Development of dehalogenases by molecular evolution technology : Application of production of useful materials and bioremediation of environments

通过分子进化技术开发脱卤酶：有用材料生产和环境生物修复的应用

• 批准号: 11558084
• 负责人: TATSUO Kurihara
• 金额: $ 6.98万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11558084/
• 关键词: organohalogen compounds; L-2-haloacid dehalogenase; DL-2-haloacid dehalogenase; fluoroacetate dehalogenase; Burkholderia属細菌; 立体構造モデリング; 炭素-フッ素結合; ハロ酸デハロゲナーゼ; Methylobacterium属細菌; クロロプロピオン酸

The aim of this project is to develop dehalogenases that can be used for bioremediation of environments and production of useful compounds. The research results are as follows.1. We isolated a dehalogenase-producing bacterium, Methylobacterium sp.CPA1, from Lake Sanaru, Hamamatsu, Shizuoka using DL-2-chloropropionate as the sole carbon source for screening. We purified DL-2-haloacid dehalogenase from the cell extract, and cloned and sequenced its gene. The enzyme acted on both D- and L-2-chloropropionate to catalyze the release of the halide ion. Chloroacetate and bromoacetate also served as the substrates, but fluoroacetate was not the substrate. The enzyme also acted on 2-chloropropionamide.2. We isolated a soil bacterium, Burkholderia sp.FA1, producing fluoroacetate dehalogenase, which catalyzes hydrolytic defluorination of fluoroacetate. The enzyme acted much better on fluoroacetate than on chloroacetate and bromoacetate. The gene coding for the enzyme was isolated, and the primary structure of the enzyme was determined.3. We analyzed the reaction mechanism of DL-2-haloacid dehalogenase, and revealed that a water molecule directly attacks the α-carbon atom of the substrate to produce the corresponding 2-hydroxyalkanoic acid. This mechanism is different from that of other dehalogenases : in the reactions of other dehalogenases, an aspartate residue of the enzyme attacks the α-carbon atom of the substrate to produce an ester intermediate, and this intermediate is subsequently hydrolyzed by a water molecule.4. We analyzed the reaction mechanism of L-2-haloacid dehalogenase D10N mutant enzyme, and found that Asn 10 functions as a catalytic residue and a β-cyanoalanine residue is produced as an intermediate structure.

该项目的目的是开发可用于生物修复环境和产生有用化合物的脱核酶。研究结果如下1。我们使用DL-2-氯丙酸酯作为筛查的唯一碳源，从Sanaru湖，Hamamatsu，shimaoka分离了一种脱核酶的细菌，Sp.Cpa1，shimaoka。我们从细胞提取物中纯化了DL-2-半酸脱脂酶，并克隆并测序了其基因。该酶作用于D-和L-2-氯丙酸酯，可催化卤化物离子的释放。氯乙酸和溴乙酸也是底物，但氟乙酸不是底物。该酶还作用于2-氯丙二硫酰胺2。我们分离了一种土壤细菌Burkholderia sp.fa1，产生氟乙酸脱核酶，从而催化氟乙酸盐的水解偏氟化。该酶在氟乙酸酯上的作用要比在氯乙酸和溴乙酸氯乙酸酯上好得多。分离为酶的编码基因，并确定酶的主要结构。3。我们分析了DL-2-甲醛酸脱核酶的反应机理，并揭示了水分子直接攻击底物的α-碳原子以产生相应的2-羟基羟基烷酸。该机制与其他去核酶不同：在其他去核酶的反应中，酶的天冬氨酸保留攻击了底物的α-碳原子以产生酯中间体，并且该中间体随后被水分子水解。我们分析了L-2-卤代酸脱核酶D10N突变酶的反应机制，发现ASN 10作为催化局部功能和β-氰丙氨酸保留率作为中间结构产生。

项目成果

Susumu Ichiyama et al.: "Novel catalytic mechanism of nucleophilic substitution by asparagine residue in volving cyanoalanine intermediate revealed by mass spectrometric monitoring of an enzyme reaction"The Journal of Biological Chemistry. 275(52). 40804-

Susumu Ichiyama 等人：“通过酶反应的质谱监测揭示了涉及氰丙氨酸中间体的天冬酰胺残基亲核取代的新催化机制”生物化学杂志。

Tatsuo Kurihara et al.: "Bacterial 2-haloacid dehalogenases : structures and reaction mechanisms"J.Mol.Catal.. 10. 57-65 (2000)

Tatsuo Kurihara 等：“细菌 2-卤酸脱卤酶：结构和反应机制”J.Mol.Catal.. 10. 57-65 (2000)

Vincenzo Nardi-Dei et al.: "DL-2-Haloacid dehalogenase from Pseudomonas sp.113 is a new class of dehalogenase catalyzing hydrolytic dehalogenation not involving enzynte-substrate ester intermediate"J.Biol.Chem.. 274. 20977-20981 (1999)

Vincenzo Nardi-Dei 等人：“来自假单胞菌 sp.113 的 DL-2-卤酸脱卤酶是一类新的脱卤酶，催化水解脱卤，不涉及酶-底物酯中间体”J.Biol.Chem.. 274. 20977-20981（

Vincenzo Nardi-Dei et al.: "DL-2-Haloacid dehalogenase from Pseudomonas sp.113 is a new class of dehalogenase catalyzing hydrolytic dehalogenation not involving enzyme-substrate ester intermediate"The Journal of Biological Chemistry. 274(30). 20977-20981

Vincenzo Nardi-Dei 等人：“来自假单胞菌 sp.113 的 DL-2-卤酸脱卤酶是一类新型脱卤酶，催化水解脱卤，不涉及酶-底物酯中间体”《生物化学杂志》。

Tatsuo Kurihara et al.: "Bacterial 2-haloacid dehalogenases : structures and reaction mechanisms"Journal of Molecular Catalysis B : Enzymatic. 10. 57-65 (2000)

Tatsuo Kurihara 等人：“细菌 2-卤酸脱卤酶：结构和反应机制”分子催化杂志 B：酶学。