Computational protein design methods were used to predict five variants of monofunctional Escherichia coli chorismate mutase expected to maintain catalytic activity. The variants were tested experimentally and three active site mutants exhibited catalytic activity similar to or greater than the wild-type enzyme. One mutant, Ala32Ser, showed increased catalytic efficiency

Keeffe, Jennifer R.

Lassila, Jonathan Kyle

Mayo, Stephen L.

Oelschlaeger, Peter

English

Caltech Authors - Main

COMMUNICATION
Computationally designed variants of
Escherichia coli chorismate mutase
show altered catalytic activity
Jonathan Kyle Lassila1, Jennifer R.Keeffe1,
Peter Oelschlaeger2 and Stephen L.Mayo2,3,4
1Biochemistry and Molecular Biophysics Option, 2Division of Biology and
3Division of Chemistry and Chemical Engineering, Howard Hughes Medical
Institute, California Institute of Technology, Pasadena, CA 91125, USA
4To whom correspondence should be addressed.
E-mail: steve@mayo.caltech.edu
Computational protein design methods were used to pre-
dict five variants of monofunctional Escherichia coli
chorismate mutase expected to maintain catalytic activity.
The variantswere tested experimentally and three active site
mutants exhibited catalytic activity similar to or greater
than the wild-type enzyme. One mutant, Ala32Ser,
showed increased catalytic efficiency.
Keywords: enzyme design/chorismate mutase/protein design
Introduction
The Claisen rearrangement of chorismate to prephenate
(Figure 1) is a rare enzyme-catalyzed pericyclic reaction that
proceeds through the same mechanism uncatalyzed in solution.
Chorismate mutases from various organisms provide rate
enhancements of around 106 despite strong dissimilarities in
three-dimensional fold (Chook et al., 1993; Xue et al., 1994;
Lee et al., 1995). The metabolic importance of chorismate as
the key branch point in the shikimate pathway has prompted
extensive experimental investigation of the chorismate-
prephenate rearrangement since the 1960s (Gibson and
Gibson, 1964) and has driven complementation experiments
to probe the structural determinants of enzyme catalysis
(Woycechowsky and Hilvert, 2004). The concerted, unimolecu-
lar nature of the rearrangement and the lack of covalent protein
contacts have encouraged numerous theoretical studies of
the catalyzed and uncatalyzed reactions. The question of how
chorismate mutases achieve rate enhancement has been act-
ively discussed in recent years (Crespo et al., 2003; Guimar~aes
et al., 2003; Hur and Bruice, 2003; Strajbl et al., 2003;
Ranaghan et al., 2004).
We used computational protein design techniques to identify
mutations within the active site of the chorismate mutase
domain (EcCM) of Escherichia coli chorismate mutase-
prephenate dehydratase (‘P-protein’) (Stewart et al., 1990)
consistent with catalytic activity. The objective of the study
was to evaluate the viability of a rotamer-based approach with
an empirical mechanics force field in modeling the active site.
We report three active site mutations that permit catalytic
activity at or above the level of the wild-type enzyme. One
of these appears to have enhanced catalytic efficiency relative
to wild-type.
Materials and methods
An ab initio calculated transition-state structure (Wiest and
Houk, 1994) was modeled at the position of a transition
state analog in the EcCM crystal structure, pdb code 1ecm
(Lee et al., 1995). Translation (60.2 Å each axis) and rotation
(65 each axis) of the transition-state structure was allowed
and all amino acids except glycine, proline, cysteine and
methionine were permitted in the following positions: 28, 32,
35, 39, 46, 47, 48, 51, 52, 55, 81, 84, 85, 88 (chain A); 7, 11, 14,
18 (chain B). Residues from both chains constitute each of
the two active sites in this symmetric homodimer. A backbone-
dependent side chain rotamer library (Dunbrack and Cohen,
1997) was used with expansion by one standard deviation
about c1 and c2 for all amino acids except arginine and lysine.
The energy function used in the ORBIT protein design soft-
ware (Dahiyat and Mayo, 1997a) was based on the DREIDING
force field (Mayo et al., 1990) and includes a scaled van der
Waals term (Dahiyat and Mayo, 1997b), hydrogen bonding and
electrostatic terms (Dahiyat et al., 1997) and a surface-area
based solvation potential (Street and Mayo, 1998). Transition-
state partial atomic charges were obtained as reported previ-
ously (Wiest and Houk, 1994). An additional energy penalty
was applied to effectively eliminate from consideration all
sequences that could not maintain key contacts between the
transition-state structure and Arg11 and Arg28. These contacts
were previously demonstrated to be necessary for catalysis
(Liu et al., 1996). The HERO rotamer optimization method
was used to obtain the minimum energy amino acid sequence
and conformations (Gordon et al., 2003). Six mutations were
predicted in the optimized structure: Leu7Ile, Ala32Ser,
Val35Ile, Asp48Ile, Ile81Leu and Val85Ile. Visual inspection
and subsequent calculations indicated that four of the muta-
tions were independent, so they were treated separately.
The gene encoding EcCM residues 1–109 was amplified
from genomic DNA (ATCC 700926D) and inserted into the
pTYB11 vector from the IMPACT-CN intein fusion system
(New England Biolabs). Inverse PCR mutagenesis (HemsleyFig. 1. Claisen rearrangement of chorismate (1) to prephenate (2).
Protein Engineering, Design & Selection vol. 18 no. 4 pp. 161–163, 2005
Published online April 8, 2005 doi:10.1093/protein/gzi015
ª The Author 2005. Published by Oxford University Press. All rights reserved. 161
For Permissions, please e-mail: journals.permissions@oupjournals.org
et al., 1989) was used to construct five variants: Leu7Ile,
Ala32Ser, Val35Ile, Asp48Ile and Ile81Leu/Val85Ile. Variant
and wild-type proteins were expressed in E.coli BL21(DE3)
and purified by chitin affinity chromatography. Further puri-
fication by gel filtration followed self-cleavage of the affinity
tag. Protein characterization followed procedures recently
reported for the same construct (Zhang et al., 2003). Protein
concentration was determined by the Bradford assay using
BSA as a standard. Chorismate mutase activity was determined
by following the disappearance of chorismate with UV absorb-
ance at 275 nm. Activity assays were conducted at 37C with
20 nM protein in 50 mM Tris pH 7.8, 2.5 mM EDTA, 20 mM
b-mercaptoethanol and 0.01% BSA. Initial velocities were
buffer corrected and were determined with <6% depletion of
initial substrate concentration. All proteins were initially tested
using a substrate concentration range of about 50–500 mM. The
wild-type and the Ala32Ser mutant were further assayed with
substrate ranges of 20–2000 mM and a minimum of five trials
including two separate protein preparations each. Kinetic para-
meters were determined by non-linear fitting to the Michaelis–
Menten equation.
Results and discussion
As can be seen in Table I, three of the five variants showed
catalytic efficiency similar to or greater than that of the wild-
type enzyme. The Ala32Ser mutation results in a slightly more
efficient catalyst than wild type owing to both a decrease in
KM and an increase in kcat. The fact that substrate binding is
enhanced in addition to catalysis is consistent with the obser-
vation that factors stabilizing the transition state also contribute
to ground-state stabilization in the catalyzed rearrangement
(Strajbl et al., 2003). The rate enhancement corresponds to a
change in activation energy of <1 kcal/mol, making a detailed
structural explanation unwarranted. However, it should be
noted that in the predicted structure of this mutant, Ser32 is
capable of hydrogen bonding with Gln88 (Figure 2), a residue
that makes an essential contact with the ether oxygen of the
breaking bond (Liu et al., 1996).
Val35Ile shows increased kcat but also increased KM, result-
ing in kcat/KM similar to that for wild type. The Leu7Ile
mutation also does not have a significant effect on catalytic
efficiency. Both mutations create slightly different hydropho-
bic packing environments near the essential residue Arg11 in
the predicted structures. The double mutant Ile81Leu/Val85Ile
was predicted to alter packing against the hydrophobic ring
face of the reacting molecule. This rearrangement of hydro-
phobic amino acids does not result in a substantial change
in kcat. However, the mutations result in increased KM and
reduced catalytic efficiency. The relative insensitivity of the
enzyme to changes in some amino acids is not surprising given
the recent finding that the reaction is efficiently catalyzed by a
protein exhibiting all the characteristics of a molten globule
(Vamvaca et al., 2004).
The Asp48Ile mutation abolished measurable catalysis. This
position makes backbone contacts to the hydroxyl group of the
transition-state analog in the EcCM crystal structure. Hydroxyl
contacts to a negatively charged residue were suggested to
create a favorable electrostatic gradient in the Bacillus subtilis
enzyme (Kast et al., 1996). However, in the E.coli structure the
Asp48 side chain points away from the active site and is distant
from the transition-state analog. Molecular dynamics simula-
tions provide some insight into a possible function of Asp48
that this mutant lacks. Averaged structures from equilibrated
systems (see Supplementary data available at PEDS Online)
show Asp48 hydrogen bonding to Arg11 in both the unligan-
ded and inhibitor-bound enzyme. In simulations of unliganded
Asp48Ile mutants, however, Arg11 is dramatically displaced
into solution, suggesting that one role of Asp48 may be to
stabilize this key active site side chain in a conformation com-
patible with substrate binding and catalysis.
While the choice of mutations in this experiment was based
solely on computational modeling and no sequence alignment
information was used in the process, a BLAST search (Altschul
et al., 1997) using the EcCM sequence as the query showed that
sequence variations corresponding to the Ala32Ser, Val35Ile,
Val85Ile and Ile81Leu mutations were observed in chorismate
mutases from related organisms.
Our design procedure stabilizes a static active site config-
uration with a bound transition-state structure. Although the
substrate of the reaction is not explicitly considered, we expect
that modeling interactions using the structure and charges of
the transition state should promote some degree of differential
stabilization of the transition state relative to substrate. The pre-
sent study demonstrates that this approach can be used effect-
ively to represent the active site of a natural enzyme. In this
case, the predicted mutations were in residues not directly
contacting the reacting molecule. The favorable result from
Table 1. Kinetic parameters of wild-type and mutant EcCMa
kcat (min
1) KM (mM) kcat/KM
(min1 mM1)
%WT
kcat/KM
WT 2332 6 306 304 6 52 7.8 6 1.0 100
Ala32Ser 2708 6 364 220 6 29 12.4 6 1.0 159
Val35Ileb 3046 6 172 365 6 59 8.5 6 1.1 109
Leu7Ileb 2193 6 291 249 6 54 9.1 6 2.4 117
Ile81Leu/Val85Ileb 2004 6 241 669 6 165 3.1 6 0.6 40
Asp48Ilec – – – –
aReported errors are standard deviations from at least three trials.
bThree mutants were assayed with a limited substrate concentration range; see
text.
cReaction rates with the Asp48Ile mutant were within error the same as for the
uncatalyzed solution reaction.
Fig. 2. Predicted hydrogen bonding in the Ala32Ser chorismate mutase
variant. Interactions in the wild-type crystal structure are the same except for
position 32A.
J.K.Lassila et al.
162
the Ala32Ser mutation suggests that such secondary contacts
are important in the enzyme design process. The complete loss
of catalytic activity from the Asp48Ile mutation implies that
improved treatment of electrostatics and consideration of the
unbound enzyme could offer some benefit in future design
efforts.
Acknowledgements
This work was supported by the Howard Hughes Medical Institute, the
Ralph M.Parsons Foundation, the Defense Advanced Research Projects
Agency, the Institute for Collaborative Biotechnologies (ARO) and an IBM
Shared University Research Grant.
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Received March 2, 2005; accepted March 4, 2005
Edited by Don Hilvert
Designed variants of E.coli chorismate mutase
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Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity

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