Dennis M, Kolattukudy P E
Ohio State Biotechnology Center, Ohio State University, Columbus 43210.
Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5306-10. doi: 10.1073/pnas.89.12.5306.
The final step in hydrocarbon biosynthesis involves loss of CO from a fatty aldehyde. This decarbonylation is catalyzed by microsomes from Botyrococcus braunii. Among the several detergents tested for solubilizing the decarbonylase, octyl beta-glucoside (0.1%) was found to be the most effective and released 65% of the enzyme activity in soluble form. FPLC of the solubilized enzyme preparation with Superose 6 followed by ion-exchange FPLC with Mono Q resulted in 200-fold increase in specific activity with 7% recovery. The purified enzyme released nearly 1 mol of CO for each mol of hydrocarbon. SDS/PAGE of the enzyme preparation showed two protein bands of equal intensity at 66 and 55 kDa. The absorption spectrum of the enzyme with bands at 410 nm, 425 nm, 580 nm, and 620 nm suggests the presence of a porphyrin. Electron microprobe analysis revealed that the enzyme contained Co. Purification of the decarbonylase from B. braunii grown in 57CoCl2 showed that 57Co coeluted with the decarbonylase. These results suggest that the enzyme contains Co that might be part of a Co-porphyrin, although a corrin structure cannot be ruled out. Co-protoporphyrin IX itself caused decarbonylation of octadecanal at 60 degrees C, whereas the metal ion or protoporphyrin alone, or several other metal porphyrins, did not cause decarbonylation. These results strongly suggest that biosynthesis of hydrocarbons is effected by a microsomal Co-porphyrin-containing enzyme that catalyzes decarbonylation of aldehydes and, thus, reveal a biological function for Co in plants.
碳氢化合物生物合成的最后一步涉及从脂肪醛中去除一氧化碳。这种脱羰反应由布朗葡萄藻的微粒体催化。在测试的几种用于溶解脱羰酶的去污剂中，发现β-辛基葡萄糖苷（0.1%）最有效，可释放出65%的可溶性酶活性。用Superose 6对溶解的酶制剂进行快速蛋白质液相色谱（FPLC），随后用Mono Q进行离子交换FPLC，比活性提高了200倍，回收率为7%。纯化后的酶每摩尔碳氢化合物释放出近1摩尔一氧化碳。该酶制剂的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）显示在66 kDa和55 kDa处有两条强度相等的蛋白条带。该酶在410 nm、425 nm、580 nm和620 nm处有吸收峰的吸收光谱表明存在卟啉。电子微探针分析显示该酶含有钴。从在57CoCl2中生长的布朗葡萄藻中纯化脱羰酶表明，57Co与脱羰酶共洗脱。这些结果表明，该酶含有钴，可能是钴-卟啉的一部分，尽管不能排除钴胺素结构。在60℃下，钴原卟啉IX本身可导致十八醛脱羰，而单独的金属离子或原卟啉，或其他几种金属卟啉均不能导致脱羰。这些结果有力地表明，碳氢化合物的生物合成是由一种含微粒体钴-卟啉的酶催化醛脱羰来实现的，从而揭示了钴在植物中的生物学功能。
