Schultz, P.G.

J. Am. Chem. Soc. 2008, 130, 13194-13195, 10.1021/ja804653f

An E. coli catabolite activator protein (CAP) has been converted into a sequence-specific DNA cleaving protein by genetically introducing (2,2′-bipyridin-5-yl)alanine (Bpy-Ala) into the protein. The mutant CAP (CAP-K26Bpy-Ala) showed comparable binding affinity to CAP-WT for the consensus operator sequence. In the presence of Cu(II) and 3-mercaptopropionic acid, CAP-K26Bpy-Ala cleaves double-stranded DNA with high sequence specificity. This method should provide a useful tool for mapping the molecular details of protein−nucleic acid interactions.

Schultz, P.G.

Science 1987, 238, 1401-1403, 10.1126/science.3685986

The relatively nonspecific single-stranded deoxyribonuclease, staphylococcal nuclease, was selectively fused to an oligonucleotide binding site of defined sequence to generate a hybrid enzyme. A cysteine was substituted for Lys116 in the enzyme by oligonucleotide-directed mutagenesis and coupled to an oligonucleotide that contained a 3'-thiol. The resulting hybrid enzyme cleaved single-stranded DNA at sites adjacent to the oligonucleotide binding site.

Schultz, P.G.

J. Am. Chem. Soc. 1990, 112, 9414-9415, 10.1021/ja00181a065

The specificity and diversity of the immune system have recently been exploited in the generation of antibodies that catalyze a wide variety of chemical reactions.1·2 Several general strategies for the design of catalytic antibodies have emerged, including the use of antibody binding energy to enhance the chemical reactivity of a cofactor or to position a cofactor and a substrate in close proximity.3,4 An intriguing target for antibody-cofactor catalysis is the oxidative reactions characteristic of heme proteins. Here we report that antibodies specific for A-methylmesoporphyrin IX bind iron(III) mesoporphyrin IX and that the resulting complex catalyzes the oxidation of several substrates. These studies are a first step toward the development of selective antibody-heme monooxygenase catalysts.

Arold, S.T.; Eppinger, J.; Groll, M.

ACS Catal. 2019, 9, 11371-11380, 10.1021/acscatal.9b02896

Artificial metalloenzymes (ArMs) have high potential in biotechnological applications as they combine the versatility of transition-metal catalysis with the substrate selectivity of enzymes. An ideal host protein should allow high-yield recombinant expression, display thermal and solvent stability to withstand harsh reaction conditions, lack nonspecific metal-binding residues, and contain a suitable cavity to accommodate the artificial metal site. Moreover, to allow its rational functionalization, the host should provide an intrinsic reporter for metal binding and structural changes, which should be readily amendable to high-resolution structural characterization. Herein, we present the design, characterization, and de novo functionalization of a fluorescent ArM scaffold, named mTFP*, that achieves these characteristics. Fluorescence measurements allowed direct assessment of the scaffold’s structural integrity. Protein X-ray structures and transition metal Förster resonance energy transfer (tmFRET) studies validated the engineered metal coordination sites and provided insights into metal binding dynamics at the atomic level. The implemented active metal centers resulted in ArMs with efficient Diels–Alderase and Friedel–Crafts alkylase activities.