Formation of Indigo by Recombinant Mammalian Cytochrome P450 1 Elizabeth M. J. Gillam,* ,2 Anna Marie A. Aguinaldo,† Lisa M. Notley,* Donghak Kim,‡ Ralf G. Mundkowski,‡ Alexander A. Volkov,† Frances H. Arnold,* Pavel Souc ˇek,§ James J. DeVoss, ¶ and F. Peter Guengerich‡ ,2 *Department of Physiology and Pharmacology and ¶ Department of Chemistry, University of Queensland, Brisbane, Queensland, Australia 4072; †Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125; ‡Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and §National Institute of Public Health, Center of Occupational Diseases, Srobarova 48, Praha 10, 10042, Czech Republic Received October 5, 1999 The development of bicistronic systems for coexpres- sion of recombinant human cytochrome P450 enzymes (P450s) with their redox partner, NADPH-cytochrome P450 reductase (NPR), has enabled P450 activity to be reconstituted within bacterial cells. During expression of recombinant P450 2E1 and some other forms, we ob- served the formation of a blue pigment in bacterial cul- tures. The pigment was extracted from cultures and shown to comigrate with standard indigo on TLC. UV- visible spectroscopy and mass spectrometric analysis provided further support for identification of the pig- ment as indigo. Indigo is known to form following the spontaneous oxidation of 3-hydroxyindole. Accordingly, we speculated that indole, formed as a breakdown prod- uct of tryptophan in bacteria, was hydroxylated by the P450 system, leading to indigo formation. Bacterial membranes containing recombinant P450 2E1 and hu- man NPR were incubated in vitro with indole and shown to catalyze formation of a blue pigment in a time- and co- factor-dependent manner. These studies suggest potential applications of mammalian P450 enzymes in industrial in- digo production or in the development of novel colorimet- ric assays based on indole hydroxylation. © 1999 Academic Press P450 enzymes constitute a superfamily of hemopro- teins catalyzing the monoxygenation of a diverse range of chemicals in a wide variety of organisms (2). The functional significance of P450-mediated reactions to individual organisms is similarly diverse; P450s are involved in clearance of xenobiotic chemicals, biosyn- thesis of hormones and other signaling molecules, ho- meostatic mechanisms, and other organism-specific phenomena such as pigmentation (3) and regulation of feeding behavior (4). Most P450 enzymes act in a con- certed fashion with one or more redox partners. In the case of mammalian P450s involved in xenobiotic me- tabolism, the major accessory enzyme is NPR. Our laboratories have been engaged in the study of human P450 enzymes expressed as recombinant en- zymes in a bacterial host. Recently we have established systems in which individual P450 forms are coexpressed with hNPR using a bicistronic expression vector (5). In the course of work with several bicistronic P450 expres- sion systems, we observed production of blue pigments in bacterial cultures. The following report describes the characterization of the major pigment produced in these cultures and its identification as indigo. MATERIALS AND METHODS Bacterial cultures. Bicistronic plasmids for expression of human P450 2E1 and NPR were prepared as described previously (5). The monocistronic expression construct for P450 2A6 was prepared by PCR amplification of the 2A6 coding sequence using primers encoding minor alterations to the 5' nucleotide sequence of the P450 2A6 coding region (resulting in only a single change to the resultant amino acid sequence, namely substitution of Ala in the second position) and inserting a 5' NdeI site and 3' SalI site flanking the P450 cDNA. The PCR product was then subcloned into the cognate sites of pCW'/NF14 (6) (replacing the P450 3A4 cDNA) and sequenced. One nucleotide difference was observed from the reported 2A6 wild type sequence, leading to mutation of Ile (ATA) at position 246 to Val (GTA) in the predicted amino acid sequence. The resultant mutant 2A6 protein appeared to retain cata- Abbreviations used: P450, cytochrome P450 (also termed heme thiolate protein 450 (1)); NPR, NADPH-P450 reductase (hNPR de- notes human); PCR, polymerase chain reaction; IPTG, isopropyl-- D-thiogalactoside; TB, Terrific Broth; DMF, N,N-dimethylformamide. 1 This work was supported in part by National Health and Medical Research Council (Australia) Grant 951135 and Australian Research Council Grant A09937199 (E.M.J.G.), Maxygen Corp. (F.H.A.), Grant IGA 1850-5 of the Internal Grant Agency of the Czech Minis- try of Health (P.S.), and Grants R35 CA44353 and P30 ES00267 from the National Institutes of Health (F.P.G.). 2 Address correspondence to either of these authors. (E.M.J.G.) Fax: 61-7-3365-1766. E-mail: gillam@plpk.uq.edu.au. (F.P.G.) Fax: (1) 615-322-3141. E-mail: guengerich@toxicology.mc.vanderbilt.edu. Biochemical and Biophysical Research Communications 265, 469 – 472 (1999) Article ID bbrc.1999.1702, available online at http://www.idealibrary.com on 469 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.