Introduction

Homoisocitrate dehydrogenase (HICDH) catalyzes the fourth step reaction in the novel α-aminoadipate (AAA) lysine biosynthetic pathway; together with the paralogous enzymes, isocitrate dehydrogenase (ICDH) and 3-isopropylmalate dehydrogenase (IPMDH), are called three brothers forming the divergent β-decarboxylating dehydrogenase family. HICDH is involved in lysine biosynthesis, while ICDH and IPMDH are involved in the TCA cycle and leucine biosynthesis, respectively. Each β-decarboxylating dehydrogenase should have evolved from a common ancestor, which possesses broad substrate specificity to catalyze the various metabolic reactions in the early stage of life. The broad substrate specificity and evolutionary relationship of β-decarboxylating dehydrogenase provide several interesting implications regarding the overall process of gene family evolution by gene duplication and functional divergence from ancestral genes.

1. Characterization of β-decarboxylating dehydrogenases from various organisms

Phylogenetic analyses from various organisms have shown the enzymes in β-decarboxylating dehydrogenase family can be separated into three groups: IPMDH group; ICDH group; and ICDH/IPMDH/HICDH-mixed group. Characterization of ICDH and IPMDH from Escherichia coli and Thermus thermophilus has shown that ICDH and IPMDH strictly discriminate their substrates from each other; however, HICDH activity is detectable for most enzymes. This suggests that HICDH activity may be retained as an evolutionary leftover in substrate-specific ICDH or IPMDH of bacteria and archaea. The existence of HICDH activity in ICDH or IPMDH from various organisms suggests that the common ancestor of β-decarboxylating dehydrogenase would have HICDH activity.

2. Evolutionary analysis of chemically synthesized common ancestor of β-decarboxylating dehydrogenase

Phylogenetic and molecular evolutionary analyses were conducted for ICDH, IPMDH and HICDH from a large set of microorganisms. To examine the catalytic feature of ancestral-type β-decarboxylating dehydrogenase, we aligned amino acid sequence of ICDHs, IPMDHs and all the β-decarboxylating dehydrogenases, and designed ancestral-type ICDH, IPMDH, and β-decarboxylating dehydrogenases, each with the ancestral-type sequence inferred by maximum likelihood method. The genes for the ancestral enzymes of ICDH group, IPMDH group and the common ancestor of all three groups were chemically synthesized and expressed in E.coli cells. Although the ancient-type IPMDH was accumulated as inclusion bodies in E.coli cells, other two proteins were produced in soluble fractions. ICDH ancestor did not show activity for isocitrate, 3-isopropylmalate, nor homoisocitrate; however, the chemically synthesized common ancestor of all three groups exhibited distinct enzymatic activity for 3-isopropylmalate using NAD+ as a coenzyme. Furthermore, trace amount of 2-oxoglutarate was detected by overnight reaction with the ancestral-type enzyme using NADP+ as a coenzyme, although activity detection by monitoring an increase in NADH was impossible due to extremely low activity. These results suggest that ancient β-decarboxylating dehydrogenase possessed dual functions at least. Complete loss of activity for homoisocitrate might suggest that a large loop to accommodate larger γ-moiety of homoisocitrate in the substrate-binding pocket was removed in the common ancestral-type enzyme during amino acid sequence alignment.

3. Characterization of β-decarboxylating dehydrogenase homolog (TK0280) from T. kodakarensis

The hyperthermophilic archaeon Thermococcus kodakarensis is suggested to synthesize lysine through the AAA pathway starting from 2-oxoglutarate as the initial compound. Interestingly, T. kodakarensis has only a single set of the lysine biosynthetic gene cluster carrying TK0280 gene, but has no other enzymes (gene clusters) specific to the leucine, glutamate and arginine biosynthesis. Therefore, I proposed that T. kodakarensis has an ancient-type multi-functional metabolic pathway that can produce not only lysine but also leucine, glutamate, and arginine. The characterization of the chemically synthesized ancient-type enzymes could not provide fully convincing evidence for presenting the ancestral feature of broad substrate specificity (Chapter 2); however, phylogenetic analyses suggest that TK0280 is in the ICDH/IPMDH/HICDH-mixed group and present close to the root of phylogenetic tree for β-decarboxylating dehydrogenase from various microorganisms, which suggests that TK0280 would exhibit all the activities for the substrates: homoisocitrate, isocitrate and 3-isopropylmalate.

The TK0280 gene was cloned and overexpressed in E. coli and catalytic properties and subunits organization were investigated. TK0280 is a tetramer composed of four identical subunits, each with a molecular weight of about 42 kDa. TK0280 exhibits distinct activity for homoisocitrate, isocitrate, and 3-isopropylmalate (Fig. 1). This result indicates that TK0280 is a promiscuous enzyme that can recognize several related compounds as substrates, and also suggests that TK0280 is potentially involved in the synthesis of lysine, leucine and glutamate in T. kodakarensis. The patchwork hypothesis in the evolution of metabolic pathways implicates that the ancestral enzymes should possess broad substrate specificity and be involved in multi-metabolic pathways. Thus, the results above indicate that TK0280 is an ancestral-type enzyme and may be very close to the common ancestor of β-decarboxylating dehydrogenase.

4. Site-directed mutagenesis of TK0280

In HICDH from T. thermophilus, it is known that Arg85 is a key determinant for the substrate recognition, due to the fact that the single substitution of Arg85 altered its substrate specificity. According to the crystal structure of HICDH from T. thermophilus, Arg85 is located in the substrate-binding pocket and suggested to interact with the γ-carboxylate of homoisocitrate. The pairwise amino acid sequence alignment with T. thermophilus HICDH shows that, Leu83 is at the equivalent position of Arg85 of T. thermophilus HICDH. To further elucidate the key residues related to the substrate specificity of TK0280, site-directed mutagenesis was conducted for Leu83.

Enzymatic analysis using 3-isopropylmalate, isocitrate, and homoisocitrate revealed that the mutant L83S shows narrow substrate specificity (Fig. 1) with increased turnover number for the reaction using homoisocitrate as the substrate. Although the catalytic efficiency, kcat/Km, was decreased due to the increased Km value for homoisocitrate, the substrate preference of homoisocitrate to isocitrate was enhanced from ~30 fold to ~100 fold. The results above further suggest that the specificity is determined by a limited number of amino acid residues in TK0280 and that TK0280 is designed to exhibit broad substrate specificity.

Conclusion

TK0280 from hyperthermophilic archaeon T. kodakarensis, the fourth enzyme involved in AAA lysine biosynthetic pathway, possesses features of ancestral-type enzyme with broad substrate specificity and may serve for multi-metabolic pathways, which is in line with the patchwork hypothesis on the evolution of metabolic pathways. The mutation at Leu83 of TK0280 indicates that Leu83 is one of key determinants for the substrate specificity in conferring promiscuous activity. This study could provide valuable insights on the evolution of β-decarboxylating dehydrogenase family and also give a clue to the evolution of lysine, leucine and other amino acid biosynthetic pathways.

Figure 1. Substrate specificity of WT and L83S

我々人間を含む生物の共通の祖先は、現在よりも単純な遺伝子や酵素のセットを持っており、それらを用いて自らの生命活動を維持していたと考えられている。酵素の機能の進化の理論としてパッチワーク仮説が広く信じられている。この仮説によれば、原始生命に含まれている酵素は基質特異性が寛容であり、複数の化合物を基質とした反応を行い各種の必要な反応を行っていたとされている。そうした基質特異性の寛容な酵素をコードする遺伝子が重複を起こすとともに、変異が蓄積し特定の基質に対する高い特異性を獲得することにより、現在多岐に分かれた代謝・物質変換系が構成されていったとされている。しかしながら、それを直接的に証明した研究は存在していない。本論文は、β脱炭酸型脱水素酵素を対象として、酵素機能の進化の解明を目的として行ったもので、4章より構成される。

まず、序論で進化仮説やβ脱炭酸型脱水素酵素の概略が述べられた後、第1章では、β脱炭酸型脱水素として共通の祖先を持つと考えられている3-isopropylmalate dehydrogenase (IMPDH)、isocitrate dehydrogenase (ICDH)、homoisocitrate dehydrogenase (HICDH)を系統樹解析した上で、Escherichia coliおよびThermus thermophilus 由来のIPMDHとICDHについて基質特異性解析を行っている。IPMDHとICDHが本来の基質に高い親和性をもつと同時に、予想外なことに弱いながらもHICDH活性を有することが明らかにされている。

第2章では、まず183種類のβ脱炭酸型脱水素酵素のアミノ酸配列を用いて、IPMDH祖先型、ICDH祖先型、β脱炭酸型脱水素酵素全ての祖先型(全祖先型)のアミノ酸配列が推定された。ついで、それらを祖先型酵素をコードする遺伝子を大腸菌において発現、精製し、遺伝子産物である酵素の熱安定性と基質特異性が調べられた。IPMDH祖先型酵素は不溶性タンパク質生産したため、その後の解析は行われなかったが、ICDH祖先型酵素と全祖先型酵素は可溶性タンパク質として生産された。現存する生物の共通祖先は超好熱性であるという考えが支持されているが、ICDH祖先型酵素と全祖先型酵素は中温域で変性したことから、今回設計したアミノ酸配列には安定を低下させるものが含まれている可能性が考えられた。これら2つの祖先型酵素のうち、ICDH祖先型酵素は活性を示さなかったため、全祖先型酵素について活性を検討した結果、全祖先型酵素はIPMDH活性だけでなく、弱いながらもICDH活性を有することが反応産物の超高感度HPLC-MS/MS解析によって示されている。

第3章では、超好熱性古細菌であるThermococcus kodakararensisのTK0280の基質特性について調べられている。TK0280は、β脱炭酸型脱水素酵素の系統解析により、β脱炭酸型脱水素酵素ファミリーの進化の初期段階から分岐している。このことから、TK0280がβ脱炭酸型脱水素酵素ホモログの中で、最も古いタイプの酵素の性質を有していると考えられた。そこでTK0280遺伝子をE. coliにおいて発現、精製し、遺伝子産物である酵素の活性を測定したところ、3つの基質homoisocitrate、isocitrateと 3-isopropylmalateに対する明瞭な活性が検出された。第2章および第3章の結果は、β脱炭酸型脱水素酵素がかつて多機能であり、それが基質特異性を狭くすることで現在の酵素へと進化したことを示すものと考えられた。

第4章では、TK0280において基質認識に関わるアミノ酸残基を推定し、そのアミノ酸残基の改変体について基質特異性を調べている。T. thermophilusのHICDHの基質特異性決定基の一つであるArg85に相当するTK0280の残基Leu83のSer置換は、3-isopropylmalateに対する活性を失わせると同時にisocitrateに対する活性を1/5程度に低下させた。その一方で、同改変はhomoisocitrateに対する活性を2.5倍程度上昇させた。複数のアミノ酸置換の蓄積により酵素の基質特異性が徐々に変化する場合、オリジナルな基質に対する特異性を残したまま新しい基質に対する特異性が生じる中間状態を経ることがこれまでに行われた分子進化工学的研究で明らかになってきている。この事実は、基質特異性が寛容な酵素は、基質特異性が狭い酵素に比べて比較的少数のアミノ酸置換で基質特異性を変化させうることを示唆している。第5章の結果は、基質特異性が寛容なTK0280においても、同様に数少ないアミノ酸置換で基質特異性が容易に変化することを示したもので、TK0280が進化を受け入れる能力が高い祖先型の性質を保持した酵素であることを示している。

以上、本論文は、β脱炭酸型脱水素酵素の機能および進化の解明を目指し祖先型酵素の設計及び祖先型に近い酵素の性質を明らかにしたものであり、学術上、応用上貢献するところが少なくない。よって、審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。