Bioorganic Chemical Studies on the Formation Mechanisms of Bioactic C-P Compounds

生物活性C-P化合物形成机制的生物有机化学研究

• 批准号: 61470133
• 负责人: SETO Haruo
• 金额: $ 3.2万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-61470133/
• 关键词: 2-phosphinomethylmalic acid; monofluoro acetic acid; PMM synthase; C-P compounds; bialaphos; ビアラホス; PMMシンターゼ

This reseach has been made in order to reveal the formation mechanism of the C-P bond found in some microbial metabolites. As a target of this research we have selected a herbicide named bialaphos which contains a unique C-P-C bond. This metabolite is produced by Streptomyces hygroscopicus SF1293.At first, the transformation mechanism of phosphimopyruvic acid (an analog of oxalacetic acid) to demethylphosphinothricin (an analog of glutamic acid) resulting in the increase of one carbor unit of the skeleton was studied by using enzyme inhibitors of TCA cycle. Addition of monofluoro acetic acid caused the accumulation of a new phosphinic acid named 2-phosphinomethylmalic acid (PMM) which was isolated by ion exchange resin cellulose chromatograpgy and gel filtration. Its structure was determined to be an analog of citric acid by ^+H- and ^<13>C-NMR spectral analysis. The absolute stereochemistry of PMM was established to be (R) by comparing with the (S)-form isomer prepared by the use of porcine heart citrate synthase.The enzyme catalyzing this reaction was named PMM synthase and was purified to a pure protein by several chromatographic procedures. It is a homodimer consisting of 48K monomers. The N-terminal amino acid sequence up to 28 amino acids was determined. The corresponding open reading frame was found in the already cloned DNA which codes for the biosynthetic genes of bialaphos. The properties of this enzyme clearly shows that PMM synthase is very closely related to (R)-citrate synthase obtained from some anaerobic bacteria.PMM was converted to demethylphosphinothricin (<alpha>-ketoglutaric acid analog) by aconitase and isocitrate dehydrogenase; this finding suggests strongly that TCA cycle enzymes are involved in the biosynthesis of bialaphos. Thus, the detailed mechanism of bialaphos operating in the later stage has been revealed by this research work.

本研究旨在揭示某些微生物代谢产物中C-P键的形成机理。作为本研究的目标，我们选择了一种名为双丙氨磷的除草剂，它含有一个独特的C-P-C键。该代谢产物是由吸水链霉菌SF 1293产生的。首先，利用TCA循环的酶抑制剂，研究了磷酸三乙酸（Phosphimovic acid，一种类似于谷氨酸的物质）转化为去甲基膦丝菌素（Demethylphosphinothricin，一种类似于谷氨酸的物质）导致骨架增加一个碳单元的机理。加入一氟乙酸后，经离子交换树脂纤维素层析和凝胶过滤分离得到一种新的次膦酸，命名为2-膦甲基苹果酸（PMM）。经~ 1H ~+和~ 1C-NMR谱分析，确定其结构为柠檬酸的类似物<13>。通过与猪心柠檬酸合成酶的（S）-异构体的比较，确定PMM的绝对立体化学为（R），催化该反应的酶命名为PMM合成酶，并通过几种色谱方法纯化得到纯蛋白。它是由48 K单体组成的同源二聚体。测定了N-末端氨基酸序列（最多28个氨基酸）。在已克隆的编码双丙氨磷生物合成基因的DNA中发现了相应的开放阅读框架。PMM合成酶的性质表明，PMM合成酶与某些厌氧细菌的（R）-柠檬酸合成酶关系密切，PMM可<alpha>被乌头酸酶和异柠檬酸脱氢酶转化为去甲基膦丝菌素（-酮基谷氨酸类似物），这一发现有力地表明TCA循环酶参与了双丙氨磷的生物合成。因此，本研究揭示了双丙氨磷在后期的具体作用机理。

项目成果

K,Shimotohno: "Studies on the biosynthesis of bialaphos (SF-1293). 10. Comparison of 2-phosphinomethylmalic acid synthase and citrate synthase of Streptomyces hygroscopicus SF-1293,a bialaphos producing organism." Agric. Biol. Chem.

K,Shimotohno：“双丙氨磷 (SF-1293) 生物合成的研究。10. 吸水链霉菌 SF-1293（一种双丙氨磷生产生物体）的 2-膦甲基苹果酸合酶和柠檬酸合酶的比较。”

Kumiko Shimotohno: THE JOURNAL OF ANTIBIOTICS. 40. (1987)

下野久美子：抗生素杂志。

K. Shimotohno: Argic. Biol. Chem.

K. Shimotohno：银色。

H,Seto: "Studies on the biosynthesis of bialaphos (SF-1293). Production of 2-phosphinomethylmalic acid, an analogue of citric acid by Streptomyces hygroscopicus SF-1293 and its involvement in the biosynthesis of bialaphos." Agric. Biol. Chem.

H，Seto：“双丙氨膦 (SF-1293) 生物合成的研究。吸水链霉菌 SF-1293 生产柠檬酸类似物 2-膦基甲基苹果酸，及其参与双丙氨膦生物合成。”

K. Shimotohno: J. Antibiotics. 39. 1356-1359 (1986)

K. Shimotohno：J. 抗生素。