Elsevier

Journal of Biotechnology

Volume 139, Issue 1, 1 January 2009, Pages 12-18
Journal of Biotechnology

Identification of amino acid residues involved in 4-chloroindole 3-hydroxylation by cytochrome P450 2A6 using screening of random libraries

https://doi.org/10.1016/j.jbiotec.2008.09.010Get rights and content

Abstract

Cytochrome P450 (P450) 2A6 is able to catalyze indole hydroxylation to form the blue dye indigo. The wild-type P450 2A6 enzyme was randomly mutated throughout the whole open reading frame and screened using 4-chloroindole hydroxylation, a substituted indole selected from 30 indole compounds for enhanced color development. Mutants with up to 5-fold increases of catalytic efficiency (kcat/Km) and 2-fold increases in kcat were selected after two rounds of screening. Important residues located both in (e.g., Thr305) and outside the active site (e.g., Ser224) were identified. The study utilized a better substrate for “indigo assay” to obtain new information on the structure-functional relationship of P450 2A6 that was not revealed by previous mutagenesis studies with this enzyme.

Introduction

Cytochrome P450 (also termed “heme thiolate P450”) (Palmer and Reedijk, 1992) enzymes are found throughout nature, and have been of extreme interest to the pharmaceutical industry as the principle catalysts involved in the disposition of drugs and other xenobiotics. Over the years, many aspects of this group of enzymes have been well addressed (Ortiz de Montellano, 2005). However, as the most versatile redox enzymes known (Ortiz de Montellano, 2005), their use in chemical synthesis and commercial processes has certainly not been explored to its full potential. Recent progress in the extensive characterization of P450s and new technologies such as molecular breeding have offered many opportunities to use both microbial and mammalian P450s in this area (Guengerich, 2002, Guengerich, 2004, Leonard and Koffas, 2007, Li et al., 2007, Urlacher et al., 2004, van Beilen and Funhoff, 2005). In the case of human P450 2A6, a major enzyme involved in nicotine metabolism, extensive studies involving molecular breeding (Kim et al., 2005, Nakamura et al., 2001, Wu et al., 2005b) and novel protein kinase inhibitor synthesis (Guengerich et al., 2004, Wu et al., 2005a) have been done in one of our labs following the observation of blue pigment formation (indigo) from Escherichia coli cultures that expresses bicistronic cytochrome P450 (P450) 2A6 (Gillam et al., 1999, Gillam and Guengerich, 2001, Gillam et al., 2000). The self-sufficient system is able to catalyze the hydroxylation of a variety of indole compounds to form dimers, among which, are the well-known dye indigo, and strong inhibitors for important therapeutic target such as protein kinases GSK-3 and CDKs.

The biotransformation capability has been further enhanced by screening small libraries constructed from six randomized primers, each covering four positions of one of the proposed six substrate recognition sites (SRS), deduced based on the sequence alignments using the P450 SRS analysis of Gotoh (1992), using a colorimetric reaction (Nakamura et al., 2001). This screening method is generally called the “indigo assay” and considered as one of the few convenient solid phase high-throughput assays for oxygenases (Tee and Schwaneberg, 2007). During this course of the work, we found that, for P450 2A6 wild-type (WT), the blue color of indigo formation usually required additional 2-3 days at 4 °C to develop and was not well visualized for single colonies on plates. Mixed with the yellowish color of E. coli cells, indigo blue usually appeared green or even brownish, which was unfavorable for high-throughput screening in the solid phase. Thus, an alternative substrate with more distinct color formation was desired.

Here we report for the first time the use of 4-chloroindole as the substrate in the “indigo assay” for considerably improved pigment formation and the search for enhanced mutants of P450 2A6 via another approach, random libraries constructed by error-prone PCR (epPCR) followed by staggered extension process (StEP) PCR (Zhao et al., 1998). Several mutants with improved catalytic activity were selected including some located in non-SRS regions.

Section snippets

Chemicals

All of the substituted 1H-indoles used in this study were purchased from Aldrich Chemical Co. (Milwaukee, WI) or Acros Organics (Geel, Belgium). All the other chemicals and solvents were obtained from commercial suppliers and used without further purification.

Analytical methods

UV–vis spectra were recorded using an Aminco DW2a/OLIS spectrophotometer. Cytochrome P450 content was assayed according to the most generally used method of Omura and Sato (1964), which utilized the reduced-CO versus reduced difference

Screening system development

To find a compound more suitable as a substrate in “indigo assay”, indole and 29 substituted indoles (Fig. 1) were subjected to biotransformation catalyzed by P450 2A6 expressed in E. coli TRN5. Among them, 4- and 7-nitroindoles could be tolerated at a concentration of 1 mM when being added at the beginning of the incubation, while 5- and 6-nitroindoles hindered the cell growth significantly at the same conditions and only worked when they were introduced 3–5 h after incubation. All the

Discussion

It is well documented that some monooxygenases and dioxygenases are able to catalyze the oxidation of indole to indoxyl and isatin, with subsequent coupling to produce indigo and indirubin pigments. The “indigo assay” is based on this principle and has been, in several cases, used as a general activity detection method for oxygenases, such as the high-throughput screening approaches (solid phase) in our laboratory and others for the direct evolution of monooxygenase P450s (Celik et al., 2005,

Acknowledgements

This work was supported by 100 Talents Program of the Chinese Academy of Sciences (Z.-L.W.), Sichuan Province Science Foundation for Young Scholars 08ZQ026-023 (Z.-L.W.) and U.S. Public Health Service Grants R37 CA90426 and P30 ES00267 (F.P.G.).

References (36)

  • A. Celik

    Identification of broad specificity P450cam variants by primary screening against indole as substrate

    Chem. Commun.

    (2005)
  • E.M. Gillam et al.

    Exploiting the versatility of human cytochrome P450 enzymes: the promise of blue roses from biotechnology

    IUBMB Life

    (2001)
  • E.M. Gillam

    Oxidation of indole by cytochrome P450 enzymes

    Biochemistry

    (2000)
  • F.P. Guengerich

    Cytochrome P450 enzymes in the generation of commercial products

    Nat. Rev. Drug. Discov.

    (2002)
  • F.P. Guengerich

    Cytochrome P450: what have we learned and what are the future issues?

    Drug Metab. Rev.

    (2004)
  • F.P. Guengerich et al.

    Analysis and characterization of enzymes and nucleic acids

  • F.P. Guengerich

    Generation of new protein kinase inhibitors utilizing cytochrome P450 mutant enzymes for indigoid synthesis

    J. Med. Chem.

    (2004)
  • E. Leonard et al.

    Engineering of artificial plant cytochrome P450 enzymes for synthesis of isoflavones by Escherichia coli

    Appl. Environ. Microbiol.

    (2007)
  • Cited by (0)

    View full text