The spike protein S of SARS coronavirus 2 (SARS-CoV-2) binds ACE2 on host cells to initiate entry, and soluble ACE2 is a therapeutic candidate that neutralizes infection by acting as a decoy. Using deep mutagenesis, mutations in ACE2 that increase S binding are found across the interaction surface, in the N90-glycosylation motif and at buried sites. The mutational landscape provides a blueprint for understanding the specificity of the interaction between ACE2 and S and for engineering high affinity decoy receptors. Combining mutations gives ACE2 variants with affinities that rival monoclonal antibodies. A stable dimeric variant shows potent SARS-CoV-2 and -1 neutralization in vitro. The engineered receptor is catalytically active and its close similarity with the native receptor may limit the potential for viral escape.

We report that orotidine 5′-monophosphate decarboxylase (OMPDC) catalyzes exchange of the C-6 proton of uridine 5′-monophosphate (UMP) for deuterium from solvent in D 2 O at 25 °C and pD 7.0 -9.3. Kinetic analysis of deuterium exchange gives pK a ≤ 22 for carbon deprotonation of enzymebound UMP, which is at least 10 units lower than that for deprotonation of an analog of UMP in water. The observation of enzyme-catalyzed deuterium exchange via a stabilized carbanion provides convincing evidence for the decarboxylation of orotidine 5′-monophosphate (OMP) by OMPDC to give the same carbanion intermediate. The data show that yeast OMPDC stabilizes the bound vinyl carbanion by at least 14 kcal/mol. We conclude that OMPDC also provides substantial stabilization of the late carbanion-like transition state for the decarboxylation of OMP, and that this transition state stabilization constitutes a large fraction, but probably not all, of the enormous 10 17 -fold enzymatic rate acceleration.We report that orotidine 5′-monophosphate decarboxylase catalyzes exchange of the C-6 proton of uridine 5′-monophosphate (UMP) for deuterium from solvent in D 2 O at 25 °C (Scheme 1). Kinetic analysis of deuterium exchange gives pK a ≤ 22 for carbon deprotonation of enzyme-bound UMP, which is at least 10 units lower than that for deprotonation of an analog of UMP in water.Orotidine 5′-monophosphate decarboxylase (OMPDC) employs no metal ions or other cofactors but yet effects an enormous 10 17 -fold acceleration of the decarboxylation of orotidine 5′-monophosphate (OMP) to give uridine 5′-monophosphate (UMP). 1 The X-ray structure of the yeast enzyme liganded with 6-hydroxyuridine 5′-monophosphate provides strong evidence that the C-6 proton of the product UMP is derived from the terminal NH 3 + group of Lys-93. 2 The product isotope effect of unity for OMPDC-catalyzed decarboxylation of OMP in 50/50 (v/v) H 2 O/D 2 O eliminates a mechanism 3 in which proton transfer from Lys-93 to C-6 provides electrophilic push to the loss of CO 2 in a concerted reaction. (1)The values of k obsd (s −1 ) determined for enzyme-catalyzed deuterium exchange in D 2 O at pD 9.34 with [UMP] total = 2.5 -10 mM show a good fit to eq 2 that was derived for Scheme 3 (see Supporting Information), withThe data give the first-order rate constant for deuterium exchange into saturating enzyme-bound UMP at pD 9.34 as k ex = 1.15 × 10 −5 s −1 . Similar experiments using ca. 0.3 mM OMPDC (9 mg/mL) gave values of k ex (s −1 ) for the turnover of saturating UMP (2.5 -5 mM) at pD 8.13 (100 mM glycylglycine buffer), and at pD 7.58 and 7.03 (48 mM imidazole buffer), at 25 °C and I = 0.1 (NaCl). 11 in k ex (s −1 ) with increasing pD and the leveling off at pD > 8 shows that deuterium exchange is promoted by the basic form of an amino acid side chain at the active site of OMPDC. 12 We suggest that the catalytic base is the neutral form of Lys-93, 13 so that deuterium exchange arises from the reverse of the proton transfer "half reaction" that occurs in the active site ...

The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) involves a stabilized anionic intermediate, although the structural basis for the rate acceleration (k cat /k non , 7.1 × 10 16 ) and proficiency [(k cat /K M )/k non , 4.8 × 10 22 M −1 ] is uncertain. That the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC) catalyze the exchange of H6 of the UMP product with solvent deuterium allows an estimate of a lower limit on the rate acceleration associated with stabilization of the intermediate and . We investigated that hypothesis by structural and functional characterization of the D70N and D70G mutants of MtOMPDC and the D91N mutant of ScOMPDC. The substitutions for Asp 70 in MtOMPDC significantly decrease the value of k cat for decarboxylation of FOMP (a more reactive substrate analog) but have little effect on the value of k ex for exchange of H6 of FUMP with solvent deuterium; the structures of wild type MtOMPDC and its mutants are superimposable when complexed with 6-azaUMP. In contrast, the D91N mutant of ScOMPDC does not catalyze exchange of H6 of FUMP; the structures of wild type ScOMPDC and its D91N mutant are not superimposable when complexed with 6-azaUMP, with differences in both the conformation of the active site loop and the orientation of the ligand vis á vis the active site residues. We propose that the differential effects of substitutions for Asp 70 of MtOMPDC on decarboxylation and exchange provide additional evidence for a carbanionic intermediate as well as the involvement of Asp 70 in substrate destabilization. † This research was supported by NIH GM039754 (to J.P.R.) and GM065155 (to J.A.G.). Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). The X-ray coordinates and structure factors for the following structures have been deposited in the Protein Data Bank with the indicated accession codes: wild type MtOMPDC in the absence of ligands (PDB accession code 3G18), wild type MtOMPDC complexed with 6-azaUMP (3G1A), wild type MtOMPDC complexed with UMP (3G1D), wild type MtOMPDC complexed with H 2 OMP (3D1F), wild type MtOMPDC complexed with H 2 UMP (3D1H), the D70N mutant of MtOMPDC in the absence of ligands (3G1Y), the D70N mutant of MtOMPDC complexed with 6-azaUMP (3G24), the D70N mutant of MtOMPDC complexed with UMP (3G22), the D70G mutant of MtOMPDC in the absence of ligands (3G1S), the D70G mutant of MtOMPDC complexed with UMP (3G1X), the D70G mutant of MtOMPDC complexed with FOMP (3G1V), wild type ScOMPDC in the absence of ligands (3GDK), wild type ScOMPDC complexed with 6-azaUMP (3GDL), the D91N mutant of ScOMPDC in the absence of ligands (3GDR), and the D91N mutant of ScOMPDC complexed with 6-azaUMP (3GDT).

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, because of close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find that an engineered decoy receptor, sACE22.v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain followed by in vitro selection, with wild-type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild-type receptor. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.

Orotidine 5'-monophosphate decarboxylase (OMPDC) is an exceptionally proficient catalyst: the rate acceleration (k cat /k non ) is 7.1 × 10 16 and the proficiency [(k cat /K M )/k non ] is 4.8 × 10 22 M −1 . The structural basis for the large rate acceleration and proficiency is unknown, although the mechanism has been established to involve a stabilized carbanion intermediate. To provide reaction coordinate context for interpretation of the values of k cat , k cat /K M , and kinetic isotope effects, we investigated the effect of solvent viscosity on k cat and k cat /K M for the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC). For MtOMPDC, we used not only the natural OMP substrate but also a catalytically impaired mutant (D70N) and a more reactive substrate (FOMP); for ScOMPDC, we used OMP and FOMP. With MtOMPDC and OMP, k cat is independent of solvent viscosity, indicating that decarboxylation is fully rate-determining; k cat /K M displays a fractional dependence of solvent viscosity, suggesting that both substrate binding and decarboxylation determine this kinetic constant. For ScOMPDC with OMP, we observed that both k cat and k cat /K M are fractionally dependent on solvent viscosity, suggesting that the rates of substrate binding, decarboxylation, and product dissociation are similar. Consistent with these interpretations, for both enzymes with FOMP, the increases in the values of k cat and k cat /K M are much less than expected based on the ability of the 5-fluoro substituent to stabilize the anionic intermediate, i.e., substrate binding and product dissociation mask the kinetic effects of stabilization of the intermediate by the substituent.Orotidine 5'-monophosphate decarboxylase (OMPDC) that catalyzes the final step in the biosynthesis of pyrimidine nucleotides is an exceptionally proficient catalyst (Scheme 1) (2, 3). For the enzyme from Saccharomyces cerevisiae (ScOMPDC), the rate acceleration (k cat / k non ) is 7.1 × 10 16 (k cat = 20 sec −1 ; k non = 2.8 × 10 −16 sec −1 ) and the proficiency [(k cat /K M )/ † This research was supported by NIH GM039754 (to J.P.R.) and GM065155 (to J.A.G.).* To whom correspondence should be addressed: J.A.G.: Department of Biochemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, IL 61801. Phone: (217) 244−7414. Fax: (217) 244−6538. E-mail: j-gerlt@uiuc.edu.. Cleland and coworkers did not report a value for k cat /K M for wild type ScOMPDC with OMP, so the value listed in the text is the one we determined; because our value for k cat is 20 sec −1 , our value for k cat /K M should be a reliable measure of the catalytic efficiency of the enzyme used by Cleland. Our values for k cat and k cat /K M for wild type ScOMPDC with FOMP are 130 sec −1 and 2.6 × 10 7 M −1 sec −1 (vide infra).We have examined the effects of viscogens on the kinetic constants of mutants with substitutions for residues that are involved in the conformational changes that accompany ligand binding, e.g., the conserved Gln 215 in ...

Vaccine hesitancy and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) escaping vaccine-induced immune responses highlight the urgency for new COVID-19 therapeutics. Engineered angiotensin-converting enzyme 2 (ACE2) proteins with augmented binding affinities for SARS-CoV-2 spike (S) protein may prove to be especially efficacious against multiple variants. Using molecular dynamics simulations and functional assays, we show that three amino acid substitutions in an engineered soluble ACE2 protein markedly augmented the affinity for the S protein of the SARS-CoV-2 WA-1/2020 isolate and multiple VOCs: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). In humanized K18-hACE2 mice infected with the SARS-CoV-2 WA-1/2020 or P.1 variant, prophylactic and therapeutic injections of soluble ACE22.v2.4-IgG1 prevented lung vascular injury and edema formation, essential features of CoV-2-induced SARS, and above all improved survival. These studies demonstrate broad efficacy in vivo of an engineered ACE2 decoy against SARS-CoV-2 variants in mice and point to its therapeutic potential.

We report that equal yields of [6-1 H]-uridine 5′-monophosphate (50%) and [6-2 H]-uridine 5′-monophosphate (50%) are obtained from the decarboxylation of orotidine 5′-monophosphate (OMP) catalyzed by orotidine 5′-monophosphate decarboxylase in a solvent of 50/50 (v/v) H 2 O/D 2 O. This observation of an unusually small product isotope effect of unity eliminates a proposed mechanism in which proton transfer from Lys-93 1 to C-6 provides electrophilic push to the loss of CO 2 from OMP in a concerted reaction. 2,3 It provides evidence that proton transfer from the ammonium cation side chain of Lys-93 to a vinyl carbanion intermediate is faster than the bond rotation that exchanges the positions of the acidic N-L + hydrons of this side chain.Orotidine 5′-monophosphate decarboxylase (OMPDC) is a remarkable enzyme because it employs no metal ions or other cofactors but yet effects an enormous 10 17 -fold acceleration of the chemically very difficult decarboxylation of OMP to give uridine 5′-monophosphate (UMP). 4,5 It has been shown that a large fraction of the enzymatic rate acceleration results directly from utilization of the intrinsic binding energy of the remote nonreacting 5′-phosphodianion group of OMP in transition state stabilization. 6 The decarboxylation reaction is often proposed to proceed in two steps through a vinyl carbanion intermediate (Scheme 1). However, it has also been suggested that this unstable intermediate might be avoided in a concerted reaction in which decarboxylation and proton transfer to C-6 occur in a single step. 2,3 Experimental and computational studies on OMPDC have focused largely on the partly rate-determining and highly unfavorable loss of CO 2 from OMP. [7][8][9] There are few data pertaining to the proton transfer to C-6 of the pyrimidine ring. Experimental characterization of this proton-transfer step is essential for insight into the existence and lifetime of the putative enzyme-bound vinyl carbanion intermediate.OMPDC catalyzes incorporation of a hydron from solvent into the UMP product and it has been reported that the decarboxylation of saturating OMP is 30% faster in H 2 O than in D 2 O. 7 While the origin of this solvent isotope effect on k cat is unclear, it may represent a secondary solvent kinetic isotope effect (SKIE). By contrast, a product isotope effect (PIE) determined in experiments in which H and D in a mixed solvent of H 2 O/D 2 O compete for reaction with enzyme-bound OMP to form UMP labeled at C-6 (Scheme 2) would provide insight into the changes in bonding at the transferred hydron that occur on proceeding to the transition state for the product-determining step. 10 PIEs are more precise and easier to interpret than SKIEs determined as the ratio of rate constants for reactions in H 2 O and D 2 O because (1) there are no complications from any secondary SKIE when the H-and D-labeled products are formed in the same mixed H 2 O/D 2 O solvent and (2) there are no errors due to differences in the conditions for separate reactions in H 2 O and D 2 O, such as enzy...

Closure of the active site phosphate gripper loop of orotidine 5′-monophosphate decarboxylase from Saccharomyces cerevisiae (ScOMPDC) over the bound substrate orotidine 5′-monophosphate (OMP) activates the bound substrate for decarboxylation by at least 104-fold [Amyes, T. L., Richard, J. P., and Tait, J. J. (2005) J. Am. Chem. Soc. 127, 15708-15709]. The 19 residue phosphate gripper loop of the mesophilic ScOMPDC is much larger than the 9 residue loop at the ortholog from the thermophile Methanothermobacter thermautotrophicus (MtOMPDC). This difference in loop size results in a small decrease in the total intrinsic phosphate binding energy of the phosphodianion group of OMP from 11.9 to 11.6 kcal/mol, along with a modest decrease in the extent of activation by phosphite dianion of decarboxylation of the truncated substrate 1-(β-D-erythrofuranosyl)orotic acid. The activation parameters ΔH‡ and ΔS‡ for kcat for decarboxylation of OMP are 3.6 kcal/mol and 10 cal/K/mol more positive, respectively, for MtOMPDC than for ScOMPDC. We suggest that these differences are related to the difference in size of the active site loops at the mesophilic ScOMPDC and the thermophilic MtOMPDC. The greater enthalpic transition state stabilization available from the more extensive loop-substrate interactions for the ScOMPDC-catalyzed reaction is largely balanced by a larger entropic requirement for immobilization of the larger loop at this enzyme.