Evaluation of cytochrome P450 BSb reactivity against polycyclic aromatic hydrocarbons and drugs Eduardo Torres a , Heiko Hayen b , Christof M. Niemeyer a,b, * a Universita ¨ t Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, D-44227 Dortmund, Germany b ISAS-Institute for Analytical Sciences, Bunsen-Kirchhoff-Str. 11, 44139 Dortmund, Germany Received 20 January 2007 Available online 6 February 2007 Abstract The oxidation of 10 polycyclic aromatic hydrocarbons (PAH) by cytochrome P450 BSb using three different electron acceptors is reported. Three PAH were found to be substrates for the oxidation by P450 BSb , namely anthracene, 9-methyl-anthracene and azulene. The respective oxidation products were identified by reversed-phase high-performance liquid chromatography coupled to electrospray ionization-mass spectrometry. In addition, 10 drug-like compounds were investigated for their effects on the catalytic activity of P450 BSb by carrying out inhibition studies. The stability of P450 BSb against hydrogen peroxide, cumene, and ter-butyl hydroperoxide was determined. Overall, the results of this study suggested that the P450 BSb enzyme represents a powerful catalyst in terms of the cat- alytic activity and operational stability. Ó 2007 Elsevier Inc. All rights reserved. Keywords: CYPBSb; Drugs; PAH; P450 The cytochromes P450 constitute a large family of heme enzymes, which plays a key role in the oxidative transfor- mation of endogeneous and exogeneous molecules [1]. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds [2]. However, the application of P450 as catalysts for commercial purpos- es faces some important drawbacks such as low operational stability, low activity, poor enzyme production, and the need for cofactor regeneration [3]. To circumvent these lim- itations, reactivities of novel P450 enzymes are being explored, and in this regard, bacterial P450s are especially interesting because they reveal higher stabilities, higher cat- alytic activities and are more readily available in large quantities than their mammalian counterparts [4]. Among the bacterial P450s, the enzyme cytochrome P450 BSb from Bacillus subtilis appears to be a very attractive biocatalyst. First, this enzyme only requires hydrogen peroxide instead of the expensive cofactor NAD(P)H, normally required for P450 catalysis [5]. Second, its crystal structure in the sub- strate-bound form is reported [6], thus enabling elucidation of the mechanisms for the substrate binding and the hydroxylation step. In addition, the P450 BSb has a high cat- alytic turnover (1000 min À1 ) for the hydroxylation of myristic acid, and it can be conveniently produced and purified with good expression rates in Escherichia coli [7]. On the other hand, bacterial P450s are known for their lim- ited substrate diversity comprising a distinct disadvantage for industrial applications. To elucidate this topic, we here report on the screening of 10 polycyclic aromatic hydrocar- bons (PAH) by the enzyme P450 BSb . In addition, 10 drug- like compounds were investigated for their effects on the catalytic activity of P450 BSb by carrying out inhibition studies. 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.01.164 * Corresponding author. Address: Universita ¨t Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, D-44227 Dortmund, Germany. Fax: +49 (0) 231 755 7082. E-mail address: christof.niemeyer@uni-dortmund.de (C.M. Niemeyer). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 355 (2007) 286–293