Nitrogen is a primary element that is indispensable for life to constitute its body, as well as carbon, hydrogen, and oxygen, and its assimilation is an important central metabolism in all organisms.

Hydrogenobacter thermophilus TK-6 is a thermophilic, hydrogen-oxidizing, obligately autotrophic bacterium. Analysis of the 16S rRNA sequence suggested that Hydrogenobacter species are located on the deepest branch in Bacteria along with other Aquificales species. Biochemical studies on H. thermophilus have brought a number of novel findings. One of them is the reductive TCA cycle, a unique central metabolism in which carbon dioxide is assimilated. It was demonstrated that reduced ferredoxin is used as an electron donor in this cycle.

In contrast to the detailed studies on the carbon anabolism, the nitrogen anabolism has not been investigated in H. thermophilus. The objective of this study was to elucidate the nitrogen assimilatory metabolism in this bacterium. The metabolic pathways in which ammonium or nitrate is assimilated as the nitrogen source were estimated, and individual enzymes were purified and characterized in this study (Fig. 1).

Chapter 1. Screening for ammonium-assimilating enzymes

In Chapter 1, I screened for enzyme activities that can serve to assimilate ammonium, using the crude extract of H. thermophilus. In most bacteria, it is known that ammonium is assimilated by glutamate dehydrogenase (GDH) or a coupling reaction of glutamine synthetase (GS) and glutamate synthase (GOGAT) as shown in Fig. 2. GDH activity was not detected in H. thermophilus. While GS activity was detected in the crude extract, NADPH-dependent GOGAT activity, which is a common type of GOGAT in non-photosynthetic bacteria, was not detected. Several possible activities that are alternative to GDH or GOGAT were tested, such as amino acid dehydrogenases and glutamine amidotransferase. However, none of them was detected. Unexpectedly, ferredoxin-dependent GOGAT (Fd-GOGAT) activity was detected. Fd-GOGAT has been found only in cyanobacteria and plants, but not in non-photosynthetic bacteria. This aroused interests in the GS-GOGAT pathway of H. thermophilus, and therefore each enzyme was purified and characterized in Chapter 2 and Chapter 3.

Chapter 2. Glutamine synthetase

In Chapter 2, GS was purified from H. thermophilus and enzymatically characterized. Purified GS was a homomultimer (probably a homododecamer) of a 55 kDa polypeptide. The GS gene was identified and its primary sequence was homologous to those of GSI-β, one of the GS families. Kinetic parameters were determined, and they were comparable to those of known GS, except for the high Km value for Glu. It was demonstrated that H. thermophilus GS is subjected to a posttranscriptional modification, an adenylyl/deadenylyl mechanism to regulate its activity. Some GSI-βs were known to be regulated by this modification, but it was not clear when this mechanism evolved. The existence of this regulation in H. thermophilus suggests that the adenylylating regulation originated before the divergence of the Aquificales from other bacteria.

Chapter 3. Glutamate synthase

In Chapter 3, Fd-GOGAT was purified from H. thermophilus and enzymatically characterized. GOGAT is classified according to its specificity for the electron donor. Fd-GOGAT had been found only in plants and cyanobacteria, whereas the other bacteria have NADPH-dependent GOGAT.

The purified enzyme from H. thermophilus was shown to be a monomer of a 168 kDa polypeptide homologous to Fd-GOGATs from phototrophs. In contrast to known Fd-GOGATs, the H. thermophilus GOGAT exhibited glutaminase activity. Furthermore, ferredoxin specificities of this enzyme were examined by using Fd1, Fd2, and Fd3, ferredoxins from H. thermophilus. Consequently, H. thermophilus GOGAT did not react with Fd3, a plant-type ferredoxin containing a [2Fe-2S] cluster, but with Fd1 and Fd2, bacterial-type ferredoxins containing [4Fe-4S] clusters. Interestingly, the H. thermophilus GOGAT was activated by some of the organic acids in the reductive TCA cycle, the central carbon metabolic pathway of this organism. This type of activation has not been reported for any other GOGATs, and this property may enable the control of nitrogen assimilation by carbon metabolism. In the study of this chapter, it was clearly demonstrated that Hydrogenobacter thermophilus, a hydrogen-oxidizing chemoautotrophic bacterium, possess Fd-GOGAT like phototrophs. This was the first observation of an Fd-GOGAT in a non-photosynthetic organism to my knowledge.

Chapter 4. Aminotransferases

In many organisms, aminotransferase is know to serve in the synthesis and the catabolism of most amino acids, transferring the amino group of the amino acid into the 2-oxo acid. Studies in Chapter 2 and Chapter 3 indicated that the GS-GOGAT pathway assimilates ammonium into Glu in H. thermophilus. To verify Glu can be used for a nitrogen donor for other metabolites synthesis, aminotransferases from H. thermophilus were examined in Chapter 4.

Aminotransferase activities in the crude extract were assayed using Glu, Asp, Ala, Gly, and their corresponding 2-oxo acids as substrates. Consequently, the following four activities were detected (Fig. 3): glutamate:oxaloacetate aminotransferase (GOT), glutamate:pyruvate aminotransferase (GPT), glutamate:glyoxylate aminotransferase (GGT), and alanine:glyoxylate aminotransferase (AGT). In attempt to purify the enzymes with these activities, three aminotransferases, AT1, AT2, and AT3, were purified from H. thermophilus. It was shown that GOT, GPT, and AGT activities were derived from AT1, AT2, and AT3, respectively. AT1 and AT2 also had GGT activity. Kinetic parameters suggested that these three enzymes were enough efficient to serve as an aminotransferase. Interestingly, phylogenetic analysis showed that AT2 and AT3 were phylogenetically located at unusual positions when compared with known aminotransferases.

Study in this chapter demonstrated that several amino acids can be synthesized in H. thermophilus using Glu as an nitrogen donor.

Chapter 5. Nitrate and nitrite reductases

While studies in Chapter 2, 3, and 4 elucidated the metabolism where ammonium is assimilated into several amino acids, it remained unknown how nitrate was assimilated as the nitrogen source. To solve this question, nitrate- or nitrite-reducing enzymes were investigated in Chapter 5. Like GOGAT, assimilatory nitrate reductase (NAS) and nitrite reductase (NIR) are classified on the basis of their electron donor. Bacterial ferredoxin-dependent NAS and NIR were reported only in cyanobacteria with a few exceptions of the other bacteria.

Ferredoxin-dependent NAS activity was detected in the crude extract of H. thermophilus, while no NAD(P)H-dependent NAS activity was detected. An enzyme possessing this activity was purified, and it exhibited NAS activity using Fd1 as the electron donor. This clearly demonstrated that H. thermophilus has a ferredoxin-dependent NAS, which is homologous to known enzymes. In the upstream region of this NAS gene (nasB), a NIR-like gene (nirB) was found (Fig. 4). NirB harbored the "ferredoxin-binding site", which is conserved in genes encoding ferredoxin-dependent NIR. nirB was cloned into a pET-21c or pUC19 vector, and heterologously expressed in E. coli. Soluble fractions of the recombinants contained the ferredoxin-dependent NIR activity, and the recombinant protein was purified. The purified protein showed a ferredoxin-dependent NIR activity using Fd1 as the electron donor. However, this protein lacked the N-terminal region of NirB because its translation started at the GTG codon that is located downstream of the expected initial codon, and it can not be excluded at present that NirB in its native form has different enzymatic properties.

Studies in this chapter indicated that a ferredoxin-dependent enzyme is involved in the nitrate reduction of H. thermophilus, and suggested that ferredoxin might also be used in the nitrite reduction.

Conclusions

This study elucidated nitrogen assimilatory pathways in H. thermophilus. Further, detailed analyses of each enzyme provided several novel findings, such as distinctive enzymatic properties and unexpected distributions of some metabolic features in H. thermophilus. Reaction pathways revealed in this study are conserved among other bacteria. This supports the speculation that the fundamental framework of the nitrogen anabolism was established before the divergence of organisms. In contrast to reaction pathways, some of enzymatic properties or electron donors are shown to be different from those of many bacteria. It could be argued that individual enzymes continued to evolve after the divergence, adapting to the physiological environment of each organism.

One of the characteristics of nitrogen anabolism in H. thermophilus is the deep involvement of ferredoxin (Fig. 5), which is involved also in the carbon anabolism of this bacterium, as is the case with cyanobacteria. This stimulates further interests in redox metabolisms of H. thermophilus.

Fig. 1. Nitrogen assimilatory pathway in H. thermophilus. Chapter numbers indicate in which chapter each pathway or enzyme was examined.

Fig. 2. GDH, GS, and GOGAT reactions. 2-OG, 2-oxoglutarate.

Fig. 3. Aminotransferase reactions catalyzed by AT1, AT2, and AT3.

Fig. 4. Physical map of the nasB and nirB gene cluster in the H. thermophilus genome.

Fig. 5. Nitrogen and carbon anabolisms in H. thermophilus. ATases, aminotransferases; Fdox, oxidized ferredoxin; Fdred, reduced ferredoxin.

本論文で研究材料とした菌Hydrogenobacter thermophilusは進化系統上もっとも古くに分岐したバクテリアの一種と推定され、生命の起源を考える上で重要な研究対象である。また本菌は、水素を唯一のエネルギー源、二酸化炭素を唯一の炭素源とするなど、生育能に関しても特異な性質を多く有している。これらの背景から本菌の代謝系に関してはこれまで精力的に研究が行われ、数多くの新規な性質が見つかっている。特に本菌の炭素同化代謝においては、還元的TCA回路という特徴的な代謝経路を有することが明らかとなっている。

このような炭素同化代謝に関する研究の進展に対し、同様に主要な生体元素である窒素の同化代謝に関しては、申請者以前にはまったく本菌で研究がなされていなかった。本論文では、本菌における窒素同化代謝の解明を目的として研究が行われた。

本論文の第1章では、まず本菌の窒素源の一つであるアンモニアの取り込み経路を同定した。各種酵素活性の測定により、GS-GOGAT経路と呼ばれる代謝がアンモニア同化経路として機能していることが示された。この代謝経路はglutamine synthetase(GS)とglutamate synthase(GOGAT)の共役反応であり、多くの生物に分布する代謝系である。本菌のGOGATは一般的なバクテリア由来酵素と異なり、フェレドキシンを用いるという特殊な性質を有することが示唆された。そこで本経路を触媒する2つの酵素について次章以降で詳細な解析を行った。

第2章ではGSの精製、酵素学的解析を行った。GSに関しては、本菌でも既知の酵素に近いものが保存されていることが示された。また一部のバクテリアに見られる制御機構を本菌も有していることも明らかとし、この制御機構が進化系統上バクテリアの古くから存在することが支持された。

第3章ではGOGATの精製、酵素学的解析を行った。GOGATは電子供与体の種類により分類され、フェレドキシンを用いるタイプが植物やシアノバクテリアのみで見つかっており、その他のバクテリアにはNADPHを用いるタイプしか知られていなかった。本菌からGOGATの精製を行ったところ、フェレドキシン型のGOGATが得られ、本酵素が非光合成生物にも存在することが初めて示された。本菌のGOGATは既知のフェレドキシン型GOGATと共通な性質が多く見られた一方で、フェレドキシンに対する特異性や、有機酸による活性化を受けることなど、既知の酵素には報告例のない性質も複数見られた。特に後者に関しては、これらの有機酸が還元的TCA回路中の代謝物であることから、炭素同化代謝による窒素同化代謝の制御を示唆する興味深い性質だと言える。

第4章では、GS-GOGAT経路により生成するGluを窒素供与体として各種アミノ酸合成を行うaminotransferaseについて研究を行った。本菌から4種類のaminotransferase活性を検出し、Gluを基質としてAsp、Ala、Glyの生合成が可能であることが示された。さらにこれらの活性の由来となる3種類の酵素の精製に成功し、その性質を明らかにした。このうち2つの酵素は既知の酵素とは異なる進化系統に属することが示され、酵素学的にも興味深い知見が得られたと言える。

第5章では、本菌のもう一つの窒素源である硝酸の同化に関して研究を行った。一般的な生物では硝酸はnitrate reductase(NAS)とnitrite reductase(NIR)の共役反応によりアンモニアへと変換され、同化される。これらの反応の電子供与体として、NADPHを用いる酵素が大腸菌などで報告されている一方、シアノバクテリアではフェレドキシンを用いる酵素のみが存在する。本菌からNASの精製を行ったところ、フェレドキシン型NASの存在が示された。さらに、その遺伝子上流にはNIRホモログ遺伝子が存在し、異種発現によりその遺伝子産物がフェレドキシン型NIRであることが強く示唆された。

以上のように、本研究ではH.thermophilusの窒素同化経路を明らかにし、また酵素学的に新規な知見も複数得られている。さらに本菌の窒素同化代謝の特徴として、これまで植物やシアノバクテリア特異的に知られていたような、フェレドキシンの深い関与が示された。本菌の炭素同化代謝においてもフェレドキシンが必須な働きをしていることはすでに示されており、本菌の窒素・炭素両同化代謝の密接な関係を示す興味深い知見と言える。

本論文で得られた知見は他の生物種の窒素代謝、また本菌の代謝全体像の解明に大きく寄与することが期待される。よって審査委員一同は、本論文が博士(農学)の学位としてふさわしいと認めた。