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. Sinha, I. Ben-Shir, T. Shekhter, T. Ratner, T. Mejuch, E. Solel, S. Shoshani, R. Gershoni, A. Karmakar, D. Pappo, G. Parvari
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
are particularly interested in selenoenzymes, which have a central role in maintaining
cellular redox potential. These enzymes have selenylsulfide 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 selenylsulfide 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
and determined their redox potentials. In particular, the position of redox equilibrium
between Grx3(C11U-C14U) (–308 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. The 102-
to 104-fold increase in the rate of thioredoxin reduction by the seleno-Grx3
analogs indicates that oxidoreductases containing either selenylsulfide or diselenide
bonds can have physiologically compatible redox potentials and enhanced reduction
kinetics in comparison with their sulfide counterparts. This research on synthetic
enzymes is a collaboration with P.E. Dawson, Scripps Research.
A relatively unexplored opportunity in
the science of catalytic antibodies is modifying the phenotype of an organism in
vivo 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 a 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 a 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.
|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
Biomolecular Computing Devices
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 to encrypt
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 an 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).
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.
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.
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. J.
Org. Chem. 73:8772, 2008.
Pappo, D., Mejuch, T., Reany, O., Solel, E., Gurram, M., Keinan, E. Diverse functionalization of corannulene: easy access to pentagonal superstructures.
Org. Lett., in press.