Nitrogen is a basic element for life because it is an essential component of most bio-organic substances, such as amino acids, proteins, vitamins and nucleic acids. It exists in the biosphere in several redox states and interconversions of these nitrogen species are mostly attributed to biogeochemical nitrogen metabolism. The nitrogen metabolisms can be categorized into two types, assimilatory and dissimilatory metabolism, according the process and purpose in organisms. Denitrification is a typical form of dissimilatory nitrogen metabolism. In this study, we studied the enzymes involved in denitrification and nitric oxide (NO) detoxification as shown Figure 1.

Denitrification has been considered to be an exclusive process of bacteria until the fungal denitrification was discovered by Shoun et al. firstly at 1991. Denitrifying fungi can reduce nitrate (NO3-) or nitrite (NO2-) to nitrous oxide (N2O) by dissimilatory nitrate reductae (dNar) or dissimilatory nitrite reducase (dNir) and nitrous oxide reductase (P450nor) (Figure 1). The acitivities of the enzymes involved in denitrification process were detected in some denitrifying fungi. However, the corresponding genes have not been identified and isolated except the one encoding p450nor. My predecessor, Master Nakanishi, isolated a copper-containing dNir (NirK) gene from Aspergillus oryzae (A. oryzae) and gave a characterization of its recombinant protein for the first time.

NO is a common product of enzymic and nonenzymic oxidation of reduced nitrogen compounds, which ubiquitously exists in environment. NO has the potential to damage a variety of biomolecules, microorganisms living in such an environment required detoxification of NO. Flavohemoglobin (FHb) of bacteria and yeast is a main enzyme involved in NO detoxification under aerobic conditions by its NO dioxygenase (NOD) activity. Multiple FHb homologue genes are found in the genomes of fungi. But their physiological significant were not known. There is considerable interest in clarifying whether the fungal FHb homologues display NOD activities and whether the multiple proteins in the same fungus share the same properties.

In this work, I cloned and characterized two FHbs from A. oryzae, and elucidated their physiological significant. On the other hand, I kept on the study of NirK to investigate the relation between NirK and denitrification. In addition, I cloned a eucaryotic dissimilatory nitrate reductase (NapA-like) gene from A. oryzae for the first time.

1.Cloning and characterization of two FHbs.

Two FHb genes, fhb1 and fhb2, were cloned from A. oryzae. The deduced amino acid sequences of FHb1 and FHb2 showed high identity to other FHbs except the predicted mitochondrial targeting signal in the N-terminus of FHb2. Recombinant proteins of full length FHb1 and FHb2 without the putative mitochondrial targeting signal were expressed in E. coli and purified to homogeneity. The recombinant proteins displayed similar absorption spectra to other FHbs, but FHb2 did not contain FAD cofactor at all. FHb1 and FHb2 were estimated to be a monomer and a dimer in solution, respectively. Both of the two enzymes exhibit high NOD activities. FHb1 utilizes either NADH or NADPH as the external electron donor, whereas FHb2 can use NADH only. These results suggest that FHb1 and FHb2 are fungal counterparts of the bacterial FHbs and act as NO detoxification enzymes in cytosol and mitochondria, respectively.

2.In vivo, FHb1 and FHb2 are involved in NO detoxification in cytosol and mitochondria, respectively.

Expression responses of fhb1 and fhb2 to NO stress were investigated by real-time PCR. fhb1 in wild type was up-regulated to about 15 times by NO or NO generator, NO2-. However, the intracellular levels of fhb2 was too low to be detected under any conditions. The fhb1-deletion mutant (Δfhb1) and fhb2-deletion mutant (Δfhb2) were constructed to investigate their physiological functions. In the minimal agar medium (CD), under the normal conditions, the growth rate of Δfhb1 was obviously slower than Δfhb2 and wild type. In the liquid CD medium, Δfhb1 could not to form clumps. Whereas, there was no difference between Δfhb2 and wild type. It was suggested that the FHb1 affect the fungal morphology. On the other hands, in the rich medium (DPY), the three strains did not show any phenotypic differences in the absence of NO stress. So DPY medium was used to investigate the effects of fhbs disruption on NO stress. Δfhb1 became hypersensitive to NO stress, whereas, fhb2 deficiency did not display any obvious effects compared with wild type. It may be attributed to the little effect of the externally added NO stress on mitochondria. To investigate the expression of fhb2, I used a nirK-over expression strain, which can promote mitochondria NO production by its high mitochondrial nitrite reductase activity. As expected, the expression of fhb2 became detectable by real-time PCR in this strain which indicated fhb2 was induced by mitochondrial NO stress.

3.FHbs enhanced the oxidative stress of H2O2 probably dependent on Fenton reaction.

E. coli FHb (HMP) and S. cerevisiae FHb (YHb) were reported to participate in defense against oxidative stress. So FHbs of A. oryzae were investigated for their responses to oxidative stress from H2O2. It is interesting that both of Δfhb1 and Δfhb2 exhibited higher resistance than wild type to H2O2. Meanwhile, the strain harboring the fhb2 over-expression plasmid also showed hypersensitivity to H2O2. These findings indicated FHb expression might have deleterious effects at the presence of oxidative stress, which is obviously conflicted the conclusion from HMP and YHb. The C-terminal portion of FHbs showed homology with E. coli flavin reductae (Fre) which possesses a ferric iron reductase activity. So when the NO is absent, FHbs can reduce FAD to FADH2, which may in turn act as ferric iron reductant and drive the Fenton reaction to cause the DNA damage. The flavin reductase activities of the purified FHbs were confirmed experimentally. We are measuring and comparing the degree of DNA damage among Δfhb1, Δfhb2 and wild type in the presence of H2O2.

4.Cloning of a periplasmic nitrate reductase (NapA) homologue gene from A. oryzae .

Bacterial NapA is a periplasmic dissimilatory nitrate reductase. Bacterial NapA is used to support anaerobic metabolism as an alternative to the NarGHI pathway when nitrate concentration is low in the culture or used as an electron sink to eliminate an excess of reducing equivalents accumulated in the cytoplasm as NADH and FADH2. To date, no eukaryotic dissimilatory nitrate reductase has been isolated, yet. However, by Blast search on the fungal genomes, a napA-homologue gene was found in some strains. So I tried to clone and characterize the napA-homologue gene from A. oryzae. The full-length cDNA of napA-homologue gene was cloned and the 990-amino acids sequence of the protein was deduced. Different from bacterial NapA, no putative targeting signal peptide was found at its N-terminus. So, it may be localized into cytosol. To characterize the recombinant protein, expression systems of E. coli and A. oryzae were used. But only A. oryzae system worked. The recombinant NapA in crude extract exhibited an obvious anaerobic nitrate reductase activity. Though the expression level was very low, NapA could be distinguished as a main band at about 110 kDa after the purification by nickel affinity column. But it became denatured before the following purification by gel filtration chromatography. So optimization of the conditions of expressing and purification remain to be done in future.

5.Study on the enzymes involved in denitrification of A. oryzae.

In this chapter, I revised and supplemented some data for the characterization of the recombinant NirK which had been done previously by Nakanishi. Then I mainly investigated the relation between the nirK gene and denitrification. I also confirmed the necessity of P450nor to denitrification of A. oryzae. The recombinant NirK showed robust anaerobic nitrite reductase activity, and many properties similar to bacterial NirK. The A. oryzae strains possessing a NirK over-expression plasmid, constructed by Nakanishi priviously, showed about 6 times higher in denitrification ability than the wide-type in my experiment condition. The pellet of the homogenate from the nirK over-expression strain showed high NirK activity. So these results suggested that NirK should be anchored in mitochondria membrane by the action of extensional predicted mitochondrial targeting signal sequence existing in its N-terminus. I compared the effects of O2 and NO2- on the expression level of nirK gene, in vivo. nirK was robustly induced under denitrification conditions. I constructed nirK-deletion mutant (ΔnirK) and P450nor-deletion mutant to compare their denitrification abilities with wide-type. P450nor deficient mutant could not denitrify any more, but ΔnirK still kept denitrifying activity like wide-type. It has been reported that plant assimilatory nitrate reductase and yeast mitochondrial cytochrome oxidase are also involved in NO production. Both of the two enzymes exist in A. oryzae. So it is possible that some NO producing enzyme substitutes NirK to reduce NO2- to NO in the nirK deficient mutant. On the other hand, P450nor is vital to denitrification.

Conclusions:

The gene of a eukaryotic dissimilatory nitrate reductase (NapA-homolog) was isolated for the first time, and the recombinant protein exhibited an anaerobic Nar activity. NirK anchored in mitochondria membrane catalyzes the conversion of NO2- to NO in the denitrification pathway. P450nor reducing NO to N2O at cytosol is vital to denitrification but there may be some isoenzyme of NirK exist in A. oryzae. FHb1 and FHb2 play a role of NO detoxification in cytosol and mitochondria, respectively. In the absence of NO stress, FHb1 and FHb2 would amplify the oxidative damage of H2O2 probably by driving Fenton reaction.

Figure 1. Enzymes involved in denitrification and NO detoxification in fungi.

Figure 2. Enzymes involved in dissimilatory nitrogen metabolism studied in this work.

生命活動による無機窒素の循環は窒素サイクルと呼ばれ、その主要な過程は窒素固定、硝化、および脱窒である。この3過程以外にも、もっとも酸化された窒素形態である硝酸については多様な代謝系が存在する。植物や微生物には、窒素源として硝酸態窒素を利用するため、硝酸を亜硝酸を経てアンモニアに還元し、アミノ酸などの有機物に取込む経路が存在する。この過程は同化型硝酸還元と呼ばれる。一方硝酸は原核生物やカビにおいて、呼吸や発酵のための電子受容体として利用される。このような生命反応を異化型硝酸還元と呼び、そのうち最終産物がN2やN2Oなどのガス状窒素である場合、脱窒と呼ぶ。また、必ずしもエネルギー(ATP)産生に結びついていない窒素還元や、本論文の主課題であるフラボヘモグロビン(FHb)による酸化反応なども含め、明らかに同化が目的でない無機窒素代謝をまとめて窒素異化代謝と呼ぶ。この定義によれば同化目的の窒素固定は窒素異化代謝には含まれず、エネルギー産生が目的の硝化は含まれることになる。しかし慣例上、硝化は窒素異化代謝とは呼ばれない。

従来、窒素サイクルには原核生物のみが関わると考えられてきた。しかるに近年 Fusariumoxysporumその他のカビが脱窒することが発見され、カビ脱窒系に関して当研究室を中心に、生化学的、分子生物学的解明が進められてきた。その結果、カビ脱窒系はミトコンドリアに局在し、細菌脱窒系と同様に嫌気呼吸として働いていることが明らかとなっている。一方多くのカビゲノムが解読されてきているが、我々はわが国でゲノムの解読された麹菌Aspergillus oryzaeのゲノムにnirk、 CYP55 (P450nor)などの脱窒関連遺伝子ホモログの存在を見出した。当研究室の中西はこのnirkホモログ産物が細菌のオルソログと同様に銅含有型亜硝酸還元酵素 (NirK)をコードすることを明らかにした。A. oryzaeにはF. oxysporumなどで脱窒に関わることが示唆されているFHbや、細菌のペリプラズム局在型硝酸還元酵素(Nap)などのホモログ遺伝子も見出される。本論文ではこれらA. oryzaeに見出されたFHb 、 Nap、 NirKなどのホモログ遺伝子産物の性質、生理機能などの解明を目的とした。細菌のFHbは一酸化窒素(NO)ジオキシゲナーゼ(NOD)活性を示し、NOストレスに応答することが知られている。

A. oryzae には FHb遺伝子ホモログが二つ見出され、それぞれfhb1(遺伝子産物は FHb1)、fhb2 (同 FHb2)と命名した。FHb2にはそのN-末にミトコンドリア移送シグナルの存在が予想された。これら遺伝子をクローニング、大腸菌で発現し、組替え体タンパク質の性質を明らかにした。両者ともFHbに特徴的な吸収スペクトルを示し、NOD活性を持っていた。また両者のコファクターとしてFADを同定し、それぞれの含有量を定量した。電子供与体としてFHb1は NADHと NADPHの両者を利用できるが、FHb2はNADPHに特異的であった。この結果はFHb1およびFHb2がそれぞれサイトゾルとミトコンドリアに局在することを示唆する。

fhb1、 fhb2のNOストレスへの応答をreal-time PCRにより観察した。その結果、fhb1はNOストレスにより15倍のup-regulationが観察されたが、 fhb2の発現はさまざまな条件でも観察されなかった。両者の遺伝子破壊株(Δfhb1、 Δfhb2)を作成し、その表現系の観察から両遺伝子の生理機能の解明を試みた。その結果fhb1は細胞の形態に関わることが示され、さらにNOストレスへ応答することも明らかになったが、fhb2に関してはそのような結果は得られなかった。一方fhb2はゲノム上でnirkに隣接することから、その脱窒への関連が示唆された。そこでnirk高発現株を用い、脱窒条件の時にfhb2が初めて発現することを示した。この結果により、ミトコンドリアにNOが多く発生する時にのみfhb2が発現することが示され、fhb2もNOストレスに対応することが明らかとなった。

Δfhb1、 Δfhb2はまた、酸化ストレス(H2O2)感受性が鈍くなっていることが発見されたが、その機構の解明を試み、FHbがフェントン反応による水酸ラジカルの生成を促進する機構が提案された。

以上、本論文はカビのFHbについて詳細に解析した最初であり、また複数のFHbアイソザイム遺伝子を同一生物からクローニングし、生理的意義を解析した初めての例である。そこでは2種のアイソザイムFHb1およびFHb2について、その細胞内局在、生理的意義の違いなどを明らかにした。とくにFHb2がミトコンドリアの脱窒に関与(NOの解毒)することが強く支持されたことは大変興味深い。

FHb以外の硝酸異化代謝関連遺伝子として、napAホモログおよびnirkにも注目した.napAは細菌脱窒系に関与する硝酸還元酵素遺伝子であるが、それがカビに存在することを示せば重要な発見となる。この遺伝子をクローニング、発現し、活性を確認しているが、精製には至っていない。またnirk破壊株を作成し、脱窒との関連を調べた。得られた結果は期待通りではなかったが、妥当な結論は得ている。

以上本論文は、カビで初めてFHb遺伝子を麹菌から2種単離し、それらのNOストレスへの応答や脱窒への関与を明らかにした。さらにカビで初めてとなるnapA頃ホモログの異種宿主発現にも成功し、その生理機能解明の基礎を固めた。これら成果は学術上ならびに応用上貢献するところ大である。よって審査員一同は本論文が博士(農学)の学術論文として価値あるものと認めた。