Pseudomonad cyclopentadecanone monooxygenase displaying an uncommon spectrum of Baeyer-Villiger oxidations of cyclic ketones
Pseudomonad cyclopentadecanone monooxygenase displaying an uncommon spectrum of Baeyer-Villiger oxidations of cyclic ketones
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DOI：
10.1128/aem.72.4.2707-2720.2006
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发表时间：
2006-04-01
影响因子：
4.4
通讯作者：
Lau, PCK
中科院分区：
文献类型：
--
作者：
Iwaki, H;Wang, SZ;Lau, PCK
Baeyer-Villiger单加氧酶（BVMO）是一类具有高化学选择性、区域选择性和对映选择性的生物催化剂，在由酮合成内酯或酯的反应中具有重要的前景。在这项研究中，我们已经克隆，测序，并在大肠杆菌中过表达一个新的BVMO，环十五烷酮单加氧酶（CpdB或CPDMO），最初来自假单胞菌菌株HI-70。CpdB的601个残基一级结构显示与已知BVMO的序列同一性仅为29%至50%。一个新的序列基序，其特征在于一簇带电残基，被确定在一个子集的BVMO序列，包含一个类似的N-末端延伸60至147个氨基酸。将64-kDa CPDMO酶纯化至表观均一性，提供3.94 μ mol/min/mg蛋白质的比活性和20%的产率。CPDMO是单体和NADPH依赖性的，并且每摩尔蛋白质含有类似于1摩尔黄素腺嘌呤二核苷酸。一个缺失突变体表明N-末端54个氨基酸对CPDMO活性的重要性。此外，与野生型酶相比，Rossmann折叠基序中的Ser 261 Ala取代导致酶对NADPH的稳定性改善和亲和力增加（K-m = 8 μ M对K-m = 24 μ M）。底物分析表明，CPDMO在已知的BVMO中是不寻常的，能够容纳和氧化大环和小环底物，包括C-11至C-15酮，甲基取代的C-5和C-6酮，以及双环酮，如十氢萘酮和β-四氢萘酮。CPDMO对环十五烷酮具有最高的亲和力（K-m = 5.8 μ M）和最高的催化效率（k（cat）/K-m比为7.2 × 10（5）M-1 s（-1）），因此命名为Cpd。进行了大量的全细胞生物转化，结果发现，CPDMO具有优异的对映体选择性（E > 200），以及对2-甲基环己酮的99%的S-选择性，用于生产7-甲基-2-氧杂环庚酮，一种潜在的有价值的手性结构单元。虽然显示出适度的选择性（E = 5.8），但也证明了使用CPDMO从2-甲基环十五烷酮的动力学拆分形成15-十六烷醇的大环内酯。
Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of similar to 60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 mu mol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains similar to 1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (K-m = 8 mu M versus K-m = 24 mu M). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C-11 to C-15 ketones, methyl-substituted C-5 and C-6 ketones, and bicyclic ketones, such as decalone and beta-tetralone. CPDMO has the highest affinity (K-m = 5.8 mu M) and the highest catalytic efficiency (k(cat)/K-m ratio of 7.2 x 10(5) M-1 s(-1)) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E = 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.