Scientific Report 2005

Molecular Biology

Synthetic Enzymes, Catalytic Antibodies, Ozone Scavengers, Organic Synthesis, and Biomolecular Computing

E. Keinan, C.H. Lo, H. Han, S. Sasmal, S. Ledoux, N. Metanis, G. Sklute, E. Kossoy, M. Soreni, D. Vebenov, R. Piran, M. Sinha, A. Alt, I. Ben-Shir, R. Girshfeld, S. Yogev

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. The observation that a single catalytic group in an enzyme can catalyze 2 reactions by 2 different mechanisms supports the hypothesis that enzyme evolution is a continuum in which a new catalytic mechanism is gained while the parent activity declines gradually through small changes in the amino acid sequence of the primordial enzyme. 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. The synthetic analog of the natural 4-oxalocrotonate tautomerase was a poor catalyst of the natural 4-oxalocrotonate substrate but an efficient catalyst for a ketoamide substrate. This research on synthetic enzymes is being done in collaboration with P.E. Dawson, Department of Cell Biology.

Catalytic Antibodies

Although the solution photochemical reaction of the ketone 1 (in Fig. 1) yields only the cleavage products 2 and 3, in the presence of 20F10, an antibody to 5a and 5b, this Norrish type II reaction results in the selective formation of cis-cyclobutanol (compound 4 in Fig. 1).

Fig. 1. The photochemical Norrish type II reaction of ketone 1 produces in solution the cleavage products 2 and 3. Antibody 20F10, which was elicited against a mixture of 5a and 5b, catalyzes enantioselective formation of cis-cyclobutanol (4).

Furthermore, the fact that compound 4, which consists of 2 asymmetric centers, is obtained as a single diastereomer makes this photoproduct a valuable building block for the synthesis of natural products. Another reaction that is exclusively catalyzed by 20F10 is the photochemical formation of cyclopropanol products.

An aldolase antibody, 24H6, obtained from immunization with large diketone haptens has an active-site lysine residue with a perturbed pK_a of 7.0. This antibody catalyzes both the aldol addition and the retrograde aldol fragmentation with a broad range of substrates that differ structurally from the hapten. This observation suggests that in reactive immunization with 1,3-diketones, the hapten structure governs the chemistry but not the overall organization of the active site. Antibody 24H6 also catalyzes the oxidation of α-hydroxyketones to α-diketones. The deuterium exchange at the α position of many ketones and aldehydes is also efficiently catalyzed by aldolase antibodies 38C2 and 24H6. All reactions were carried out in deuterium oxide under neutral conditions and showed regioselectivity, chemoselectivity, and high catalytic rates.

Ozone Scavengers and Antiasthma Activity

A new hypothesis we proposed for 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 asthmatic symptoms. These results could have consequences in the management of asthma.

Organic Synthesis

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 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, Department of Molecular Biology.

Biomolecular Computing Devices

Four years ago we described the first nanoscale, programmable finite automaton with 2 symbols and 2 states that computed autonomously. All of the components of the device, including hardware, software, input, and output, were biomolecules mixed together in solution. The hardware consisted of a restriction nuclease and a ligase; the software (transition rules) 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.

Computation was carried out by processing the input molecule via repetitive cycles of restriction, hybridization, and ligation reactions to produce a final-state output in the form of a double-stranded DNA molecule. Currently, we are taking the concept of molecular computing a step further and are constructing computing devices in which the computation output is a specific biological function rather than a specific molecule.

Most recently, we markedly increased the levels of complexity and mathematical power of these automata by the design of a 3-state–3-symbol automaton, thus increasing the number of syntactically distinct programs from 765 to 1 billion. We have further amplified the applicability of this design by using surface-anchored input molecules and surface plasmon resonance technology to monitor the computation steps in real time. This technology allowed parallel computation and automatic, real-time detection with DNA chips that carry multiple input molecules and can be used as pixel arrays for image encryption.

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.

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.