution for detoxifying.
2. Fungal NH3 fermentation and genes involved in theprocess
Studies on NH3 fermentation mechanisms in F. oxysporum showed that the key reactions of NH3 fermentation are
the conversion of acetaldehyde to acetyl-CoA by acetaldehyde dehydrogenase (AddA) and the hydrolysis of acetylCoA by the ATP-forming acetate kinase (Ack). These reactions have been identified only in the obligatory anaerobe
Clostridium and they do not appear to be a feature of eukaryotic cells. Here I investigated the mechanisms by using
Aspergillus nidulans as a model fungus. Mutant strains inthe loci required for NO3 assimilation were tested for ability of NH3 fermentation. The results showed that niaD andniiA that code for NO3 and NO2 reductases, respectively
were critical for NH3 fermentation as well as for NO3 assimilation. These correspond well to the previous observation of F. oxysporum NH3 fermentation system and suggested both enzymes function in both assimilation and dissimilation of NO3.

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The activities of AddA and Ack were able to be reconstituted using cytosolic fractions of A. nidulans as well as
those of F. oxysporum. Interestingly, the loss-of function
mutant in the facA gene, which codes for acetyl CoA synthetase (Acs), completely lost Ack activity and fermentative
production of NH3. Since Ack catalyzes reverse reaction ofAcs, it is suggested that facA is responsible for Ack activity
under the NH3 fermentation conditions in A. nidulans.
3. Post-transcriptional modification of FacA regulates
enzymic reactions
Acs is widely distributed from prokaryotic to eukaryotic
cells and the reaction is shown to be reversible. Recombinant FacA protein purified from normal aerobically-growing cells of A. nidulans exhibited higher Acs activity andlittle Ack activity under the physiological concentration ofthe substrates. By contrast, FacA protein purified from theNH3-fermenting A. nidulans showed less Acs activity thanthat from aeroically growing cells. It is surprising that thelatter FacA protein exhibited considerably high Ack activity. Steady state kinetics showed that the difference of theseforms of Acs is dependent with affinity (Km values) for thesubstrates. The FacA protein from the NH3-fermenting cells
showed slightly higher molecular mass on SDS-PAGE andwas reacted more strongly with anti-acetyl lysine antibody.
These results indicates that A. nidulans acetylates some lysine residue(s) of FacA protein in response to the environmental O2 concentration to regulate its affinity for the substrates, which reversed the reaction from Acs to Ack. Detailed regulating mechanisms of the FacA protein is now
under investigation.