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含硫化合物代謝酵素の立体構造と触媒機構の研究

佐藤, 優太 東京大学 DOI:10.15083/0002006874

2023.03.24

概要





















佐藤

優太

生物は硫黄原子を含む様々な化合物を利用しており、中には独特な代謝系を用意して含硫化合
物を生産する生物も存在する。植物では、ネギ属植物が合成する化合物群システインスルフォキ
シド(CSO)が代表的である。CSO 分解生成物の中には抗酸化作用などヒトの健康に役立つ活性を
持つ化合物もあることから、食用ネギ属植物中の含硫化合物やその代謝経路には古くから関心
が寄せられてきた。また、火山性の環境に生息する微生物の一部は、この環境に豊富な元素状硫
黄などの硫黄化合物を利用するための機構を備えている。本論文は、代謝経路が未だ十分に理解
されていない CSO および元素状硫黄の代謝に着目している。本論文ではこれらの代謝経路上に
存在する三種の酵素についての構造・機能解析を通じ、酵素が持つ触媒機構の詳細ならびに酵素
反応における含硫化合物の挙動の理解を目的としており、三章から構成される。

第一章では CSO の分解に関わる酵素、タマネギ由来催涙因子合成酵素(AcLFS)の触媒機構につ
いて述べている。AcLFS は 1-propene sulfenic acid (1-PSA)という不安定なスルフェン酸化合物に
作用し、催涙因子 syn-propanethial S-oxide (PTSO)という独特な活性を持つ化合物へと変換する酵
素である。本章では、生化学的解析に結晶構造解析、計算化学的手法を組み合わせ、AcLFS の触
媒機構の解明を目指している。1-PSA のアナログ分子の存在下で精製 AcLFS を結晶化し、複合
体構造を最大分解能 1.70 Å で決定している。1-PSA 類似骨格の cis/trans-2-buten-1-ol との複合体
構造では、活性部位中の trans-2-buten-1-ol 分子は U 字型に歪んだ配向で認識されていた。この
配向を 1-PSA に当てはめて考察し、syn 型の PTSO が選択的に生成される理由と AcLFS の触媒
機構を推定している。更に、この機構が実現可能なものなのか、計算化学的アプローチを元に検
証している。MD シミュレーション、QM/MM 計算、および分子軌道計算により、活性部位で 1PSA を歪んだ形で認識し脱プロトン化を誘発するという AcLFS の独特な機構を明らかにしてい
る。

第二章では CSO の合成に関わる酵素、ニンニク由来フラビン含有モノオキシゲナーゼ
(AsFMO1)の構造・機能解析について述べている。AsFMO1 は FAD/NADPH 依存性のモノオキシ
ゲナーゼであり、ニンニクの主要 CSO アリインの合成系において、最終段階である硫黄原子の
立体選択的な酸素付加を触媒する。本章では AsFMO1 の構造・機能解析を行い、CSO の立体化
学的特徴が生み出される機構の解明を目指している。精製した AsFMO1 溶液は黄色を呈し、高

い FAD 保持率を示していた。精製 AsFMO1 溶液を用いて X 線結晶構造解析を行い、AsFMO1 の
FAD 複合体構造および FAD/NADP+複合体構造を最大分解能 2.16 Å で決定して、複合体構造中
には FAD あるいは NADP+の電子密度を明瞭に観察している。精製した AsFMO1 について、分
光測定、TLC、HPLC、ESI-MS による各種分析を行い、in vitro における AsFMO1 の酵素特性を
解析している。精製 AsFMO1 は高い FAD 保持率/NADPH 酸化活性を持つにも関わらず、アリイ
ン合成活性をほとんど示さなかった一方で、AsFMO1 の in vitro 反応では過酸化水素を生成する
活性が存在することを発見している。

第三章では、Sulfurisphaera tokodaii 由来 sulfur oxygenase reductase (StSOR)のクライオ電子顕微
鏡を用いた単粒子解析について述べている。StSOR は元素状硫黄を基質として、亜硫酸を生成す
る酸素添加反応と、硫化水素と亜硫酸を生成する不均化反応を同時に触媒する。本章ではクライ
オ電子顕微鏡を用いて StSOR の構造解析を行い、結晶構造解析とは異なる観点からの知見の獲
得を試みている。精製 StSOR を用いて観察用グリッドを作製し、加速電圧 200 kV の電子顕微鏡
Talos Arctica を用いて粒子画像を撮影している。得られた画像から StSOR 粒子を拾い上げて二次
元分類・三次元再構築し、最大分解能 2.05 Å の静電ポテンシャルマップを得て、このマップに
対してモデルを構築し StSOR 電顕構造を得ている。また、同一発現ロットの StSOR 溶液をさら
に精製し結晶構造解析を行い、分解能 1.73 Å の StSOR 結晶構造も得ている。獲得した 2 つの構
造を元に StSOR の構造的特徴を比較したところ、その球状構造において内部と外部の領域を繋
ぐチムニーと呼ばれる構造は 2 つの構造で大きな違いは観察されていない。しかし、活性部位に
存在する非ヘム鉄とその配位子においては、結晶構造では明瞭に観察されていた水分子が電顕
構造では消失しているなどの違いが観察されている。活性部位付近に存在する 3 つのシステイ
ン残基については 2 つの構造間で観察されるマップに違いが現れ、特に C31 残基側鎖の硫黄原
子の先に結晶構造では見られなかった由来不明の連続した静電ポテンシャルマップが観察され
ている。

以上、本論文により明らかにした、三種の酵素の立体構造や触媒機構についての知見は、生
物が含硫化合物を利用してきた歴史の理解に貢献すると期待できる。また、新規な含硫化合物の
設計や工業的な生産への酵素の応用も期待されることから、これらの研究成果は学術上・応用上
寄与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。

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