Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics.
Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics.
复制标题
DOI：
10.1016/j.chembiol.2008.07.014
复制
发表时间：
2008-09-22
影响因子：
--
通讯作者：
Sherman DH
中科院分区：
文献类型：
--
作者：
Anzai Y;Li S;Chaulagain MR;Kinoshita K;Kato F;Montgomery J;Sherman DH
大环内酯类抗生素是一类有价值的抗感染剂，其包含大环内酯环、至少一个附加糖单元，并且在大多数情况下还包含羟基和/或环氧基团形式的附加官能化。有大量工作来了解此类化合物的聚酮衍生糖苷配基的组装，特别是通过模块化聚酮合酶的作用产生的红霉素和匹克霉素。此外，脱氧糖和氨基糖的组装模式以及它们向糖苷配基的转移在过去十年中已有报道。然而，关于最终定制反应的信息要少得多，这些反应通常由 P450 酶介导，这些酶能够进行区域和立体特异性氧化以添加羟基或环氧化物功能。在此，我们在体外表征了来自灰红小单孢菌霉素生物合成基因簇的两种 P450 酶。 MycCI 的克隆、过表达和纯化揭示了其对 C21 甲基羟基化的选择性。这种 P450 酶的天然底物是霉素 VIII，它是 PKS 后剪裁途径中最早的大环内酯形式，在 C5 OH 处附加了去糖胺。此外，我们发现 MycCI 的最佳活性取决于天然铁氧还蛋白 MycCII。第二次和第三次氧化反应分别包括羟基化和环氧化，由MycG以霉素IV作为初始底物介导。该反应需要预先对第二个脱氧糖残基进行二甲基化（例如，6-脱氧阿洛糖转化为霉素糖），以便通过双功能 MycG P450 进行有效转化，MycG P450 是第一个天然产物生物合成单加氧酶，其特征在于能够催化羟基化和环氧化步骤。
Macrolide antibiotics are a class of valuable anti-infective agents that include a macrolactone ring, at least one appended sugar unit, and in most cases, additional functionalization in the form of hydroxyl and/or epoxide groups. There is a significant body of work to understand assembly of the polyketide derived aglycone for this class of compounds, particularly for erythromycin and pikromycin that are generated through the action of a modular polyketide synthases. In addition, the mode of assembly of deoxysugars and aminosugars as well as their transfer to the aglycone has been reported over the past decade. However, much less information exists for the final tailoring reactions, typically mediated by P450 enzymes that are capable of regio- and stereospecific oxidations to add hydroxyl or epoxide functionality. Herein, we have characterized in vitro two P450 enzymes from the mycinamicin biosynthetic gene cluster of Micromonospora griseorubida. Cloning, overexpression and purification of MycCI revealed its selectivity for C21 methyl group hydroxylation. The natural substrate for this P450 enzyme is mycinamicin VIII, the earliest macrolide form in the post-PKS tailoring pathway appended with desosamine at C5 OH. Moreover, we found the optimal activity of MycCI is dependent on the native ferredoxin MycCII. The second and third oxidation reactions including hydroxylation and epoxidation respectively are mediated by MycG with mycinamicin IV as initial substrate. This reaction requires prior dimethylation of the second deoxysugar residue (e.g., 6-deoxyallose to mycinose) for effective conversion by the dual function MycG P450, the first natural product biosynthetic monooxygenase characterized with an ability to catalyze both hydroxylation and epoxidation steps.
登录
查看更多内容
影响因子：
2.1
作者：
Anzai, Y;Salto, N;Kato, F
通讯作者：
Kato, F
DOI：
10.1007/bf00302258
发表时间：
1994-11-15
期刊：
MOLECULAR & GENERAL GENETICS
影响因子：
--
作者：
INOUYE, M;TAKADA, Y;HORINOUCHI, S
通讯作者：
HORINOUCHI, S
影响因子：
4.8
作者：
Sherman, David H.;Li, Shengying;Podust, Larissa M.
通讯作者：
Podust, Larissa M.
DOI：
10.1016/s0006-291x(03)01231-2
发表时间：
2003-08-01
影响因子：
3.1
作者：
Lamb, DC;Ikeda, H;Kelly, SL
通讯作者：
Kelly, SL
影响因子：
3.2
作者：
ANDERSEN, JF;HUTCHINSON, CR
通讯作者：
HUTCHINSON, CR