Cuatrecasas, P.

J. Biol. Chem. 1967, n/a

A spectrophotometric assay is described for staphylococcal nuclease, based on the increase in absorbance at 260 mp which accompanies deoxyribonucleic acid and RNA hy- drolysis. Initial velocities are proportional to enzyme con- centration over a 70-fold range. The enzyme has greater aflinity for DNA than for RNA, and activity is greater with heat-denatured DNA than with native DNA. No inhibitory products accumulate during the reaction. The enzyme is stable at pH values as low as 0.1, and in a concentration of 0.15 mg per ml there is no loss of activity after boiling (20 min). Dilute solutions are protected from heat inactivation by a mixture of albumin and Ca++ as well as by denatured DNA. The optimum pH for RNase and DNase activities is be- tween 9 and 10, depending on the Ca++ concentration. At higher pH values, less Ca+f is required. The inhibitory effect of high Ca+f concentrations is more pronounced at higher pH values. Considerable DNase but no RNase activity results if Ca++ is replaced by Sr+f, while Fe++ and C&f cause minimal activation. A number of heavy metal cations inhibit DNase and RNase activities competitively with Ca++; Hg++, Zn++, and Cd++ are the most potent of these. Activities resulting from combinations of DNA and RNA with Ca+f or Sr+f suggest that these substrates are hy- drolyzed by the same or closely related regions on the en- zyme. Enzyme activity toward DNA and RNA is strongly in- hibited by 5’-phosphoryl (not by 2’- or 3’-phosphoryl) deriva- tives of deoxyadenylic, adenylic, and deozythymidylic acids, and deozythymidine 3’,5’-diphosphate is the most po- tent inhibitor. High activity is obtained with polyadenylic acid compared to polyuridylic acid, polycytidylic acid, and RNA. These tidings are consistent with the known action of the enzyme (cleavage of the 5’-phosphoryl ester bond), and suggest that the differential activity toward DNA and RNA results at least in part from differences in the afhnity toward the constituent bases of these nucleic acids.

Sigman, D.S.

Science 1987, 237, 1197-1201, 10.1126/science.2820056

The tryptophan gene (trp) repressor of Escherichia coli has been converted into a site-specific nuclease by covalently attaching it to the 1,10-phenanthroline-copper complex. In its cuprous form, the coordination complex with hydrogen peroxide as a coreactant cleaves DNA by oxidatively attacking the deoxyribose moiety. The chemistry for the attachment of 1,10-phenanthroline to the trp repressor involves modification of lysyl residues with iminothiolane followed by alkylation of the resulting sulfhydryl groups with 5-iodoacetamido-1,10-phenanthroline. The modified trp repressor cleaves the operators of aroH and trpEDCBA upon the addition of cupric ion and thiol in a reaction dependent on the corepressor L-tryptophan. Scission was restricted to the binding site for the repressor, defined by deoxyribonuclease I footprinting. Since DNA-binding proteins have recognition sequences approximately 20 base pairs long, the nucleolytic activities derived from them could be used to isolate long DNA fragments for sequencing or chromosomal mapping.

Jäger, C.M.; Pordea, A.

Faraday Discuss. 2022, 10.1039/d1fd00070e

Artificial metalloenzymes (ArMs) confer non-biological reactivities to biomolecules, whilst taking advantage of the biomolecular architecture in terms of their selectivity and renewable origin. In particular, the design of ArMs by the supramolecular anchoring of metal catalysts to protein hosts provides flexible and easy to optimise systems. The use of cofactor dependent enzymes as hosts gives the advantage of both a (hydrophobic) binding site for the substrate and a cofactor pocket to accommodate the catalyst. Here, we present a computationally driven design approach of ArMs for the transfer hydrogenation reaction of cyclic imines, starting from the NADP+-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH). We tested and developed a molecular docking workflow to define and optimize iridium catalysts with high affinity for the cofactor binding site of TbADH. The workflow uses high throughput docking of compound libraries to identify key structural motifs for high affinity, followed by higher accuracy docking methods on smaller, focused ligand and catalyst libraries. Iridium sulfonamide catalysts were selected and synthesised, containing either a triol, a furane, or a carboxylic acid to provide the interaction with the cofactor binding pocket. IC50 values of the resulting complexes during TbADH-catalysed alcohol oxidation were determined by competition experiments and were between 4.410 mM and 0.052 mM, demonstrating the affinity of the iridium complexes for either the substrate or the cofactor binding pocket of TbADH. The catalytic activity of the free iridium complexes in solution showed a maximal turnover number (TON) of 90 for the reduction of salsolidine by the triol-functionalised iridium catalyst, whilst in the presence of TbADH, only the iridium catalyst with the triol anchoring functionality showed activity for the same reaction (TON of 36 after 24 h). The observation that the artificial metalloenzymes developed here lacked stereoselectivity demonstrates the need for the further investigation and optimisation of the ArM. Our results serve as a starting point for the design of robust artificial metalloenzymes, exploiting supramolecular anchoring to natural NAD(P)H binding pockets.

Sauer, D.F.; Schwaneberg, U.

Nat. Catal. 2021, 4, 814-827, 10.1038/s41929-021-00673-3

The field of artificial metalloenzymes (ArMs) is rapidly growing and ArMs are attracting increasing attention, for example, in the fields of biosensing and drug therapy. Protein-engineering methods that are commonly used to tailor the properties of natural enzymes are more frequently included in the design of ArMs. In particular, directed evolution allows the fine-tuning of ArMs, ultimately assisting in the development of their enormous potential. The integration of ArMs in whole cells enables their in vivo application and facilitates high-throughput directed-evolution methodologies. In this Review, we highlight the recent progress of whole-cell conversions and applications of ArMs and critically discuss their limitations and prospects. To focus on ArMs and their specific properties, advantages and challenges, the evolution of natural enzymes for non-natural reactions will not be covered.

Okuda, J.; Sauer, D.F.

Beilstein J. Org. Chem. 2018, 14, 2861-2871, 10.3762/bjoc.14.265

This review summarizes the recent progress of Grubbs–Hoveyda (GH) type olefin metathesis catalysts incorporated into the robust fold of β-barrel proteins. Anchoring strategies are discussed and challenges and opportunities in this emerging field are shown from simple small-molecule transformations over ring-opening metathesis polymerizations to in vivo olefin metathesis.