Studies on New Enzyme System for the Isoprenoid Biosynthesis in Bacteria

裂解乳杆菌细胞，细胞提取液在DEAE-TOYOPEL 650S离子交换柱上进行层析。观察到从法尼基二磷酸进入非极性产物的两个酶活性峰。纯化后的酶命名为酶I和酶11。在NADPH存在下，酶I与法尼基二磷酸反应生成的非极性产物为角鲨烯。法尼基二磷酸和NADPH的角鲨烯合成酶的Km值分别为0.4um和10um。相反，在NADPH存在下，[14&gt；C]法尼基二磷酸生成的放射性非极性产物与脱氢角鲨烯重合，而与角鲨烯不重合。从完整的分析混合物中去除NADPH不会影响非极性产物的放射性，主要产物是脱氢角鲨烯。酶11催化脱氢角鲨烯的双键构型为12-顺式脱氢角鲨烯。脱氢角鲨烯合成酶的Km值为0.04微米，表明酶I为角鲨烯合成酶，酶11为脱氢角鲨烯合成酶。这两种酶似乎都能催化2摩尔的法尼基二磷酸缩合，生成中间产物普奎烯二磷酸。脱氢角鲨烯合成酶是一种新的酶，角鲨烯合成酶是第一个从细菌中获得并部分纯化的酶。

The cells of Fravobacte rium okeanokoites were disrupted and the cell extracts were subjected to a chromatography on DEAE-Toyopearl 650S ion exchange column. Two peaks of the enzyme activity for ^<14>C-incorporation from[ ^<14>C]farnesyl diphosphate into non-polar products were observed. The purified enzymes obtained after rechromatography were designated Enzyme I and 11. The non-polar product formed in the reaction of Enzyme I with[ ^<14>C]farnesyl diphosphate in the presence of NADPH was squalene. Km values of the squalene synthesizing enzyme for farnesyl diphosphate and NADPH were 0.4 muM and 10 muM, respectively.In contrast, the radioactive non-polar products formed from[ ^<14>C]farnesyl diphosphate in the presence of NADPH by the catalysis of Enzyme II coincided with dehydrosqualene but not with squalene. Removal of NADPH from tlie complete assay mixture did not affect the radioactivity of the non-polar products and the main product was dehydrosqualene. The geometries of double bonds of dehydrosqualene obtained in the reaction catalyze by Enzyme 11 was 12-cis-dehydrosqualene. The Km value of the dehydrosqualene synthesizing enzyme for farnesyl diphosphate was 0.04 muM.These results indicate that Enzyme I is squalene synthase and Enzyme 11 is dehydrosqualene synthase. Both enzymes seem to catalyze the condensation of 2 moles of farnesyl diphosphate to yield presqualene diphosphate as an intermediate. Then from presqualene diphosphate the former yields squalene in the presence of NADPH and the latter produces dehydrosqualene in the presence and absence of NADPH.Dehydrosqualene synthase is a new enzyme and squalene synthase is the first enzyme obtained and partially purified from bacteria.