G. Gilardi et al.
Experimental Section
Library generation: For the generation of the library of variants contain-
ing random mutations within the haem domain of P450 BM3, the re-
[
43]
combinant construct pT7m3Z, which contains the 3147 bp gene trans-
lating to P450 BM3, was used. Part of the gene extending from À24 bp to
1
000 bp and incorporating the BamHI and BalI sites was amplified by
PCR by using the 27-mer primers 5’-CTTAACAAGTGAAGGAGG-
GATCCTATG-3’ (forward primer) and 5’-GGGAAAACGCAGGAG-
CAGTTGGCCATA-3’ (reverse primer). The amplified gene was then
subjected to error-prone PCR. The reaction mixture (40 mL) contained
Tris-HCl (10 mm, pH 8.0), potassium chloride (50 mm), magnesium chlo-
ride (3.5 mm), manganese chloride (0.5 mm), primers (0.4 mm each), three
dNTPs (1.0 mm) with the fourth at 0.2 mm, template DNA (3.1–6.2 ng),
Taq-polymerase (2 U). Four reactions were run in parallel, each having a
different dNTP triplet in excess. Reactions were pooled and the PCR
product was purified from an agarose gel. It was subsequently digested
with BamHI and MscI (an isoschisomer of BalI). The digested insert was
purified subsequently ligated to the similarly digested pT7m3Z vector, re-
placing the WT sequence.
[
22]
Alkali assay: The alkali assay was carried out as described before.
Briefly, the colonies containing the randomised pT7m3Z library were in-
À1
oculated into LB/amp (170 mL, 100 mgmL ) in a microtitre-plate well.
As a control, cells carrying the WT plasmid were also inoculated in a sep-
arate well. The microtitre plate was incubated at 378C, and when the cul-
ture reached an OD600 of approximately 0.7, protein expression was in-
duced by the addition of isopropyl b-d-1-tiogalattopiranoside (IPTG,
Scheme 1. Products performed by the catalysis of mutant A2 on diclofe-
nac, ibuprofen and tolbutamide.
1
.0 mm). After growth, the cells were harvested by centrifugation and the
pellet re-suspended into KP (100 mm, pH 8.0) to give OD600 of about
.4–0.5. By correcting the cell density before performing the assay, equal
i
0
ure 1A). The structure of the substrate-free enzyme shows
+
background NADP levels were achieved.
that the N of Gln307 is likely to participate in a hydrogen
e
The compounds to be screened for turnover were added to the cells for
2 h before the reaction was started by the addition of NADPH (1.5 mm).
The final concentration of diclofenac, ibuprofen and tolbutamide was
bond with the carboxylate oxygen Od2 of Asp300 (Fig-
ure 1C). Replacement with the charged His may cancel the
hydrogen bond and perturb the local structure. Helix J’ is
absent in other bacterial P450s, but it is present in microso-
mal cytochromes P450 where it has a role in recognition and
0
.5 mm. After incubation for 4 h at room temperature, the alkali product
+
[22]
of NADP was developed, as previously described.
spectrum at l=340–450 nm of each well was read on a SPECTRAmax
40PC microplate spectrophotometer (Molecular Devices, California).
The absorbance
3
[41]
binding of the reductase.
However, the structure of the
Controls reactions were performed where no compound was added. With
the exception of lauric acid that was dissolved in potassium carbonate
(50 mm), stocks of all compounds were in ethanol. The final ethanol con-
centration in the assay incubation did not exceed 2%. The alkali assay
was also repeated on the purified mutant. The protein concentration, de-
termined by the CO binding assay, was ꢀ1 mm. A e450 nm value of
complex of the P450 BM3 haem domain with the FMN re-
[42]
ductase domain shows that Gln307 is located far from the
reductase docking site; Gln307 does not belong to the postu-
lated pathway of electron transfer from the reductase to the
[42]
haem.
À1
À1
[44]
9
1000m cm was used to quantify the protein.
Expression and purification of P450 BM3 WT and mutant A2: P450
BM3 WT and mutant A2 were expressed in the E. coli BL21(DE3) strain
and purified essentially as described previously for the WT enzyme.
AHCTUNGTRENNUNG
[
22]
Conclusion
Substrate binding and determination of the high-spin species: Substrate
binding was monitored by using spectrophotometric titrations in a 1 cm
path-length cuvette by following the characteristic low-to-high-spin tran-
sition, as indicated by the shift in the main Soret band from l=418 to
A new biocatalyst, able to mimic the substrate specificity
and the metabolite profile of human P450 2C, has been gen-
erated through random mutagenesis applied to P450 BM3.
The mutant shows new catalytic abilities towards diclofenac,
ibuprofen and tolbutamide with respect to the WT protein.
The data shown give evidence to the fact that new biocat-
alytic capabilities can be introduced through directed evolu-
tion, an approach that allows the production of protein var-
iants with mutations in random positions, unpredictable by
rational methods, but important in maintaining the structur-
al scaffold of the protein optimal for a certain range of sub-
strates. In this specific case, only two mutations in positions
not directly involved in substrate binding or turnover in the
catalytic site are found to be sufficient to generate new spe-
cific reactions of biotechnological interest.
3
92 nm. The reaction mixture contained 0.5–1 mm of enzyme in KP
i
(100 mm, pH 8.0). Spectra were recorded from l=360 to 460 nm after
each addition to the sample cuvette. Substrates were always freshly pre-
pared. Controls with the same volume of the corresponding solvent
added to the enzyme-containing solution were also carried out. All spec-
tral titrations were carried out at 208C. Difference spectra were generat-
ed by subtraction of the substrate-free spectrum of the ones correspond-
ing to substrate-bound spectrum after each addition. KD values were de-
termined by plotting the maximal absorbance changes calculated from
each difference spectrum against the concentration of drugs and fitting
the data by using SigmaPlot 8.0.
The percentage of high-spin species was calculated assuming that the
P450 BM3 ferric iron was 100% low spin in the resting state of the
À1
À1 [45]
enzyme (e418 nm =105000m cm
)
and 100% high spin when saturated
with arachidonic acid.
3586
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 3582 – 3588