Discovery of a thermostable Baeyer-Villiger monooxygenase by genome mining.
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.
Fraaije
Marco W
MW
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. m.w.fraaije@chem.rug.nl
Wu
Jin
J
Heuts
Dominic P H M
DP
van Hellemond
Erik W
EW
Spelberg
Jeffrey H Lutje
JH
Janssen
Dick B
DB
D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016680:Genome, Bacterial; D023281:Genomics; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases