The 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety is an essential residue for the antithrombotic activities of aeruginosins, which are a class of cyanobacterial derived bioactive linear tetrapeptides. Biosynthetic pathway of Choi is still elusive. AerF was suggested to be involved in the biosynthesis of Choi, and can be assigned to the short-chain dehydrogenase/reductase (SDR) superfamily. However, both the exact role and the catalytic mechanism of AerF have not been elucidated. In this study, functional and mechanistic analyses of AerF from Microcystis aeruginosa were performed. Observation of enzymatic assay demonstrates that AerF is a NADPH-dependent alkenal double bond reductase that catalyzes the reduction of dihydro-4-hydroxyphenylpyruvate (H2HPP) to generate tetrahydro-4-hydroxyphenylpyruvate (H4HPP), which is the third step of the biosynthetic pathway from prephenate to Choi. Comparative structural analysis indicates that ligand binding-induced conformational change of AerF is different from that of the other SDR superfamily reductase using H2HPP as a substrate. Analyses of NADPH and substrate analogue binding sites combined with the results of mutagenesis analyses suggest that a particular serine residue mainly involves in the initiation of the proton transfer between the substrate and the residues of AerF, which is an uncommon feature in SDR superfamily reductase. Furthermore, based on the observations of structural and mutagenesis analyses, the catalytic mechanism of AerF is proposed and a proton transfer pathway in AerF is deduced.

中文翻译：

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AerF的结构和功能研究，AerF是一种NAEPH依赖的烯键双键还原酶，参与铜绿素酶Choi部分的生物合成。

2-羧基-6-羟基八氢吲哚（Choi）部分是铜绿素类抗血栓形成活性的必需残基，铜绿素类是一类由蓝细菌衍生的生物活性线性四肽。Choi的生物合成途径仍然难以捉摸。建议AerF参与Choi的生物合成，并且可以分配给短链脱氢酶/还原酶（SDR）超家族。但是，AerF的确切作用和催化机理都尚未阐明。在这项研究中，对铜绿微囊藻的AerF进行了功能和机理分析。酶促测定的观察结果表明，AerF是NADPH依赖性的烯键双键还原酶，可催化还原二氢-4-羟苯基丙酮酸（H2HPP）生成四氢-4-羟苯基丙酮酸（H4HPP）这是从苯酚到崔的生物合成途径的第三步。比较结构分析表明，以H2HPP为底物，配体结合诱导的AerF构象变化与其他SDR超家族还原酶不同。对NADPH和底物类似物结合位点的分析以及诱变分析的结果表明，特定的丝氨酸残基主要参与质子在底物和AerF残基之间的转移，这是SDR超家族还原酶的罕见特征。此外，基于对结构和诱变分析的观察，提出了AerF的催化机理并推导了AerF中的质子转移途径。比较结构分析表明，以H2HPP为底物，配体结合诱导的AerF构象变化与其他SDR超家族还原酶不同。对NADPH和底物类似物结合位点的分析以及诱变分析的结果表明，特定的丝氨酸残基主要参与质子在底物和AerF残基之间的转移，这是SDR超家族还原酶的罕见特征。此外，基于对结构和诱变分析的观察，提出了AerF的催化机理并推导了AerF中的质子转移途径。比较结构分析表明，以H2HPP为底物，配体结合诱导的AerF构象变化与其他SDR超家族还原酶不同。对NADPH和底物类似物结合位点的分析以及诱变分析的结果表明，特定的丝氨酸残基主要参与质子在底物和AerF残基之间的转移，这是SDR超家族还原酶的罕见特征。此外，基于对结构和诱变分析的观察，提出了AerF的催化机理并推导了AerF中的质子转移途径。对NADPH和底物类似物结合位点的分析以及诱变分析的结果表明，特定的丝氨酸残基主要参与质子在底物和AerF残基之间的转移，这是SDR超家族还原酶的罕见特征。此外，基于对结构和诱变分析的观察，提出了AerF的催化机理并推导了AerF中的质子转移途径。对NADPH和底物类似物结合位点的分析以及诱变分析的结果表明，特定的丝氨酸残基主要参与质子在底物和AerF残基之间的转移，这是SDR超家族还原酶的罕见特征。此外，基于对结构和诱变分析的观察，提出了AerF的催化机理并推导了AerF中的质子转移途径。