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The Skaggs Institute
for Chemical Biology
Synthetic Enzymes, Catalytic Antibodies, Biomolecular Computing, and Synthetic Capsids
E. Keinan, O. Reany, N. Metanis, E. Kossoy, M. Soreni, R. Piran, M.K. Sinha, I. Ben-Shir, T. Ratner, T. Shekhter, T. Mejuch, E. Solel, A. Karmakar, S. Shoshani,
M.K. Pandey, G. Parvari
Synthetic Enzymes
Efforts
to generate new enzymatic activities from existing protein scaffolds may not only
provide biotechnologically useful catalysts but also lead to a better understanding
of the natural process of evolution. Enzymes are usually characterized as catalyzing
a specific reaction by a unique chemical mechanism. However, small changes in the
amino acid sequence of some enzymes can markedly alter the catalytic properties
of the enzymes, affecting the substrate selectivity and subtle aspects of the catalytic
mechanism. The catalytic promiscuity displayed in these enzymes may be an important
factor in the natural evolution of new catalytic activities and in the development
of new catalysts through protein engineering methods.
We
profoundly changed the catalytic activity and mechanism of 4-oxalocrotonate tautomerase
by means of a rationally designed and synthetically produced amino acid substitution.
For example, we found that the substrate specificity can be altered in a predictable
way by electrostatic manipulation of the enzyme's active site. A total of 1,
2, or all 3 active-site arginine residues were replaced by citrulline analogs to
maintain the steric features of the active site of 4-oxalocrotonate tautomerase
while changing its electronic properties. These synthetic changes revealed that
the wild-type enzyme binds the natural substrate predominantly through electrostatic
interactions. This and other mechanistic insights led to the design of a modified
enzyme specific for a new substrate that had different electrostatic properties
and that bound the enzyme via hydrogen-bonding complementarity rather than by electrostatic
interactions. The synthetic analog of 4-oxalocrotonate tautomerase was a poor catalyst
of the natural 4-oxalocrotonate substrate but an efficient catalyst for a ketoamide
substrate.
Selenoenzymes
have a central role in maintaining cellular redox potential. These enzymes have
selenenylsulfide bonds in their active sites that catalyze the reduction of peroxides,
sulfoxides, and disulfides. The selenol-disulfide exchange reaction is common to
all of the enzymes, and the active-site redox potential reflects the ratio between
the forward and reverse rates of this reaction. The preparation of enzymes containing
selenocysteine is experimentally challenging. As a result, little is known about
the kinetic role of selenols in enzyme active sites, and the redox potential of
a selenenylsulfide or diselenide bond in a protein has not been experimentally determined.
To fully evaluate
the effects of selenocysteine on oxidoreductase redox potential and kinetics, we
chemically synthesized glutaredoxin 3 (Grx3) and all 3 selenocysteine variants of
its conserved 11CXX14C active site. Grx3, Grx3(C11U), and
Grx3(C14U) had redox potentials of –194, –260, and –275 mV, respectively.
The position of redox equilibrium between Grx3(C11U-C14U) (–309 mV) and thioredoxin
(–270 mV) suggests a possible role for diselenide bonds in biological systems.
Kinetic analysis showed that the lower redox potentials of the selenocysteine variants
are due primarily to the greater nucleophilicity of the active-site selenium rather
than to its role as either a leaving group or a central atom in the
exchange reaction. The 102- to 104-fold increase in the rate
of thioredoxin reduction by the seleno-Grx3 analogs indicates that oxidoreductases
containing either selenenylsulfide or diselenide bonds can have physiologically
compatible redox potentials and enhanced reduction kinetics in comparison with their
sulfide counterparts. This research was done in collaboration with P.E. Dawson,
Scripps Research.
Catalytic Antibodies
A relatively
unexplored opportunity in the science of catalytic antibodies is modifying the phenotype
of an organism by incorporating the gene for a catalytic antibody into the genome
of that organism. An attractive application of this concept would be the expression
of such a catalyst in transgenic plants to provide a beneficial trait. For example,
introduction of herbicide-resistance trait in commercial plants is highly desirable
because plants with the trait could be grown in the presence of a nonselective herbicide
that affects only weeds and other undesired plant species.
We have shown
that herbicide-resistant plants can be engineered by designing both herbicide and
a catalytic antibody that destroys the herbicide within the plants. Such a transgenic
plant was achieved via a 3-step maneuver: (1) development of a new carbamate herbicide,
one that can be catalytically destroyed by the aldolase antibody 38C2; (2) separate
expression of the light chain and half of the heavy chain (Fab) of the catalytic
antibody in the endoplasmic reticulum of 2 plant lines of Arabidopsis thaliana;
and (3) cross-pollination of these 2 transgenic plants to produce a herbicide-resistant
F1 hybrid (Fig. 1). In vivo expression of catalytic antibodies could
become a useful, general strategy to achieve desired phenotype modifications not
only in plants but also in other organisms.
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| Fig. 1. Influence of a new
herbicide (1) on the rooting and development of A thaliana plant lines. The control plants are shown in A, C, and E; the hybrid plants (F1) expressing
both light and heavy chains, in B, D, and F. Plantlets grown on medium without the herbicide are shown in A and B; those grown with the herbicide, in C and F. |
Biomolecular Computing Devices
In fully autonomous
molecular computing devices, all components, including input, output, software,
and hardware, are specific molecules that interact with each other through a cascade
of programmable chemical events, progressing from the input molecule to the molecular
output signal. DNA molecules and DNA enzymes have been used as convenient, readily
available components of such computing devices because the DNA materials have highly
predictable recognition patterns, reactivity, and information-encoding features.
Furthermore, DNA-based computers can become part of a biological system, generating
outputs in the form of biomolecular structures and functions.
Our previously
reported 2-symbol–2-state finite automata computed autonomously, and all of
their components were soluble biomolecules mixed in solution. The hardware consisted
of 2 enzymes, an endonuclease and a ligase, and the software and the input were
double-stranded DNA oligomers. More recently, we designed and created 3-symbol–3-state
automata that can carry out more complex computations. In addition, we found that
immobilization of the input molecules on chips allowed parallel computation, a system
that can be used to encrypt information.
The main advantage
of autonomous biomolecular computing devices compared with electronic computers
is the ability of the devices to interact directly with biological systems. No interface
is required because all components of molecular computers, including hardware, software,
input, and output, are molecules that interact in solution along a cascade of programmable
chemical events. We showed for the first time that the output of a molecular finite
automaton can be a visible bacterial phenotype. Our 2-symbol–2-state finite
automaton uses linear double-stranded DNA inputs prepared by inserting a string
of 6-bp symbols into the lacZ gene on plasmid pUC18. The computation resulted
in a circular plasmid that differed from the original pUC18 by either a 9-bp (accepting
state) or 11-bp (unaccepting state) insert within the lacZ gene. Upon transformation
and expression of the resultant plasmids in Escherichia coli, either blue
colonies or white colonies, respectively, were formed (Fig. 2).
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| Fig. 2. Computation with aaba input in the presence (A) and absence (B) of transition molecules results in white bacteria when the transition molecules are present. Computation with abba
input in the presence (C) and absence (D) of transition molecules results in blue bacteria when the transition molecules are present. |
Synthetic Capsids
Stable structures
of icosahedral symmetry can have numerous functional roles, including chemical microencapsulation
and delivery of drugs and biomolecules, a way to observe encapsulated reactive intermediates,
presentation of epitopes for efficient immunization, synthesis of nanoparticles
of uniform size, and formation of structural elements for molecular supramolecular
constructs and molecular computing. By examining physical models of spherical virus
assembly, we developed a general synthetic strategy for producing chemical capsids
at sizes between fullerenes and spherical viruses. Such capsids can be formed by
self-assembly from a class of novel symmetric molecules developed from a pentagonal
core. By designing chemical complementarity into the 5 interface edges of the molecule,
we can produce self-assembling stable structures of icosahedral symmetry.
We
considered 3 different binding mechanisms: hydrogen bonding, metal binding, and
formation of disulfide bonds. These structures can be designed to assemble and disassemble
under controlled environmental conditions. We have conducted molecular dynamics
simulation on a class of corannulene-based molecules to demonstrate the characteristics
of self-assembly and to aid in the design of the molecular subunits. This research
was done in collaboration with A.J. Olson, Scripps Research.
Publications
Ben-Shir, I., Sasmal, S., Mejuch, T., Sinha, M.K., Kapon, M., Keinan, E. Repulsive interaction can be a key design element of molecular rotary motors. Chem. Eur. J.,
in press.
Kossoy, E., Lavid, N., Soreni-Harari, M., Shoham, Y., Keinan, E. A programmable biomolecular computing machine with bacterial phenotype output. Chembiochem
8:1255, 2007.
Lo, H.C., Han, H., D'Souza, L.J., Sinha, S.C., Keinan, E. Rhenium(VII) oxide catalyzed heteroacylative ring-opening dimerization of tetrahydrofuran.
J. Am. Chem. Soc. 129:1246, 2007.
Lo, H.C., Iron, M.A., Martin, J.M.L., Keinan, K. Proton walk in the aqueous platinum complex [TpPtMeCO] via a sticky σ-methane
ligand. Chem. Eur. J. 13:2812, 2007.
Olson, A.J., Hu, Y.H., Keinan, E. Chemical mimicry of viral capsid self-assembly. Proc. Natl. Acad. Sci. U. S. A. 104:20731, 2007.
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