Table 1 The activity of wild-type (WT) cytochrome P450cam and active site mutants for the oxidation of chlorinated benzenes. ND: no product observed by
HPLC. The products of the reactions are shown in Fig. 2
WT
F87W–Y96F
F87W–Y96F–V247L
F87W–Y96F–F98W
1
,2-dichlorobenzene (1,2-DCB)
Binding constant K
/amM
NADH turnover rate
D
3.0
20
2.0
408
0.9
391
1.2
158
Product formation rate (k
Coupling efficiency
2
)b
0.45
106
83
78
c
2.3%
2.50 3 103
26%
8.83 3 105
21%
1.54 3 106
49%
1.08 3 106
21
21
(k
2 D
/K )/M
s
1
,3,5-trichlorobenzene (1,3,5-TCB)
Binding constant K
NADH turnover rate
D
/mM
3.9
6.5
3.0
224
2.0
308
1.8
121
Product formation rate (k
2
)
0.07
115
175
119
Coupling efficiency
1.1%
3.00 3 102
51%
6.39 3 105
57%
1.46 3 106
97%
1.10 3 106
21
21
(k
2 d
/K )/M
s
Pentachlorobenzene (PeCB)
NADH turnover rate
Product formation rate
Coupling efficiency
2.4
ND
—
100
2.3
2.3%
229
5.5
2.4%
43
3
7%
a
Given as nanomoles of NADH consumed per nanomole of P450cam per minute and the average of at least 3 experiments with all the data within 10% of
the means. Incubation mixtures (1.70 ml) contained 50 mM Tris.HCl, pH 7.4, 1 mM P450cam, 10 mM putidaredoxin, 2 mM putidaredoxin reductase and 200
mM KCl. Both 1,2-DCB and 1,3,5-TCB (200 mM) were added as a 0.1 M stock in ethanol. The mixture was incubated at 30 °C for 2 min after the addition
b
of NADH (100 mM) and the reaction initiated by the addition of substrate. NADH absorbance at 340 nm was monitored over the course of the reaction. The
total amount (in nanomoles) of chlorinated phenol products formed per nanomole of P450cam per minute. After the addition of 100 ml of an internal standard
to a turnover incubation, organics were extracted by solid phase methods using Varian Bond-Elut columns and products were analysed by reverse phase
HPLC. To obtain quantitative results, mixtures containing known concentrations of a product and all of the incubation components except NADH were
c
extracted and analyzed as described above. Linear calibration plots that passed through the origin were obtained for all of the products. The coupling
efficiency is the ratio of the total amount of products formed to the amount of NADH consumed and is expressed as a percentage.
phenol (PCP), but two small peaks (ca. 2% each) were ascribed
by co-elution experiments to 2,3,5,6- and 2,3,4,5-tetrachlor-
ophenols formed by oxidative dehalogenation.
mutant represents an excellent compromise between reasonable
rate and tight coupling, especially for the in vivo oxidation of
polychlorinated benzenes, where very fast turnover could lead
to the build up of polychlorinated phenols to toxic levels.
All the chlorophenol products in Fig. 2 are known to be
degraded by various micro-organisms,4 and therefore the
mutants can be the basis of novel bioremediation systems for
polychlorinated benzenes by genetically introducing the genes
encoding the three proteins of the P450cam system into
chlorophenol-degrading micro-organisms such as Pseudomo-
nas bacteria. The F87W–Y96F–F98W mutant can be used for
the particularly inert 1,2-DCB and 1,3,5-TCB. The coupling for
PeCB and HCB can be improved by further active site
mutations, and then even these highly inert compounds can be
efficiently degraded.
As shown in Table 1, the wild-type (WT) had low rates ( < 0.5
2
1
min ) and couplings (1–2%) for the oxidation of the
chlorinated benzenes compared to the totally coupled (100%)
2
1
camphor oxidation rate of 1050 min under identical condi-
tions. The mutants all had 2–3 orders of magnitude faster
chlorinated benzene oxidation rates than the WT. The NADH
turnover rates were up to 400 min21, and the coupling
efficiencies were also much higher. The 50% coupling for
1
,2-DCB oxidation by the F87W–Y96F–F98W mutant is a
dramatic improvement, and the near total coupling for
,3,5-TCB oxidation is truly remarkable because the structure
1
of this molecule is completely different from that of camphor.
Very importantly, the results also showed that the low solubility
of PeCB in water was not a problem, in that a very reasonable
We thank HEFCE and BBSRC for support of this work.
J. P. J. thanks the EPSRC for a Studentship.
2
1
NADH turnover rate of 229 min could be attained although
the coupling was low. The data show that the rationale for active
site redesign, whilst empirical and qualitative in nature, was
very successful indeed.
Notes and references
1 S. Fetzner and F. Lingens, Microbiol. Rev., 1995, 58, 641.
The strength of binding and catalytic efficiency of the
P450cam enzymes for 1,2-DCB and 1,3,5-TCB oxidation were
investigated. The dissociation constants (K ) in Table 1 show
D
that, as expected, the binding of these compounds was
strengthened by the mutations, but by no more than a factor of
2 S. Beil, B. Happe, K. N. Timmis and D. H. Pieper, Eur. J. Biochem.,
1
997, 247, 190; T. Potrawfke, K. N. Timmis and R. M. Wittich, Appl.
Environ. Microbiol., 1998, 64, 3798; B. E. Haigler, C. A. Pettigrew and
J. C. Spain, Appl. Environ. Microbiol., 1992, 58, 2237.
3
4
A. H. Neilson, Int. Biodeterior. Biodegrad., 1996, 3; M. Haggblom,
FEMS Microbiol. Rev., 1992, 103, 29.
I. P. Solyanikova and L. A. Golovleva, Biochemistry (Engl. Transl.),
three. On the other hand, the substrate oxidation rates (k
showed 2–3 orders of magnitude increases. There was no direct
correlation between the values of K and the NADH turnover
2
)
1
999, 64, 365.
D
5 P. J. Loida and S. G. Sligar, Biochemistry, 1993, 32, 11530.
6 J.-A. Stevenson, A. C. G. Westlake, C. Whittock, C. and L.-L. Wong,
J. Am. Chem. Soc., 1996, 118, 12836.
rates or, notably, the coupling efficiency, which is a stringent
measure of the enzyme–substrate fit. Nevertheless, it is
7
8
9
D. P. Nickerson, C. F. Harford-Cross, S. R. Fulcher and L.-L. Wong,
FEBS Lett., 1997, 405, 153.
P. A. England, C. F. Harford-Cross, J.-A. Stevenson, D. A. Rouch and
L.-L. Wong, FEBS Lett., 1998, 424, 271.
instructive to compare the relative specificity (kcat/K
mutants. It has been suggested that the k /K ratio is a fair
approximation for the kcat/K ratio for P450cam
M
) of the
2
D
10
M
.
2 D
The k /K
ratios in Table 1 again highlight the very dramatic accelerating
effects achieved by the mutations although they are well short of
the near-optimal ratio for camphor oxidation by the WT enzyme
T. L. Poulos, B. C. Finzel and A. J. Howard, J. Mol. Biol., 1987, 195,
6
87.
1
0 W. M. Atkins and S. G. Sligar, J. Biol. Chem., 1988, 269, 18842.
2
1
7
21
(
s
K
D
= 0.27 mM, k
2
= 1050 min , k
2
/K
D
= 6.5 3 10 M
2
1
). However, we conclude that the F87W–Y96F–F98W
Communication a909536e
248
Chem. Commun., 2000, 247–248
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