Scientific Report 2006

Molecular Biology

Synthetic Enzymes, Catalytic Antibodies, Ozone Scavengers in Asthma, Organometallic Chemistry, and Biomolecular Computing

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

We focus on synthetically modified enzymes, antibody-catalyzed reactions, anticancer and antiasthma agents, and biomolecular computation, as illustrated in the following examples.

Synthetic Enzymes

Efforts to generate new enzymatic activities from existing protein scaffolds may not only provide biotechnologically useful catalysts but also lead to better understanding of the natural process of evolution. We profoundly changed the catalytic activity and mechanism of the enzyme 4-oxalocrotonate tautomerase by means of rationally designed synthetic mutations. For example, a single amino acid substitution that corresponds to a mutation in a single base pair led to a dramatic change in the catalytic activity. Although the wild-type enzyme catalyzes only the tautomerization of 4-oxalocrotonate, the mutant P1A catalyzes both the original tautomerization reaction via a general acid-base mechanism and the decarboxylation of oxaloacetate via a nucleophilic mechanism.

We also showed that the electrostatic manipulation of an enzyme’s active site can alter the substrate specificity of the enzyme in a predictable way. We replaced 1, 2, or all 3 active-site arginine residues with 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 that was specific for a new substrate that had different electrostatic properties and that bound the enzyme via hydrogen-bonding complementarity rather than electrostatic interactions. This research on synthetic enzymes is being done in collaboration with P.E. Dawson, Department of Cell Biology.

Catalytic Antibodies

Engineering herbicide resistance in crops facilitates control of weed species, particularly weeds that are genetically related to the crop, and may be useful in selecting lines that have undergone multiple transformation events. We showed that herbicide-resistant plants can be engineered by designing both a herbicide and a catalytic antibody that destroys the herbicide within the plants. First, we developed a carbamate herbicide that can be catalytically destroyed by the aldolase antibody 38C2. Then we targeted the light chain and half of the heavy chain (Fab) of the catalytic antibody to the endoplasmic reticulum in 2 lines of Arabidopsis thaliana transformants. Finally, we crossed the 2 transgenic plants to produce a herbicide-resistant F₁ hybrid (Fig. 1). Our results suggest that in vivo expression of catalytic antibodies could become a general strategy to achieve phenotype modifications not only in plants but also in other organisms.

Fig. 1. Influence of herbicide (1) on the rooting and development of seedlings of F1 hybrids and control A thaliana plants. The control plants are shown in A and C; the hybrid plant lines (F1) expressing both light and heavy chains of the catalytic antibody 38C2, in B and D. Plantlets grown on medium without the herbicide are shown in A and B; those grown with the herbicide are shown in C and D.

Ozone Scavengers and Antiasthma Activity

A new hypothesis we proposed on the mechanism of asthmatic inflammation has led to an ozone-scavenging compound that prevents bronchial obstruction in rats with asthma. Previously, scientists at Scripps Research discovered that ozone can be generated not only via the antibody-mediated water oxidation pathway but also by antibody-coated activated white blood cells during inflammatory processes. This finding led us to speculate that the pulmonary inflammation in asthma might be caused by ozone production by white blood cells in lungs and that inhalation of electron-rich olefins, which are known ozone scavengers, might have antiasthmatic effects. In experiments in rats, inhalation of such a compound, limonene, caused a significant improvement in signs of asthma. These results could have consequences in the management of asthma.

Organometallic Chemistry

Rhenium oxide, which is known primarily as a strong oxidant, is a highly selective Lewis acid catalyst that affects the heteroacylative dimerization of tetrahydrofuran at room temperature. This multicomponent reaction, which involves tetrahydrofuran, trifluoroacetic anhydride, and a carboxylic acid, produces a nonsymmetrical diester (compound 3 in Fig. 2) in high yields. The proposed catalytic cycle (Fig. 2) involves a multistep sequence of nucleophilic attacks, metal-oxygen bond metathesis, and electrophilic cleavage by trifluoroacetic anhydride. This synthetically useful reaction highlights the unique, frequently avoided Lewis acidity of transition-metal oxides.

Fig. 2. The catalytic cycle of the rhenium oxide–catalyzed heteroacylative dimerization of tetrahydrofuran (THF), which is proposed on the basis of isotope-labeling experiments, starts with an attack of a rhenium oxo ligand on a coordinated tetrahydrofuran, then an attack of the resultant alkoxide ligand on a second coordinated tetrahydrofuran, nucleophilic addition of the resultant alkoxide ligand to the coordinated carboxylic acid, and finally, electrophilic cleavage of the other coordinated alkoxide by trifluoroacetic anhydride (TFAA).

In study with the platinum complex TpPt(CO)CH₃ (Tp = hydridotrispyrazolylborate), we found that the proton exchange between water and the methyl group involves the formation and deprotonation of a “sticky” σ-methane ligand. The efficiency of this nontrivial process is attributed to the spatial orientation of functional groups that operate in concert to achieve a multistep proton walk. The key role played by the free pyrazolyl nitrogen, acting as a proton carrier, is reminiscent of the dual functionality of the histidine in the catalytic triad of natural serine proteases.