Yam, the tuber of Dioscorea spp., is an important staple food in many tropical countries. More than 600 species of yam have been planted all over the world. In China, yam has been traditionally used as a health food and Chinese herbal medicine. It also receives much attention for its functional properties and pharmaceutical potential. Some of the beneficial properties of yam have been attributed to the storage protein dioscorin.

Dioscorin is the major storage protein of the yam tuber and accounts for approximately 80-85% of the total soluble proteins. It has been reported that the protein has carbonic anhydrase (CA), trypsin inhibitor (TI), dehydroascorbate (DHA) reductase, and monodehydroascorbate (MDA) reductase activities. CAs are zinc metalloenzymes that catalyze the interchange of CO2 and HCO3-. In plants, they play an important role in photosynthesis and other biosynthetic reactions. Proteinaceous protease inhibitors are important in regulating and controlling endogenous proteases and in acting as protective agents against insect and pathogen proteases in plants. Both of DHA reductase and MDA reductase are important enzymes in the glutathione-ascorbate cycle, which is a part of the antioxidative system for protecting plants from the toxicity of reactive oxygen species (ROS).

Although the functional properties of dioscorin have been well explored, the mechanism for exerting the diverse functions remains unclear due to the lack of information about the structure-function correlation. In an effort to accelerate the structural and biochemical characterization of dioscorin, we cloned, expressed, purified and crystallized dioscorin from Dioscorea japonica.

Firstly, dioscorin was cloned, and the DNA sequencing showed that four similar genes of dioscorin were obtained. The dioscorins were expressed in E. coli and purified using affinity resin and anion-exchange chromatography. The recombinants were compared with native dioscorin using SDS-PAGE and CD measurement, and no obvious structure change was detected (Fig. 1).

Recombinant dioscorins obtained clearly showed CA, TI, DHA reductase and MDA reductase activities. These activities were not affected by the amino acid substitutions, which suggest that these dioscorins were isoforms of one another. Although the recombinants had the same CA activity and TI activity as native dioscorin, The DHA and MDA reductase activities of recombinants decreased greatly (Fig. 2). Since glycosylation is a post-translational process and very rarely occurs in bacteria, the recombinant dioscorins were not glycosylated. In order to investigate the activities of native dioscorin without glycans, PNGase F was used to eliminate the N-glycans, and neuraminidase and endo-α-N-acetylgalactosaminidase were used to eliminate the O-glycans from native dioscorin. After the cleavage by the deglycosyslation enzymes and the purification using a Superdex75 gel filtration column, the DHA reductase activity and MDA reductase activity of deglycosylated dioscorin were tested again. The results showed that the DHA reductase activity and the MDA reductase activity of both N-deglycosyslation and O-deglycosyslation dioscorins decreased greatly, and the differences between the deglycosylated and the recombinant dioscorins were not significant (Fig. 2), which means the glycans are closely related to the DHA reductase activity and the MDA reductase activity of native dioscorin. We also confirmed that native dioscorin is an N- and O-linked glycoprotein using enhanced concanavalin A-peroxidase staining method and N- / O-deglycosyslation treatments.

Dioscorin was crystallized, and after several rounds of optimization, a single crystal with dimensions of 0.5 × 0.3 × 0.06 mm was obtained using the reservoir solution consisting of 0.1M CAPS pH 10, 0.2 M lithium sulfate, 1.8 M ammonium sulfate at 278 K for 6 days. An X-ray diffraction data set was collected to 2.11 A using a synchrotron X-ray source (PF NW12). The crystal belonged to the centered orthorhombic type and the space group was C2221, with unit-cell parameters a =83.5 A, b = 156.8 A, c = 83.6 A. A total of 222 696 reflections in the resolution range of 20.0-2.11 A were collected with 99.3% completeness and an Rsym of 3.3%. After phase determination and refinement, the structure of dioscorin was resolved. Dioscorin has one big β-sheet that consists of nine β-strands, which was surrounded by six α-helices, two small β-strands and several loops (Fig. 3). Although dioscorin and CA from N. gonorrhoeae only share 32% sequence identity and two of the active site residues are altered (Q114 mismatch, H95 different orientation), the three-dimensional structures are quite similar for the two proteins (Fig.3). Q114H mutant changed dioscorin to a typical α-CA and the CA activity was increased greatly in the presence of zinc ions.

The structures of dioscorin-DHA complex (2.28 A) and dioscorin-ascorbate complex (2.20 A) were also obtained using cocrystallization method, and some miner structure changes (side chain movements of His69 and Arg93) were identified, which were considered to be the structure changes happened during the reaction process. As a result of glycosylation site prediction using YinOYang 1.2 server, two possible O-glycosylation sites (Thr188 and Thr228) were identified, and Thr188 was located near the active site of dioscorin. It is possible that the loss of O-glycosylation of Thr188 caused the change of structure, and then the DHA and MDA reductase activities of dioscorin were reduced. Based on the typsin docking model of dioscorin obtained from the Cluspro 2.0 server, point mutations were introduced. After the TI activity determination of the mutants, the inhibitory loop (loop α2-α3) of dioscorin was identified.

Fig.1 SDS-PAGE (A) and CD spectra (B) of native and recombinant dioscorins

Fig. 2 DHA reductase (A) and MDA reductase(B) activity of native and recombiant dioscorins

Fig. 3 Structure comparison between dioscorin (green) and CA form N. gonorrhoeae (cyan)

本論文では、ヤムの主要な貯蔵タンパク質dioscorinを同定し、活性測定とX線結晶構造解析を行い、dioscorinの炭酸脱水素酵素、デヒドロアスコルビン酸(DHA)還元酵素及びトリプシン阻害剤としての構造基盤について述べている。本論文は5章からなる。

第一章では、ヤムの塊根の液胞に主に存在しているdioscorinが有する多機能性について述べている。Dioscorinは炭酸脱水素酵素、トリプシン阻害剤並びにDHA還元酵素活性をもつことが報告されている。炭酸脱水素酵素は植物の光合成と呼吸に重要である。トリプシン阻害剤は植物の内在性プロテアーゼの調節と外来プロテアーゼの抑制に機能する。DHA還元酵素は植物の抗酸化システムの重要な酵素である。また、dioscorinは抗酸化、抗高血圧、免疫賦活という様々な作用をもつことが説明されている。dioscorinがこうした植物生理学や食品科学の研究対象として非常に興味深いことを説明している。

第二章では、dioscorinのクローニング、発現と精製について述べている。ヤム塊根からtotal RNAを抽出し、逆転写によりcDNAを合成した。DNA配列解析の結果、配列が類似した4種類のdioscorin(D1, D2, D3, D4)を取得した。これらのdioscorinを大腸菌で発現・精製し、ヤムから抽出・精製した天然型dioscorinと構造的特徴の比較を行なった結果、組換え型dioscorin は天然型と同様の二次構造を形成していることを明らかにしている。以上、dioscorinを組換え型として大量調製する系の構築に成功した。

第三章では、組み換え型と天然型のdioscorinの活性比較について述べている。活性測定の結果、組換え型dioscorinは天然型と同様の炭酸脱水素酵素、トリプシン阻害剤活性を持つことを明らかにしている。一方、DHA還元酵素活性については、天然型よりも著しく低下していた。また、糖鎖切断反応(N-/O-結合型)と糖鎖染色の結果、天然型はN-及びO-結合型糖鎖を持ち、糖鎖切断反応よりDHA還元酵素活性が組換え型と同レベルまで低下した。以上、dioscorinのDHA還元酵素活性には糖鎖が寄与していることを明らかにした。

第四章では、dioscorinの立体構造と炭酸脱水素酵素としての構造基盤について述べている。dioscorin(D1)を結晶化し、分子置換法を用いて分解能2.11Aの構造解析に成功した。その結果、dioscorinの立体構造は1つのβシートを分子中央にもち、それを取り囲むようにαヘリックスとループが分子表面に位置していることを明らかにした。また、dioscorinの活性部位は2つのHis残基と1つのGln残基で形成され、3つのHis残基で活性部位が形成される他の炭酸脱水素酵素と異なっていた。変異体解析より、Q114H変異によっても炭酸脱水素酵素活性を示したが、その活性はZn2+依存的に変化したことを明らかにしている。これは、これまでの炭酸脱水素酵素では見られないdioscorinに特徴的な活性であると言える。

第五章では、複合体構造解析とドッキングシミュレーションによるDHA還元酵素及びトリプシン阻害剤としての構造基盤について述べている。dioscorinと基質(DHA)及び産物(ascorbate, ASC)を共結晶化し、分解能2.28Aと2.20 Aの構造解析に成功している。構造比較から、次のような反応機構モデルを提案している。(1) His95はAsp70との相互作用により水分子からプロトンを引き抜く。(2) このHis95はプロトンをDHAの3位カルボニル基の酸素原子に供給する。(3) プロトンが3位から2位カルボキシル基の酸素原子に渡される。(4) His95はもう1つの水分子を使って、3位カルボニル基の酸素原子にプロトンを再度供給する。こうしてDHAがASCに変換される。一方、dioscorinのトリプシン阻害活性については、Cluspro 2.0 serverを使ってdioscorinとtrypsinのドッキングシミュレーションにより結合モデルを提案している。変異体解析により、C28-C187ジスルフィド結合が相互作用するループの形成に重要であること、trypsinの活性ポケットに入ると予想されたArg43が実際にtrypsin阻害活性に重要であることを明らかにしている。

以上のように、本論文は多機能タンパク質としてのdioscorinの構造・機能解析に成功し、多機能性を説明するユニークな構造基盤を明らかにしており、得られた知見は学術上、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。