title: Altering the regioselectivity of cytochrome P450 CYP102A3 of Bacillus subtilis by using a new versatile assay system.
abstract: A novel monooxygenase (CYP102A3) has been discovered within the Bacillus subtilis genome that reveals a similarity of 76 % to the well-known cytochrome P450 BM-3 of B. megaterium (CYP102A1). Both enzymes are natural fusion proteins consisting of a heme domain and a FAD/FMN-reductase domain. Because of their high turnover rates, these biocatalysts are of special interest for industrial applications, but show only limited regioselectivity. In this work, the regioselectivity of CYP102A3 was changed by directed evolution and protein design to hydroxylate substrates not only in different subterminal, but also to a high extent, in terminal carbon chain positions. To enable a high-throughput screening procedure, a very versatile assay was developed that is capable of discriminating between terminal and subterminal hydroxylation of carbon chains. A double mutant of CYP102A3 was obtained that produces 48 % octan-1-ol as the main product of the reaction.
authors.affiliation: Institute for Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Lentz
authors.forename: Anton
authors.initials: A
authors.lastname: Feenstra
authors.forename: Tilo
authors.initials: T
authors.lastname: Habicher
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hauer
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
authors.forename: Vlada B
authors.initials: VB
authors.lastname: Urlacher
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D000442:Octanols / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D000442:Octanols; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C445019:CYP102A3 protein, Bacillus subtilis