Substitution of a Single Amino Acid Reverses the Regiospecificity of the Baeyer-Villiger Monooxygenase PntE in the Biosynthesis of the Antibiotic Pentalenolactone.
Substitution of a Single Amino Acid Reverses the Regiospecificity of the Baeyer-Villiger Monooxygenase PntE in the Biosynthesis of the Antibiotic Pentalenolactone.
复制标题
单一氨基酸的取代可逆转抗生素五烯内酯生物合成中拜尔-维利格单加氧酶 PntE 的区域特异性。
DOI：
10.1021/acs.biochem.6b01040
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发表时间：
2016-12-06
期刊：
影响因子：
2.9
通讯作者：
Zhu, Dongqing
中科院分区：
文献类型：
--
作者：
Chen, Ke;Wu, Shiwen;Zhu, Lu;Zhang, Chengde;Xiang, Wensheng;Deng, Zixin;Ikeda, Haruo;Cane, David E.;Zhu, Dongqing
在戊烯内酯（1）的生物合成中，PenE和PntE是来源于脱落链霉菌和S. arenae分别催化1-脱氧-11-氧代戊二烯酸（4）的黄素依赖性Baeyer-Villiger氧化为内酯戊二烯内酯D（5），其中较少取代的亚甲基碳已经迁移。相比之下，来自S.阿维菌素催化4氧化为新戊烯内酯D（6），其中更多取代的甲烷取代物发生迁移。我们报告的设计和分析的13个单一和多个突变体的PntE突变体，以确定关键的氨基酸，有助于这两个类别的Baeyer-Villiger单加氧酶的区域特异性。PntE中的L185 S突变逆转了观察到的PntE的区域特异性，使得所有携带该L185 S突变的重组PntE突变体获得PtlE的特征性区域特异性，催化4转化为6作为主要产物。携带R484 L的重组PntE突变体表现出降低的区域特异性，产生含有超过17%的6的内酯的混合物。这些体外结果通过对S.阿伐他汀ΔptlED双缺失突变体与pntE突变体的混合物中，含有L185 S的pntE突变体产生6作为主要产物，而ΔptlED缺失突变体与含有R484 L突变的pntE突变体的互补产生6作为总内酯产物混合物的33%以上。
In the biosynthesis of pentalenolactone (1), PenE and PntE, orthologous proteins from Streptomyces exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer-Villiger oxidation of 1-deoxy-11-oxopentalenic acid (4) to the lactone pentalenolactone D (5), in which the less-substituted methylene carbon has migrated. By contrast, the paralogous PtlE enzyme from S. avermitilis catalyzes the oxidation of 4 to neopentalenolactone D (6), in which the more substituted methane substitution has undergone migration. We report the design and analysis of 13 single and multiple mutants of PntE mutants to identify the key amino acids that contribute to the regiospecificity of these two classes of Baeyer-Villiger monooxygenases. The L185S mutation in PntE reversed the observed regiospecificity of PntE such that all recombinant PntE mutants harboring this L185S mutation acquired the characteristic regiospecificity of PtlE, catalyzing the conversion of 4 to 6 as the major product. The recombinant PntE mutant harboring R484L exhibited reduced regiospecificity, generating a mixture of lactones containing more than 17% of 6. These in vitro results were corroborated by analysis of the complementation of the S. avermitilis ΔptlED double deletion mutant with pntE mutants, such that pntE mutants harboring L185S produced 6 as the major product, whereas complemention of the ΔptlED deletion mutant with pntE mutants carrying the R484L mutation gave 6 as more than 33% of the total lactone product mixture.
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DOI：
10.1073/pnas.0404538101
发表时间：
2004-09-07
影响因子：
11.1
作者：
Malito, E;Alfieri, A;Mattevi, A
通讯作者：
Mattevi, A
影响因子：
3.6
作者：
Kayser, Margaret M.;Clouthier, Christopher M.
通讯作者：
Clouthier, Christopher M.
影响因子：
4.4
作者：
Iwaki, H;Wang, SZ;Lau, PCK
通讯作者：
Lau, PCK
影响因子：
2.9
作者：
Jiang, Jiaoyang;Tetzlaff, Charles N.;Takamatsu, Satoshi;Iwatsuki, Masato;Komatsu, Mamoru;Ikeda, Haruo;Cane, David E.
通讯作者：
Cane, David E.
影响因子：
4.4
作者：
Kamerbeek, NM;Olsthoorn, AJJ;Janssen, DB
通讯作者：
Janssen, DB