Hornung E, Walther M, Kühn H, Feussner I
Institute of Plant Biochemistry, D-06120 Halle, Germany.
Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):4192-7. doi: 10.1073/pnas.96.7.4192.
Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.
黄瓜脂质体脂氧合酶的多序列比对以及该酶与底物相互作用的结构建模表明，组氨酸608是位置特异性的主要决定因素。用空间占位较小的缬氨酸取代该氨基酸，改变了这种亚油酸13 -脂氧合酶的位置特异性，有利于9 -脂氧合作用。这些改变可能是由于H608V突变可能使R758带正电荷的胍基暴露，这反过来可能迫使脂肪酸底物呈现相反的头对尾方向。R758L + H608V双突变体的反应速率大幅降低，且具有随机的位置特异性。缺乏游离羧基的三亚油酸甘油酯被野生型酶和亚油酸9 -脂氧合的H608V突变体氧化为相应的(13S)-氢(过)氧衍生物。这些数据表明，通过单点突变可将亚油酸13 -脂氧合酶完全转化为9 -脂氧合酶。据推测，H608V交换可能改变底物在活性位点的方向和/或其空间构型，使得在C - 9处的立体特异性双加氧插入可能单独发生。
