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The Skaggs Institute
for Chemical Biology

Scientific Report 2006

Catalytic Antibodies, Synthetic Enzymes, Biomolecular Computing, and Peroxide Explosives

E. Keinan, O. Reany, C.H. Lo, S. Bauer, N. Metanis, E. Kossoy, M. Soreni, R. Piran, M. Sinha, I. Ben-Shir, S. Shoshani, T. Ratner, T. Shekhter, T. Mejuch, E. Solel

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 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 and F.

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-disufide exchange reaction is common to all of these 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 is being done in collaboration with P.E. Dawson, the Skaggs Institute.

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).

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.

Peroxide Explosives

We are using both x-ray crystallography and electronic structure calculations to study the explosives triacetone triperoxide (TATP) and diacetone diperoxide. The structure, vibrational spectrum, and thermal decomposition of TATP were calculated by using functional density theory.

The calculated thermal decomposition pathway of the TATP molecule was a complicated multistep process with several highly reactive intermediates, including singlet molecular oxygen and various biradicals. Of note, the calculations predict formation of acetone and ozone as the main decomposition products and not the intuitively expected oxidation products.

The key conclusion from this study is that the explosion of TATP is not a thermochemically highly favored event. Rather, the explosion involves entropy burst, which is the result of formation of 4 gas-phase molecules from every molecule of TATP in the solid state. Quite unexpectedly, the 3 isopropylidene units of the TATP molecule do not play the role of fuel that can be oxidized and release energy during the explosion. Instead, these units function as molecular scaffolds that hold the 3 peroxide units close together spatially in the appropriate orientation for the decomposition chain reaction. This research was done in collaboration with R. Kosloff and Y. Zeiri, Hebrew University of Jerusalem, Jerusalem, Israel.


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., in press.

Lo, H.C., Iron, M.A., Martin, J.M.L., Keinan, E. Proton walk in the aqueous platinum complex TpPtMeCO via a sticky α-methane ligand. Chem. Eur. J., in press.

Metanis, N., Keinan, E., Dawson, P.E. Synthetic seleno-glutaredoxin 3 analogues are highly reducing oxidoreductases with enhanced catalytic efficiency. J. Am. Chem. Soc. 128:16684, 2006.

Tuttle, T., Keinan, E., Thiel, W. Understanding the enzymatic activity of 4-oxalocrotonate tautomerase and its mutant analogues: a computational study. J. Phys. Chem. B Condens. Matter Mater. Surf. Interfaces Biophys. 110:19685, 2006.

Weiss, Y., Rubin, B., Shulman, A., Ben Shir, I., Keinan, E., Wolf, S. Determination of plant resistance to carbamate herbicidal compounds inhibiting cell division and early growth by seed and plantlets bioassays. Nat. Protoc. 1:2282, 2006.

Weiss, Y., Shulman, A., Ben Shir, I., Keinan, E., Wolf, S. Herbicide-resistance conferred by expression of a catalytic antibody in Arabidopsis thaliana. Nat. Biotechnol. 24:713, 2006.


Ehud Keinan, Ph.D.
Adjunct Professor

Keinan Web Site