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A Dual Anchoring Strategy for the Directed Evolution of Improved Artificial Transfer Hydrogenases Based on Carbonic Anhydrase.

ABSTRACT: Artificial metalloenzymes result from anchoring a metal cofactor within a host protein. Such hybrid catalysts combine the selectivity and specificity of enzymes with the versatility of (abiotic) transition metals to catalyze new-to-nature reactions in an evolvable scaffold. With the aim of improving the localization of an arylsulfonamide-bearing iridium-pianostool catalyst within human carbonic anhydrase II (hCAII) for the enantioselective reduction of prochiral imines, we introduced a covalent linkage between the host and the guest. Herein, we show that a judiciously positioned cysteine residue reacts with a p-nitropicolinamide ligand bound to iridium to afford an additional sulfonamide covalent linkage. Three rounds of directed evolution, performed on the dually anchored cofactor, led to improved activity and selectivity for the enantioselective reduction of harmaline (up to 97% ee (R) and >350 turnovers on a preparative scale). To evaluate the substrate scope, the best hits of each generation were tested with eight substrates. X-ray analysis, carried out at various stages of the evolutionary trajectory, was used to scrutinize (i) the nature of the covalent linkage between the cofactor and the host as well as (ii) the remodeling of the substrate-binding pocket.

SUBMITTER: Stein A

PROVIDER: S-EPMC8620556 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Proton transfer function of carbonic anhydrase: Insights from QM/MM simulations.

Project description:Recent QM/MM analyses of proton transfer function of human carbonic anhydrase II (CAII) are briefly reviewed. The topics include a preliminary analysis of nuclear quadrupole coupling constant calculations for the zinc ion and more detailed analyses of microscopic pK(a) of the zinc-bound water and free energy profile for the proton transfer. From a methodological perspective, our results emphasize that performing sufficient sampling is essential to the calculation of all these quantities, which reflects the well solvated nature of CAII active site. From a mechanistic perspective, our analyses highlight the importance of electrostatics in shaping the energetics and kinetics of proton transfer in CAII for its function. We argue that once the pK(a) for the zinc-bound water is modulated to be in the proper range (approximately 7.0), proton transfer through a relatively well solvated cavity towards/from the protein surface (His64) does not require any major acceleration. Therefore, although structural details like the length of the water wire between the donor and acceptor groups still may make a non-negligible contribution, our computational results and the framework of analysis suggest that the significance of such "fine-tuning" is likely secondary to the modulation of pK(a) of the zinc-bound water. We encourage further experimental analysis with mutation of (charged) residues not in the immediate neighborhood of the zinc ion to quantitatively test this electrostatics based framework; in particular, Phi analysis based on these mutations may shed further light into the relative importance of the classical Grotthus mechanism and the "proton hole" pathway that we have proposed recently for CAII.

| S-EPMC6787916 | biostudies-literature

Neutron structure of human carbonic anhydrase II: implications for proton transfer.

Project description:Human carbonic anhydrase II (HCA II) catalyzes the reversible hydration of carbon dioxide to form bicarbonate and a proton. Despite many high-resolution X-ray crystal structures, mutagenesis, and kinetic data, the structural details of the active site, especially the proton transfer pathway, are unclear. A large HCA II crystal was prepared at pH 9.0 and subjected to vapor H-D exchange to replace labile hydrogens with deuteriums. Neutron diffraction studies were conducted at the Protein Crystallography Station at Los Alamos National Laboratory. The structure to 2.0 A resolution reveals several interesting active site features: (1) the Zn-bound solvent appearing to be predominantly a D(2)O molecule, (2) the orientation and hydrogen bonding pattern of solvent molecules in the active site cavity, (3) the side chain of His64 being unprotonated (neutral) and predominantly in an inward conformation pointing toward the zinc, and (4) the phenolic side chain of Tyr7 appearing to be unprotonated. The implications of these details are discussed, and a proposed mechanism for proton transfer is presented.

| S-EPMC2893723 | biostudies-literature

Tuning the Dual Inhibition of Carbonic Anhydrase and Cyclooxygenase by Dihydrothiazole Benzensulfonamides.

Project description:A novel series of of 4-[(3-phenyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides (EMAC10111a-g) was synthesized and assayed toward both human carbonic anhydrase isozymes I, II, IX, and XII and cyclooxygenase isoforms. The majority of these derivatives preferentially inhibit hCA isoforms II and XII and hCOX-2 isozyme, indicating that 2,3,4-trisubstituted 2,3-dihydrothiazoles are a promising scaffold for the inhibition of hCA isozymes and of hCOX-2 enzyme. The nature of the substituent at the dihydrothiazole ring position 4 influenced the activity and selectivity toward both enzyme families. EMAC10111g resulted as the best performing compound toward both enzyme families and exhibited preferential activity toward hCA XII and hCOX-2 isozymes.

| S-EPMC6187410 | biostudies-literature

carbonic anhydrase (1 or 2) simple or double mutants-Comparative transcriptome analysis between carbonic anhydrase mutants and wild type Arabidopsis

Project description:cea05-01_carbonic-anhydrase - carbonic anhydrase (1 or 2) simple or double mutants - Identification of genes differentially expressed in leaves of single CA1 and CA2 T-DNA insertional mutants and in the corresponding double mutant vs wild type - CA1 (At3g01500) and CA2 (At5g14740) T-DNA insertional mutant lines, the double (CA1+CA2) mutant and wild type Arabidopsis seeds were sown in soil in a phytotron. Leaves were harvested 40 days later for RNA extraction

2012-03-25 | GSE36625 | GEO

carbonic anhydrase (1 or 2) simple or double mutants-Comparative transcriptome analysis between carbonic anhydrase mutants and wild type Arabidopsis

2012-03-25 | E-GEOD-36625 | biostudies-arrayexpress

Chemical rescue of enzymes: proton transfer in mutants of human carbonic anhydrase II.

Project description:In human carbonic anhydrase II (HCA II), the mutation of position 64 from histidine to alanine (H64A) disrupts the rate limiting proton transfer (PT) event, resulting in a reduction of the catalytic activity of the enzyme as compared to the wild-type. Potential of mean force (PMF) calculations utilizing the multistate empirical valence bond (MS-EVB) methodology for H64A HCA II yields a PT free energy barrier significantly higher than that found in the wild-type enzyme. This high barrier, determined in the absence of exogenous buffer and assuming no additional ionizable residues in the PT pathway, indicates the likelihood of alternate enzyme pathways that utilize either ionizable enzyme residues (self-rescue) and/or exogenous buffers (chemical rescue). It has been shown experimentally that the catalytic activity of H64A HCA II can be chemically rescued to near wild-type levels by the addition of the exogenous buffer 4-methylimidazole (4MI). Crystallographic studies have identified two 4MI binding sites, yet site-specific mutations intended to disrupt 4MI binding have demonstrated these sites to be nonproductive. In the present work, MS-EVB simulations show that binding of 4MI near Thr199 in the H64A HCA II mutant, a binding site determined by NMR spectroscopy, results in a viable chemical rescue pathway. Additional viable rescue pathways are also identified where 4MI acts as a proton transport intermediary from the active site to ionizable residues on the rim of the active site, revealing a probable mode of action for the chemical rescue pathway.

| S-EPMC4120857 | biostudies-literature

Water networks in fast proton transfer during catalysis by human carbonic anhydrase II.

Project description:Variants of human carbonic anhydrase II (HCA II) with amino acid replacements at residues in contact with water molecules in the active-site cavity have provided insights into the proton transfer rates in this protein environment. X-ray crystallography and (18)O exchange measured by membrane inlet mass spectrometry have been used to investigate structural and catalytic properties of variants of HCA II containing replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The rate constants for transfer of a proton from His64 to the zinc-bound hydroxide during catalysis were 4 and 9 ?s(-1) for Y7F and Y7F/N67Q, respectively, compared with a value of 0.8 ?s(-1) for wild-type HCA II. These higher values observed for Y7F and Y7F/N67Q HCA II could not be explained by differences in the values of the pK(a) of the proton donor (His64) and acceptor (zinc-bound hydroxide) or by the orientation of the side chain of the proton shuttle residue His64. They appeared to be associated with a reduced level of branching in the networks of hydrogen-bonded water molecules between proton shuttle residue His64 and the zinc-bound solvent molecule as observed in crystal structures at 1.5-1.6 Å resolution. Moreover, Y7F/N67Q HCA II is unique among the variants studied in having a direct, hydrogen-bonded chain of water molecules between the zinc-bound solvent and N(?) of His64. This study provides the clearest example to date of the relevance of ordered water structure to rate constants for proton transfer in catalysis by carbonic anhydrase.

| S-EPMC3540201 | biostudies-literature

Chemical Optimization of Whole-Cell Transfer Hydrogenation Using Carbonic Anhydrase as Host Protein.

Project description:Artificial metalloenzymes combine a synthetic metallocofactor with a protein scaffold and can catalyze abiotic reactions in vivo. Herein, we report on our efforts to valorize human carbonic anhydrase II as a scaffold for whole-cell transfer hydrogenation. Two platforms were tested: periplasmic compartmentalization and surface display in Escherichia coli. A chemical optimization of an IrCp* cofactor was performed. This led to 90 turnovers in the cell, affording a 69-fold increase in periplasmic product formation over the previously reported, sulfonamide-bearing IrCp* cofactor. These findings highlight the versatility of carbonic anhydrase as a promising scaffold for whole-cell catalysis with artificial metalloenzymes.

| S-EPMC6503580 | biostudies-literature

Imaging of carbonic anhydrase IX with an 111In-labeled dual-motif inhibitor.

Project description:We developed a new scaffold for radionuclide-based imaging and therapy of clear cell renal cell carcinoma (ccRCC) targeting carbonic anhydrase IX (CAIX). Compound XYIMSR-01, a DOTA-conjugated, bivalent, low-molecular-weight ligand, has two moieties that target two separate sites on CAIX, imparting high affinity. We synthesized [111In]XYIMSR-01 in 73.8-75.8% (n = 3) yield with specific radioactivities ranging from 118 - 1,021 GBq/?mol (3,200-27,600 Ci/mmol). Single photon emission computed tomography of [111In]XYIMSR-01 in immunocompromised mice bearing CAIX-expressing SK-RC-52 tumors revealed radiotracer uptake in tumor as early as 1 h post-injection. Biodistribution studies demonstrated 26% injected dose per gram of radioactivity within tumor at 1 h. Tumor-to-blood, muscle and kidney ratios were 178.1 ± 145.4, 68.4 ± 29.0 and 1.7 ± 1.2, respectively, at 24 h post-injection. Retention of radioactivity was exclusively observed in tumors by 48 h, the latest time point evaluated. The dual targeting strategy to engage CAIX enabled specific detection of ccRCC in this xenograft model, with pharmacokinetics surpassing those of previously described radionuclide-based probes against CAIX.

| S-EPMC4741798 | biostudies-other

Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas.

Project description:Carbonic anhydrase (CA) is one of nature's fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme's sensitivity to the harsh process conditions. Using directed evolution, the properties of a β-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.

| S-EPMC4246266 | biostudies-literature

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