Tijet N, Schneider C, Muller B L, Brash A R
Department of Pharmacology, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA.
Arch Biochem Biophys. 2001 Feb 15;386(2):281-9. doi: 10.1006/abbi.2000.2218.
A novel member of the plant cytochrome P450 CYP74 family of fatty acid hydroperoxide metabolizing enzymes has been cloned from melon fruit (Cucumis melo). The cDNA is comprised of 1,446 nucleotides encoding a protein of 481 amino acids. The homology at the amino acid level to other members of the CYP74 family is 35-50%, the closest relatives being allene oxide synthases. The cDNA was expressed in Escherichia coli, and the corresponding protein was purified by affinity column chromatography. The native enzyme showed a main Soret band at 418 nm, indicative of a low spin ferric cytochrome P450, and a 447-nm peak appeared in the CO-difference spectrum. Using [U-14C]radiolabeled substrate, HPLC, UV, and GC-MS, the products of conversion of 9S-hydroperoxy-linoleic acid were identified as 9-oxo-nonanic acid and 3Z-nonenal. Kinetic analysis of this hydroperoxide lyase showed the highest rate of reaction with 9-hydroperoxy-linolenic acid followed by 9-hydroperoxy-linoleic acid and then the corresponding 13-hydroperoxides. Overall, the newly characterized cytochrome P450 enzyme is a fatty acid hydroperoxide lyase with a preference, but not absolute specificity for the 9-positional hydroperoxides of linoleic and linolenic acids.
一种植物细胞色素P450 CYP74家族中新型的脂肪酸氢过氧化物代谢酶已从甜瓜果实（黄瓜）中克隆出来。该cDNA由1446个核苷酸组成，编码一个481个氨基酸的蛋白质。其氨基酸水平与CYP74家族其他成员的同源性为35%-50%，最接近的亲属是丙二烯氧化物合酶。该cDNA在大肠杆菌中表达，相应的蛋白质通过亲和柱层析法纯化。天然酶在418nm处显示出一个主要的Soret带，表明是一种低自旋铁细胞色素P450，并且在CO差光谱中出现了一个447nm的峰。使用[U-14C]放射性标记底物、高效液相色谱、紫外光谱和气相色谱-质谱联用技术，确定了9S-氢过氧化亚油酸转化产物为9-氧代壬酸和3Z-壬烯醛。对这种氢过氧化物裂解酶的动力学分析表明，其与9-氢过氧化亚麻酸反应的速率最高，其次是9-氢过氧化亚油酸，然后是相应的13-氢过氧化物。总体而言，新鉴定的细胞色素P450酶是一种脂肪酸氢过氧化物裂解酶，对亚油酸和亚麻酸的9-位氢过氧化物具有偏好性，但并非绝对特异性。
