Kandzia Romy, Stumpe Michael, Berndt Ekkehardt, Szalata Marlena, Matsui Kenji, Feussner Ivo
Institute of Plant Biochemistry-Halle/Saale, Weinberg 3, D-06120 Halle/Saale, Germany.
J Plant Physiol. 2003 Jul;160(7):803-9. doi: 10.1078/0176-1617-01026.
Fatty acid hydroperoxide lyase (HPL) is a membrane associated P450 enzyme that cleaves fatty acid hydroperoxides into aldehydes and omega-oxo fatty acids. One of the major products of this reaction is (3Z)-hexenal. It is a constituent of many fresh smelling fruit aromas. For its biotechnological production and because of the lack of structural data on the HPL enzyme family, we investigated the mechanistic reasons for the substrate specificity of HPL by using various structural analogues of HPL substrates. To approach this 13-HPL from Arabidopsis thaliana was cloned and expressed in E. coli utilising a His-Tag expression vector. The fusion protein was purified by affinity chromatography from the E. coli membrane fractions and its pH optimum was detected to be pH 7.2. Then, HPL activity against the respective (9S)- and (13S)-hydroperoxides derived either from linoleic, alpha-linolenic or gamma-linolenic acid, respectively, as well as that against the corresponding methyl esters was analysed. Highest enzyme activity was observed with the (13S)-hydroperoxide of alpha-linolenic acid (13alpha-HPOT) followed by that with its methyl ester. Most interestingly, when the hydroperoxy isomers of gamma-linolenic acid were tested as substrates, 9gamma-HPOT and not 13gamma-HPOT was found to be a better substrate of the enzyme. Taken together from these studies on the substrate specificity it is concluded that At13HPL may not recognise the absolute position of the hydroperoxy group within the substrate, but shows highest activities against substrates with a (1Z4S,5E,7Z)-4-hydroperoxy-1,5,7-triene motif. Thus, At13HPL may not only be used for the production of C6-derived volatiles, but depending on the substrate may be further used for the production of Cg-derived volatiles as well.
脂肪酸氢过氧化物裂解酶（HPL）是一种与膜相关的P450酶，可将脂肪酸氢过氧化物裂解为醛类和ω-氧代脂肪酸。该反应的主要产物之一是（3Z）-己烯醛。它是许多清新果香的成分之一。鉴于其生物技术生产以及HPL酶家族缺乏结构数据，我们使用HPL底物的各种结构类似物研究了HPL底物特异性的机制原因。为实现这一目标，从拟南芥中克隆了13-HPL，并利用His-Tag表达载体在大肠杆菌中进行表达。通过亲和色谱从大肠杆菌膜部分纯化融合蛋白，检测其最适pH为7.2。然后，分析了HPL对分别源自亚油酸、α-亚麻酸或γ-亚麻酸的相应（9S）-和（13S）-氢过氧化物以及对相应甲酯的活性。观察到对α-亚麻酸的（13S）-氢过氧化物（13α-HPOT）的酶活性最高，其次是对其甲酯的活性。最有趣的是，当测试γ-亚麻酸的氢过氧异构体作为底物时，发现9γ-HPOT而非13γ-HPOT是该酶的更好底物。综合这些关于底物特异性的研究可以得出结论，At13HPL可能无法识别底物中氢过氧基的绝对位置，但对具有（1Z4S,5E,7Z）-4-氢过氧-1,5,7-三烯基序的底物表现出最高活性。因此，At13HPL不仅可用于生产C6衍生的挥发物，而且根据底物的不同，还可进一步用于生产Cg衍生的挥发物。
