Welford Richard W D, Clifton Ian J, Turnbull Jonathan J, Wilson Stuart C, Schofield Christopher J
Chemical Research Laboratory, Department of Chemistry and Oxford Centre for Molecular Sciences, Mansfield Road, Oxford OX1 3TA, UK.
Org Biomol Chem. 2005 Sep 7;3(17):3117-26. doi: 10.1039/b507153d. Epub 2005 Aug 1.
During the biosynthesis of the tricyclic flavonoid natural products in plants, oxidative modifications to the central C-ring are catalysed by Fe(ii) and 2-oxoglutarate dependent oxygenases. The reactions catalysed by three of these enzymes; flavone synthase I, flavonol synthase and anthocyanidin synthase (ANS), are formally desaturations. In comparison, flavanone 3beta-hydroxylase catalyses hydroxylation at the C-3 pro-R position of 2S-naringenin. Incubation of ANS with the unnatural substrate (+/-)-naringenin results in predominantly C-3 hydroxylation to give cis-dihydrokaempferol as the major product; trans-dihydrokaempferol and the desaturation product, apigenin are also observed. Labelling studies have demonstrated that some of the formal desaturation reactions catalysed by ANS proceed via initial C-3 hydroxylation followed by dehydration at the active site. We describe analyses of the reaction of ANS with 2S- and 2R-naringenin substrates, including the anaerobic crystal structure of an ANS-Fe-2-oxoglutarate-naringenin complex. Together the results reveal that for the 'natural' C-2 stereochemistry of 2S-naringenin, C-3 hydroxylation predominates (>9 : 1) over desaturation, probably due to the inaccessibility of the C-2 hydrogen to the iron centre. For the 2R-naringenin substrate, desaturation is significantly increased relative to C-3 hydroxylation (ca. 1 : 1); this is probably a result of both the C-3 pro-S and C-2 hydrogen atoms being accessible to the reactive oxidising intermediate in this substrate. In contrast to the hydroxylation-elimination desaturation mechanism for some ANS substrates, the results imply that the ANS catalysed desaturation of 2R-naringenin to form apigenin proceeds with a syn-arrangement of eliminated hydrogen atoms and not via an oxygenated (gem-diol) flavonoid intermediate. Thus, by utilising flavonoid substrates with different C-2 stereochemistries, the balance between C-3 hydroxylation or C-2, C-3 desaturation mechanisms can be altered.
在植物中三环类黄酮天然产物的生物合成过程中，中心C环的氧化修饰由铁（II）和2-氧代戊二酸依赖性加氧酶催化。其中三种酶，即黄酮合酶I、黄酮醇合酶和花青素合酶（ANS）催化的反应形式上是去饱和反应。相比之下，黄烷酮3β-羟化酶催化2S-柚皮素C-3 pro-R位的羟基化反应。将ANS与非天然底物（±）-柚皮素一起温育，主要导致C-3羟基化，生成顺式二氢山奈酚作为主要产物；还观察到反式二氢山奈酚和去饱和产物芹菜素。标记研究表明，ANS催化的一些形式上的去饱和反应是通过最初的C-3羟基化，然后在活性位点脱水进行的。我们描述了对ANS与2S-和2R-柚皮素底物反应的分析，包括ANS-铁-2-氧代戊二酸-柚皮素复合物的厌氧晶体结构。结果共同表明，对于2S-柚皮素的“天然”C-2立体化学，C-3羟基化比去饱和占主导（>9:1），这可能是由于C-2氢原子无法接近铁中心。对于2R-柚皮素底物，去饱和相对于C-3羟基化显著增加（约1:1）；这可能是由于该底物中的C-3 pro-S和C-2氢原子都可被反应性氧化中间体接近。与一些ANS底物的羟基化-消除去饱和机制不同，结果表明ANS催化2R-柚皮素去饱和形成芹菜素的过程中，消除的氢原子以顺式排列进行，而不是通过氧化（偕二醇）类黄酮中间体。因此，通过使用具有不同C-2立体化学的类黄酮底物，可以改变C-3羟基化或C-2、C-3去饱和机制之间的平衡。
