The Skaggs Institute
for Chemical Biology

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

E. Keinan, C.H. Lo, S. Ledoux, C. Bauer, N. Metanis, E. Kossoy, M. Soreni, R. Piran, M. Sinha, A. Alt, I. Ben-Shir, R. Girshfeld, T. Ratner, T. Shekhter, T. Mejuch

Catalytic Antibodies

A relatively unexplored opportunity in the science of catalytic antibodies is modifying an organism’s phenotype 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 would affect 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 destroy 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–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 single amino acid substitution that corresponds to a mutation in a single base pair. Although the wild-type enzyme catalyzes only the tautomerization of oxalocrotonate to 2-oxo-3E-hexenedioate, the mutant P1A catalyzes 2 reactions: the original tautomerization reaction via a general acid-base mechanism and the decarboxylation of oxaloacetate via a nucleophilic mechanism. The observation that a single catalytic group in an enzyme can catalyze 2 reactions by 2 different mechanisms supports the theory that new enzymatic activity can evolve in a continuous manner.

Highly evolved enzymes are optimized not only to catalyze a desired reaction but also to avoid undesired processes. Mutation of active-site residues designed to decrease the optimized catalytic activity may also enhance alternative reaction pathways. Thus, even a minor change in the active-site residues could result in a dramatic change in the delicately optimized balance of their chemical reactivities. We showed that the mutant P1A, which catalyzes the isomerization of the double bond in 4-oxalocrotonate, also undergoes specific 1,4-addition to the tautomerization product to form a stable covalent adduct. This research is being done in collaboration with P.E. Dawson, the Skaggs Institute.

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.

Drug Discovery

Annonaceous acetogenins, particularly those with adjacent bis-tetrahydrofuran rings, have remarkable cytotoxic, antitumor, antimalarial, immunosuppressive, pesticidal, and antifeedant activities. More than 350 different acetogenins have been isolated from only 35 of 2300 plants of the family Annonaceae. We developed synthetic approaches that can be used to generate chemical libraries of stereoisomeric acetogenins. These efforts have resulted in the total synthesis of several naturally occurring acetogenins, including asimicin, bullatacin, trilobacin, rolliniastatin, solamin, reticulatacin, rollidecins C and D, goniocin, cyclogoniodenin, and mucocin, and many nonnatural stereoisomers. A substituted photoactive derivative of asimicin has been prepared for photoaffinity labeling of the target protein subunit in the mitochondrial complex I. This research is being done in collaboration with S.C. Sinha, the Skaggs Institute.

Experimental evidence from studies in a rat model supports the hypothesis that the pulmonary inflammation in asthma may involve a vicious cycle of ozone production and recruitment of white blood cells, which produce more ozone. Accordingly, electron-rich olefins, such as volatile, unsaturated monoterpenes, which are known ozone scavengers, might be useful for prophylaxis for asthma. Both pulmonary function tests and data from pathologic studies strongly support this hypothesis. These results suggest that a new pharmaceutical model should be considered in which appropriately designed ozone scavengers are used to control asthma, as well as other inflammatory diseases.

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 (Fig. 2).

Fig. 2. A biomolecular computing machine made of molecules. The hardware consists of a restriction nuclease and a ligase; the input, transition molecules (software), and detection molecules are all made of double-stranded DNA.

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 for encryption of information. We also developed an advanced computing device in which the input is a molecule but the output is a biological phenomenon.

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.

Publications

Dubnikova, F., Kosloff, R., Almog, J., Zeiri, Y., Boese, R., Itzhaky, H., Alt, A., Keinan, E. Decomposition of triacetone triperoxide is an entropic explosion. J. Am. Chem. Soc. 127:1146, 2005.

Keinan, E., Alt, A., Amir, G., Bentur, L., Bibi, H., Shoseyov, D. Natural ozone scavenger prevents asthma in sensitized rats. Bioorg. Med. Chem. 13:557, 2005.

Matteo, C., Jonoska, N., Yogev, S., Piran, R., Keinan, E., Seeman, N.C. Biomolecular implementation of computing devices with unbounded memory. In: DNA Computing: 10th International Workshop on DNA Computing, DNA10, Milan, Italy, June 7-10, 2004, Revised Selected Papers. Ferretti, C., Mauri, G., Zandron, C. (Eds.). Springer-Verlag, New York, 2005, p. 35. Lecture Notes in Computer Science, Vol. 3384.

Metanis, N., Keinan, E., Dawson, P.E. A designed synthetic analogue of 4-OT is specific for a non-natural substrate. J. Am. Chem. Soc. 127:5862, 2005.

Saphier, S., Hu, Y., Sinha, S.C., Houk, K.N., Keinan, E. The origin of selectivity in the antibody 20F10-catalyzed Yang cyclization. J. Am. Chem. Soc. 127:132, 2005.

Soreni, M., Yogev, S., Kossoy, E., Shoham, Y., Keinan, E. Parallel biomolecular computation on surfaces with advanced finite automata. J. Am. Chem. Soc. 127:3935, 2005.