Fraaije Marco W, Wu Jin, Heuts Dominic P H M, van Hellemond Erik W, Spelberg Jeffrey H Lutje, Janssen Dick B
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Appl Microbiol Biotechnol. 2005 Jan;66(4):393-400. doi: 10.1007/s00253-004-1749-5. Epub 2004 Oct 27.
Baeyer-Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer-Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (k(cat) = 1.9 s(-1), K(M) = 59 microM). The enzyme exhibits moderate enantioselectivity with alpha-methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer-Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52 degrees C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.
拜耳-维利格单加氧酶是有用的生物催化工具，因为它们能催化一些用化学方法难以实现的反应。然而，只有有限数量的这类非典型单加氧酶以重组形式存在。利用最近描述的蛋白质序列基序，在嗜热放线菌栖热放线菌（Thermobifida fusca）的基因组中鉴定出一种假定的拜耳-维利格单加氧酶（BVMO）。在大肠杆菌中对相应蛋白质进行异源表达并随后进行酶学表征表明，它确实是一种BVMO。这种依赖NADPH且含FAD的单加氧酶对多种芳香酮具有活性，脂肪族底物也能被转化。目前发现的最佳底物是苯丙酮（k(cat)=1.9 s(-1)，K(M)=59 microM）。该酶对α-甲基苯丙酮表现出中等对映选择性（对映体比例为7）。除了拜耳-维利格反应外，该酶还能够进行硫氧化反应。与所有已知的BVMO不同，这种新鉴定的生物催化剂相对耐热，在52℃下活性半衰期为1天。这项研究表明，利用有效的注释工具，可以有效地将基因组作为新型BVMO的来源加以利用。
