Cloning, expression and characterization of a fast self-sufficient P450: CYP102A5 from Bacillus cereus Puneet K. Chowdhary, Mussie Alemseghed, Donovan C. Haines * Department of Chemistry, University of Texas at Dallas, P.O. Box 830688 M/S BE26, Richardson, TX 75083-0688, USA Received 14 August 2007, and in revised form 10 September 2007 Available online 19 September 2007 Abstract CYP102s represent a family of natural self-sufficient fusions of cytochrome P450 and cytochrome P450 reductase found in some bacteria. One member of this family, named CYP102A1 or more traditionally P450BM-3, has been widely studied as a model of human P450 cytochromes. Remarkable detail of P450 structure and function has been revealed using this highly efficient enzyme. The recent rapid expansion of microbial genome sequences has revealed many relatives of CYP102A1, but to date only two from Bacillus subtilis have been characterized. We report here the cloning and expression of CYP102A5, a new member of this family that is very closely related to CYP102A4 from Bacillus anthracis. Characterization of the substrate specificity of CYP102A5 shows that it, like the other CYP102s, will metabolize saturated and unsaturated fatty acids as well as N-acylamino acids. CYP102A5 catalyzes very fast substrate oxidation, showing one of the highest turnover rates for any P450 monooxygenase studied so far. It does so with more specificity than other CYP102s, yielding primarily x-1 and x-2 hydroxylated products. Mea- surement of the rate of electron transfer through the reductase domain reveals that it is significantly faster in CYP102A5 than in CYP102A1, providing a likely explanation for the increased monooxygenation rate. The availability of this new, very fast fusion P450 will provide a great tool for comparative structure–function studies between CYP102A5 and the other characterized CYP102s. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Cytochrome P450; Cytochrome P450 reductase; Fatty acid metabolism; Enzymology; Bacillus cereus; CYP102A1; CYP102A5; Anthrax Cytochrome P450s (P450 or CYP) constitute the largest superfamilies of monooxygenase enzyme proteins [1]. Dis- covered in 1958 by Klingenberg, P450 genes are widely dis- tributed in prokaryotes and are present in all eukaryotic organisms [2]. Nearly 7000 P450 genes have been sequenced [3], and genome sequencing projects continue to reveal many new P450 members. Much of the interest in P450 enzymes stems from their extensive roles in Phase I drug metabolism (‘xenobiotic’ metabolism) and in the metabolism of sterols and other lipid derived hormones. P450s interact with a wide variety of substrates [4]. They reductively cleave molecular oxygen to produce an oxygen- ated organic product and water. They perform regiospecific and stereospecific oxidation of non-activated hydrocarbons at physiological temperature. Because of the high reactivity of the active intermediate, P450s have been compared to a blowtorch [4]. Most P450s show monooxygenase activity typically resulting in substrate hydroxylation or olefin epoxidation, although N-, O-, and S-dealkylation, intermo- lecular oxygen transfer, organic peroxide isomerization and several other less common reactions are also catalyzed by specific P450 enzymes. One of the most widely studied P450s is CYP102A1 (commonly referred to as P450BM-3), which is found in the gram-positive soil borne bacterium Bacillus megateri- um. CYP102A1 is catalytically self-sufficient thanks to the natural fusion of the P450 with flavoprotein domains homologous to cytochrome P450 reductase, the electron donor in mammalian endoplasmic reticulum bound 0003-9861/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.09.010 * Corresponding author. Fax: +1 972 883 2925. E-mail address: Haines@utdallas.edu (D.C. Haines). www.elsevier.com/locate/yabbi Available online at www.sciencedirect.com Archives of Biochemistry and Biophysics 468 (2007) 32–43 ABB