COMMUNICATION
Identification of broad specificity P450CAM variants by primary
screening against indole as substrate
Ayhan C¸ elik, Robert E. Speight and Nicholas J. Turner*
Received (in Cambridge, UK) 4th May 2005, Accepted 24th June 2005
First published as an Advance Article on the web 5th July 2005
DOI: 10.1039/b506156c
combinatorially randomised, was screened for the ability to
catalyse hydroxylation of indole 1 to 3-hydroxy indole 2, which
subsequently undergoes spontaneous air oxidation to produce
the insoluble dye indigo 3 (Scheme 1). This dye can easily be
detected at very low levels and can be used as an indication of
enzyme activity either on LB-agar or liquid media. During the
screening of P450CAM mutant enzymes, which were functionally
co-expressed with their natural redox partners in E. coli for
activities towards various substrates (e.g. diphenylmethane), we
observed that some of the mutant enzymes produced a dark
blue pigment within the biotransformation wells. Although
similar observations of this kind have been reported previously
for several mono- and di-oxygenase enzymes, including styrene
monooxygenase,4 naphthalene dioxygenase,5 cytochrome
High-throughput screening of cytochrome P450CAM libraries,
for their ability to oxidise indole to indigo and indirubin, has
resulted in the identification of variants with activity towards
the structurally unrelated substrate diphenylmethane.
Cytochrome P450 monooxygenases (P450s) are a diverse super-
family of enzymes present in plants, animals and microorganisms.
The function of these enzymes varies depending on the host
organisms. For example, in plants they are involved in both
primary and secondary metabolism. In mammals, P450s play a
central role in detoxification pathways. Bacterial P450s are
involved in the degradation of various natural substrates. The
most extensively studied P450 monooxygenase is cytochrome
P450CAM from Pseudomonas putida. This enzyme, which catalyses
the stereoselective hydroxylation of camphor to 5-exo-hydroxy
camphor,1 serves as a model system for studying the mechanism of
other P450 monooxygenases. Typically (type I) three separate
proteins are required for functional activity, namely a cytochrome
P450 reductase (putidadoxin reductase), an iron–sulfur electron
transfer protein (putidaredoxin) and the cytochrome P450
monooxygenase enzyme. Thus, unlike cytochrome P450BM3
from Bacillus megaterium, which contains both a haem-containing
P450 domain and a cytochrome P450 reductase domain in a
fused form, P450CAM is not a catalytically self sufficient enzyme.
For applications in the synthesis of fine chemicals, bioremediation
and the development of herbicide resistant plants, an efficient
enzyme system is clearly desirable. For P450CAM the three
proteins have been engineered into a fused system.2 However,
in comparison to the wild type system, the activity was found to
be quite low. Moreover, the lack of effective high throughput
screening methods has limited the number of variants that
can be screened in order to identify modified enzymes with
altered properties (e.g. broadened substrate specificity or
enhanced rate of electron transfer between P450CAM and
putidaredoxin).
6
P450s2A6 and 2E1 and P450BM3,7 there are no previous reports of
the involvement of P450CAM in the pigment formation. We
subsequently identified the pigments formed in bacterial cultures
containing a functional recombinant P450CAM system and
examined the ability of different P450CAM variants to catalyze
indigo formation.
A library of P450CAM mutants8 (Tyr96/Phe98) was screened for
hydroxylation of various substrates in a 96-well microtitre plate
format (only 48 wells were used), in which each well contained a
mutant of P450CAM (at least one amino acid mutation).
Generation of the blue pigment was observed in several wells
(Fig. 1, Panel A). Addition of exogenous indole (1 mM) resulted in
enhanced colour formation with the same variants (Fig. 1, Panel
B). Pigment formation in the absence of added indole is not
surprising, since it was previously demonstrated that a tryptopha-
nase in E. coli converts tryptophan to indole.9 Wild type P450CAM
does not form the pigment regardless of the presence or absence
Recently we reported an approach for identifying P450CAM
variants based upon generating fully randomised active site
libraries coupled with GC-MS screening for product formation.3
The use of GC-MS allows only ca. 500 variants per day to be
screened and hence places limitations on the size of libraries that
can be evaluated. Hence we sought to develop an alternative
approach that would be able to handle much larger libraries.
The basis for this method is that a library of P450CAM mutants,
in which the active site residues of Tyr96 and Phe98 were
School of Chemistry, University of Edinburgh, King’s Buildings, West
Mains Road, Edinburgh, UK EH9 3JJ. E-mail: n.j.turner@ed.ac.uk;
Fax: +44 131 6504719
Scheme 1
3652 | Chem. Commun., 2005, 3652–3654
This journal is ß The Royal Society of Chemistry 2005