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Ground-state destabilization by electrostatic repulsion is not a driving force in orotidine-5′-monophosphate decarboxylase catalysis

Abstract

The origins of enzyme catalysis have been attributed to both transition-state stabilization as well as ground-state destabilization of the substrate. For the latter paradigm, the enzyme orotidine-5′-monophosphate decarboxylase (OMPDC) serves as a reference system as it contains a negatively charged residue at the active site that is thought to facilitate catalysis by exerting an electrostatic stress on the substrate carboxylate leaving group. Snapshots of how the substrate binds to the active site and interacts with the negative charge have remained elusive. Here we present crystallographic snapshots of human OMPDC in complex with the substrate, substrate analogues, transition-state analogues and product that defy the proposed ground-state destabilization by revealing that the substrate carboxylate is protonated and forms a favourable low-barrier hydrogen bond with a negatively charged residue. The catalytic prowess of OMPDC almost entirely results from the transition-state stabilization by electrostatic interactions of the enzyme with charges spread over the substrate. Our findings bear relevance for the design of (de)carboxylase catalysts.

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Fig. 1: Proposed mechanism and catalytic strategies of OMPDC.
Fig. 2: Structural snapshots of substrate binding in OMPDC.
Fig. 3: Structure of the transition-state analogue BMP bound to human OMPDC.
Fig. 4: Structure of the product UMP bound to human OMPDC and implications for the mechanism.

Data availability

The refined structural protein models and corresponding structure-factor amplitudes are deposited under PDB accession codes 7OUZ (wild-type, BMP, crystal 1), 7OTU (wild-type, BMP, crystal 2), 6ZWY (wild-type, UMP, crystal 1), 7ASQ (wild-type, UMP, crystal 2), 6ZX1 (wild-type, aza-UMP), 7OV0 (wild-type, resting state), 6ZX2 (carboxamido-UMP), 6ZX3 (thiocarboxamido-UMP), 6ZWZ (variant K314AcK, resting state), 6YWU (variant K314AcK, UMP), 6YVK (variant K314AcK, 2 min soaking with OMP, 0.71 MGy dose), 6YVL (variant K314AcK, 2 min soaking with OMP, 1.42 MGy dose), 6YVM (variant K314AcK, 2 min soaking with OMP, 2.13 MGy dose), 6YVN (variant K314AcK, 2 min soaking with OMP, 2.84 MGy dose), 6YVO (variant K314AcK, 2 min soaking with OMP, 3.55 MGy dose), 6YWT (variant K314AcK, BMP), 7OQF (variant K314AcK, 5 min soaking with OMP), 7OQI (variant K314AcK, 10 min soaking with OMP), 7OQK (variant K314AcK, 15 min soaking with OMP), 7OQM (variant K314AcK, 20 min soaking with OMP), 7OQN (variant K314AcK, 30 min soaking with OMP), 7AM9 (variant K314AcK, 2 min soaking with OMP, merged dataset), 6ZX0 (variant K314AcK, OMP), 7Q1H (variant D312N, 2 min soaking with OMP) (Supplementary Table 1). The results of the quantum chemical calculations have been deposited in the GRO.data repository at https://doi.org/10.25625/6OOHE5. All other data are available on request.

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Acknowledgements

This study was supported by the Max-Planck Society and the DFG-funded Göttingen Graduate Center for Neurosciences, Biophysics, and Molecular Biosciences GGNB. We acknowledge access to beamline P14 at DESY/EMBL. We thank M. Rudolph for providing a plasmid for recombinant expression of human OMPDC and H. Neumann for providing the AMBER suppression system for the expression of acetyllysine residues. We thank J. Chin, G. Howe and A. Pearson for discussion. We thank G. Bricogne and his team from Global Phasing Limited for discussion regarding anisotropic data processing.

Author information

Authors and Affiliations

Authors

Contributions

K.T., R.A.M. and U.D. designed and coordinated the project. S.R. expressed, purified, crystallized and enzymatically characterized proteins under the supervision of K.T. S.R. collected crystallographic datasets with support from A.C., G.B. and T.S. S.R. and F.R.v.P. refined the structures with support from A.C., G.B., T.S. and K.T. L.L.K. expressed, purified and crystallized variant Asp312Asn. S.R., F.R.v.P. and K.T. interpreted the crystallographic data. J.U. carried out the electronic structure calculations and molecular dynamics calculations. A.B. carried out molecular dynamics calculations under the supervision of J.U. J.U. and R.A.M. interpreted the calculations. M.K. and T.S. chemically synthesized the substrate and transition-state analogues under the supervision of U.D. S.R., J.U., R.K., U.D, R.A.M. and K.T. discussed the enzymatic reaction mechanism. S.R., J.U., T.S., R.A.M. and K.T. wrote the paper with input from all the other authors.

Corresponding authors

Correspondence to Ricardo A. Mata or Kai Tittmann.

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Extended data

Extended Data Fig. 1 Suggested mechanisms of OMPDC-catalysed decarboxylation of OMP.

Suggested mechanisms of OMPDC-catalysed decarboxylation of OMP. (a) protonation at 2-oxo, (b) attack of a nucleophile at C5, (c) protonation at 4-oxo, (d) electrophilic displacement, (e) protonation at C5. Scheme adapted from3. The currently accepted mechanism invoking a combination of transition-state stabilisation and ground-state destabilisation is shown in Fig. 1a of the main manuscript. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate.

Extended Data Fig. 2 Structure of human OMPDC variant Lys314Ac-Lys in complex with product UMP at a resolution of 1.10 Å.

Structure of human OMPDC variant Lys314Ac-Lys in complex with product UMP at a resolution of 1.10 Å. (a) Structure of the active site showing the bound product and interacting protein groups. UMP and mutated residue AcLys314 are highlighted in yellow color. The structural model is superposed with the corresponding 2mF-DFc electron density map at a contour level of 4σ (UMP: blue, protein residues: grey). H-bonding interactions are indicated. (b) Superposition of the active sites of human OMPDC wild-type in complex with UMP (green, this study) with variant Lys314AcLys (yellow). Shown are the bound product UMP and interacting protein groups. Note the conserved binding mode of the product and interactions with protein groups except for a flip of the Cδ-Cε bond of the Lys side-chain in the variant. (c) Superposition of the backbone of human OMPDC wild-type in complex with UMP (green, this study) with variant Lys314AcLys (yellow). The RMSD of the Cα carbons amounts to 0.15 Å. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate; UMP, uridine-5’-monophosphate; RMSD, root mean square deviation.

Extended Data Fig. 3 Structures of human OMPDC wild-type in complex with substrate analogs.

Structures of human OMPDC wild-type in complex with substrate analogs. (a) Structure of human OMPDC in complex with substrate analog 6-thio-carboxyamido-UMP showing the active site with the bound analog and interacting protein groups. The structural model of the analog is super-posed with the corresponding 2mFo-DFc electron density map at a contour level of 3.3σ. Hydrogen-bonding interactions are indicated. Note the H-bond interaction between the N7 atom of the analog with Asp312 similar to the genuine substrate (see Fig. 2a of the main manuscript). (b) Physical distortion of the C6-C7 bond of the analog relative to the base ring plane shown in grey and tilt of the thio-carboxamido plane relative to the base ring plane shown in grey. (c) Structure of human OMPDC in complex with substrate analog 6-carboxyamido-UMP showing the active site with the bound analog and interacting protein groups. The structural model of the analog is superposed with the corresponding 2mFo-DFc electron density map at a contour level of 3σ. Hydrogen-bonding interactions are indicated. Note the H-bond interaction between the N7 atom of the analog with Asp312 similar to the genuine substrate (see Fig. 2a of the main manuscript). (d) Physical distortion of the C6-C7 bond of the analog relative to the base ring plane shown in grey and tilt of the carboxamido plane relative to the base ring plane shown in grey. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase.

Extended Data Fig. 4 Dose-dependent structure analysis of human OMPDC in complex with substrate OMP.

Dose-dependent structure analysis of human OMPDC in complex with substrate OMP. Datasets of crystals of OMPDC variant Lys31AcLys soaked with OMP for 2 min were collected at beamline P14 at DESY/EMBL Hamburg depositing calibrated doses as indicated. The refined structural models of bound substrate and interacting residue Asp312 are shown for both active sites of the homodimer. The structural models are superposed with the corresponding 2mFo-DFc electron density maps at a contour level of 1σ. No evidence for a radiation-induced decarboxylation of either the substrate or side chain of Asp312 was obtained. Refinements were carried out in space group P21 (containing a functional homodimer in the asymmetric unit) for all datasets. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate.

Extended Data Fig. 5 Time-resolved structural snapshots of OMPDC-catalysed conversion of substrate OMP into product UMP.

Time-resolved structural snapshots of OMPDC-catalysed conversion of substrate OMP into product UMP. Crystals of human OMPDC variant Lys314AcLys were soaked with substrate OMP for different reaction times as indicated ranging from 2–30 min at 6 °C. The refined structural models of bound substrate and interacting residue Asp312 are shown for both active sites of the homodimer. The structural models are superposed with the corresponding 2mFo-DFc electron density maps at a contour level of 1σ (in blue). The refined occupancies of substrate OMP are shown. The difference electron density relative to the dataset obtained at 2 min is shown for all later datasets at a contour level of 3σ in red colour indicating the progressive decarboxylation of substrate OMP over time. Refinements were carried out in space group P21 (containing a functional homodimer in the asymmetric unit) for all datasets. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate; UMP, uridine-5’-monophosphate.

Extended Data Fig. 6 Detection of cooperativity in human OMPDC catalysis.

Detection of cooperativity in human OMPDC catalysis. (a) Steady-state kinetic analysis of OMPDC-catalysed conversion of OMP into UMP by isothermal titration calorimetry showing the raw data thermogram after a single injection of 0.2 mM OMP to a solution containing 1 µM OMPDC in 20 mM HEPES/NaOH, pH 7.4 at 25 °C and the therefrom calculated v_S plot. The data were fitted with the Hill equation (fit shown in red). Note the estimated Hill coefficient of nH = 2.0 that indicates positive cooperativity between the two active sites in the homodimeric enzyme. All measurements were carried out in triplicate and are shown as mean ± s.d. (b) Thermodynamic analysis of binding of product UMP to human OMPDC using isothermal titration calorimetry showing the raw data thermogram and the integrated heats. The data were fit with a two-site binding model as detailed in the methods section (fit shown in red). The observation of two binding sites suggests a negative cooperativity between the two active sites in the homodimeric enzyme. All experiments were carried out as triplicates with almost identical results. The fitted kinetic and thermodynamic constants along with the associated calculated standard deviation are shown for a representative experiment. (c) Putative communication wires in human OMPCase that link the two remote active sites of the homodimer. Shown is the structure of human OMPDC variant Lys314AcLys in complex with substrate OMP highlighting the two active sites with the bound substrate molecules and two potential signaling pathways involving Asp317*-W1-W2-W3-W4-W5-Asp317 and/or His283-Glu311-Asp285 from both subunits and several water molecules. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate; UMP, uridine-5’-monophosphate.

Extended Data Fig. 7 Structure of human OMPDC in complex with transition-state analog BMP.

Structure of human OMPDC in complex with transition-state analog BMP. Close-up of the active site showing the local interactions of residue Asp317’. The structural model is superposed with the corresponding 2mFo-DFc electron density map (blue, contour level 5.3σ). Peaks in the H-omit mFo-DFc difference electron density map (magenta, contour level 3σ) indicate the positions of hydrogen atoms of the analog and interacting protein groups. The structural data suggest that the side chain Asp317’ is ionized and interacts with Lys314 (-NH3+), the backbone amide of Ile318’ and the 2’-OH group of BMP. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; BMP, 6-hydroxy-UMP.

Extended Data Fig. 8 Structure of human OMPDC in complex with transition-state analog 6-aza-UMP.

Structure of human OMPDC in complex with transition-state analog 6-aza-UMP. (a) Close-up of the active site showing the bound analog, interacting protein groups and water molecules (W). Hydrogen bonds and the bond lengths of the C2-O2 and C4-O4 bonds are indicated. Note the syn-conformation of the base that places the 2-oxo group into the vicinity of the catalytic tetrad rather than Gln430 as observed for all other ligands, which bind in the anti-conformation (see panel b). The structural models are superposed with the corresponding 2mFo-DFc electron density maps at a contour level of 5.3σ (in blue). Crystallographic statistics are provided in Supplementary Table 1. (b) Structural superposition of OMPDC in complex with 6-aza-UMP (in yellow) and UMP (in grey) showing the active site including the bound ligand and selected active site residues. Note the different orientation of the the 2-oxo function for both ligands. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; UMP, uridine-5’-monophosphate.

Extended Data Fig. 9 Functional and structural analysis of human OMPDC variant Asp312Asn.

Functional and structural analysis of human OMPDC variant Asp312Asn. The variant exhibits no measurable enzymatic activity for conversion of OMP. (a) Thermodynamic analysis of binding of product UMP to variant Asp312Asn using isothermal titration calorimetry showing the raw data thermogram and the integrated heats (inset). The data were fit with a 1:1 binding model and yielded a dissociation constant of KDapp = 49 ± 6 µM and a stoichiometry N of 0.59 ± 0.01 indicative for half-of-the-sites reactivity. (b) Thermodynamic analysis of binding of substrate OMP to variant Asp312Asn using isothermal titration calorimetry showing the raw date thermogram and the integrated heats (inset). The data were fit with a two-sites binding model and yielded dissociation constants of KD1 = 0.58 ± 0.56 µM KD2 = 0.19 ± 0.09 µM indicative for positive cooperativity. (c) Structure of variant Asp312Asn in complex with substrate showing the active site with bound OMP and interacting residues. The structural model of OMP is superposed with the 2mFo-DFc electron density map (in blue) at a contour level of 2σ. Hydogen-bond interactions of OMP with active-site residues and relative occupancies for residues with alternative conformations are indicated. Note that the carboxylate portion of OMP is interacting with both Lys281 as well as Lys314 forming a stable (anticatalytic) enzyme: substrate complex as shown in the accompanying scheme. All experiments were carried out as triplicates with almost identical results. The fitted kinetic and thermodynamic constants along with the associated calculated standard deviation are shown for a representative experiment. Crystallographic statistics are provided in Supplementary Table 1. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate; UMP, uridine-5’-monophosphate.

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Rindfleisch, S., Krull, M., Uranga, J. et al. Ground-state destabilization by electrostatic repulsion is not a driving force in orotidine-5′-monophosphate decarboxylase catalysis. Nat Catal 5, 332–341 (2022). https://doi.org/10.1038/s41929-022-00771-w

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