- 著者
- 田口 貴章 小澤 誠 Kimberley Meriel R. Booker-Milburn Kevin I. Stephenson G. Richard 海老塚 豊 市瀬 浩志
- 出版者
- 天然有機化合物討論会
- 雑誌
- 天然有機化合物討論会講演要旨集
- 巻号頁・発行日
- no.44, pp.235-240, 2002-09-01
A class of Streptomyces aromatic polyketide antibiotics, the benzoisochromanequinone (BIQs) antibiotics all show trans stereochemistry at C-3 and C-15 in the pyran ring. The opposite stereochemical control is found in actinorhodin (3S, 15R, ACT) from S. coelicolor A3(2) and dihydrogranaticin (3R, 15S, DHGRA) from S. violaceoruber Tu22. A common bicyclic intermediate, which is produced by the early biosynthetic genes encoding a type II minimal polyketide synthase, C-9 ketoreductase (KR), aromatase, and cyclase, was postulated to undergo stereospecific reduction to provide either (S)-DNPA or (R)-DNPA. In the ACT biosynthesis, RED1 encoded by act VI-ORF 1 was proved to reduce C-3 of bicyclic intermediate to determine the 3-(S)-configuration of DNPA. Although the homolog of act VI-ORF 1 was not found in the gra cluster, RED2 was suggested to reduce bicyclic intermediate. An explored RED-2 coding gene, gra-6, was subjected to updated BLAST analysis. The gra-6 product, a putative short-chain alcohol dehydrogenase, has virtually no sequence similarity with RED1. Functional analysis of RED1/2 was made from the following points. 1) Introduction of gra-ORF 6 and gra-ORF 5 under translational coupling (gra-5+6) into the act VI-ORF 1 mutant, S. coelicolor B22, led to ACT-like pigmentation, demonstrating gra-ORF 6 to complement the function of act VI-ORF 1 possibly under unnatural stereochemical control. 2) Combinations of the ketoreductase genes were co-expressed with the early biosynthetic genes required for the bicyclic intermediate formation. gra-ORF6 was essential to produce (R)-DNPA in DHGRA biosynthesis. gra-5+6 led to the most efficient production of (R)-DNPA, implying a possible unique cooperative function as RED2. 3) A series of synthetic analogues was applied to the biotransformations based on ketosynthase-deficient recombinants of S. coelicolor carrying either RED1 or RED2. In all cases for RED1, the β-keto ester substrates were reduced with good to excellent enantioselectivity. However, the simpler substrates were not accepted by RED2, indicating the significant difference in substrate specificity between the two reductases. 4) 3D structures of RED1 and RED2 were predicted based on homology modeling (FAMS) using the templates, L-3-hydroxyacyl-CoA dehydrogenase from human heart (for RED1) and tropinone reductase II (for RED2). Catalytically key amino acid residues were revealed for the both enzymes. 5) RED1 and RED2 were overexpressed in E. coli, and in vitro assay system were successfully established. Optimization of the system, purification of both enzymes and site directed mutagenesis to the suggested key residues are in progress.
本研究は、多様な生理活性物質の生産者として放線菌に注目し、代表的ポリケタイド二次代謝産物アクノロジン(ACT)を取り上げ、その生合成に必要な20段階の酵素反応を試験管内で完全再構成することを基盤とする。諸反応中には、単機能型縮合酵素による反復的炭素鎖構築反応、酵素反応中間体の支持タンパク質からの遊離機構および基本骨格修飾反応における酵素基質認識、単機能型酵素による多段階反応の制等が含まれる。本研究では、ACT生合成再構成系を材料として、酵素学的解析、各種機器分析を用いた酵素反応の精密解析、タンパクモデリング法を含む情報科学的解析を駆使した上記問題の網羅的解明を目的とする。本年度の研究計画として、ACT基本骨格形成反応(反復的炭素鎖構築反応)の再構成系の構築を設定し、以下の成果を得た。ACTの基本骨格形成に必須のタンパク群、すなわちActtI-ORF1(ケト縮合酵素α)、ActII-ORF2(ケト縮合酵素β)、ActI-ORF3(アシルキャリアタンパク)、ActIII(ケト還元酵素)、sfp(フォスホパンテニルトランスフェラーゼ)、fabD(アシルトランスフェラーゼ)の発現を放線菌および大腸菌の宿主を用いて実施し、組換精製タンパクとして調製した。これらのタンパクを用い、マロニルCoAを基質としたin Vitroでの生合成反応の再構築を試みたところ、7段階のケト縮合反応を経て生成するオクタケタイド鎖の閉環生成物であるSEK4/SEK4bとともにオクタケタイド鎖の炭素9位還元後の閉鎖生成物であるmutactinの生成がHPLCおよびLC/MSで確認された。この結果は、ACT生合成に関わる炭素骨格形成反応に関わる単機能縮合酵素による反復的炭素鎖構築反応を再現したものであり、本年度の研究計画の一部を達成することができた。