OMP Decarboxylase Research Articles

In Silico Approach Triterpene Glycoside of H. atra Targeting Orotidine 5-Monophosphate Decarboxylase Protein (PfOMPDC) in P. falciparum Infection Mechanism.

This study accessed the potential antimalarial activity of triterpene glycoside of H. atra through targeting orotidine 5-monophosphate decarboxylase protein (PfOMPDC) in P. falciparum by molecular docking. Nine triterpene glycosides from H. atra extract modeled the structure by the Corina web server and interacted with PfOMPDC protein by using Hex 8.0.0. The docking results were visualized and analyzed by Discovery Studio version 21.1.1. 17-Hydroxyfuscocineroside B showed the lowest binding energy in PfOMPDC interaction, which was -1,098.13 kJ/mol. Holothurin A3, echinoside A, and fuscocineroside C showed low binding energy. Nine triterpene glycosides of H. atra performed interaction with PfOMPDC protein at the same region. Holothurin A1 posed interaction with PfOMPDC protein by 8 hydrogen bonds, 3 hydrophobic interactions, and 8 unfavorable bonds. Several residues were detected in the same active sites of other triterpene glycosides. Residue TYR111 was identified in all triterpene glycoside complexes, except holothurin A3 and calcigeroside B. In summary, the triterpene glycoside of H. atra is potentially a drug candidate for malaria therapeutic agents. In vitro and in vivo studies were required for further investigation.

Expression and Enzyme Kinetics of Fused Plasmodium falciparum Orotate Phosphoribosyltransferase and Orotidine 5′-monophosphate Decarboxylase in Different Escherichia Coli

Background: Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inverse order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5′-monophosphate decarboxylase (OMPDC), as OPRT-OMPDC, has been described in many organisms. Objective: The study aimed to select the optimum host cell and temperature for expressing the recombinant fusion OMPDC-OPRT having the enzymatic activity. Methods: We constructed gene fusions of the human malaria parasite Plasmodium falciparum OMPDC-OPRT (1,836 bp) in the pTrcHisA vector and expressed it as a 6xHis-tag bifunctional protein in three Escherichia coli strains (BL21(DE3), TOP10, Rosetta) at 18°C and 25°C. The recombinant bifunctional protein was partially purified by Ni-nitrilotriacetic acid affinity chromatography and confirmed via Western blot and LC-MS/MS. The enzyme kinetics of OPRT and OMPDC was assessed. Results: Specific enzymatic activities of both OPRT and OMPDC domains expressed in E. coli BL21(DE3) cells were approximately eight-to-nine-fold higher than those in the TOP10 cells at 18°C. However, the specific activities of both domains expressed in the TOP10 cells were twice higher than those of the BL21(DE3) cells at 25°C. Very low and no enzymatic activities were observed when the constructed vector was expressed in the Rosetta cells at both induction temperatures. The bifunctional enzyme had specific activities of the OPRT and OMPDC domains in a ratio of 1:2. Kinetic study values of the OPRT domain in the bifunctional OMPDC-OPRT enzyme were found to be relatively low at µM level and at the perfect catalytic efficiency (kcat/Km). Conclusion: The recombinant fusion of OMPDC-OPRT exhibited a high expression level of E. coli BL21(DE3) at 18°C. The kinetic parameter is greater than 108M-1s-1.

Aldol Cleavage in Water and the Power of Citrate Lyase as a Catalyst.

Citrate lyase allows Klebsiella aerogenes to grow anaerobically on citrate as the sole carbon source. Arrhenius analysis of experiments at high temperatures indicates that citrate is cleaved nonenzymatically to acetate and oxaloacetate with a t1/2 of 6.9 million years in neutral solution at 25 °C, while malate cleavage occurs even more slowly (t1/2 = 280 million years). However, t1/2 is only 10 days for the nonenzymatic cleavage of 4-hydroxy-2-ketoglutarate, indicating that the introduction of an α-keto group enhances the rate of aldol cleavage of malate by a factor of 1010. The aldol cleavages of citrate and malate, like the decarboxylation of malonate (t1/2 = 180 years), are associated with a near-zero entropy of activation, and their extreme differences in rate reflect differences between their heats of activation. Citrate lyase enhances the rate of substrate cleavage 6 × 1015-fold, comparable in magnitude with the rate enhancement produced by OMP decarboxylase, although these enzymes are strikingly different in their mechanisms of action.

Studi Penambatan Maya dan Prediksi Toksisitas Turunan 5-Fluorouridin Monofosfat Pada Enzim Orotidin-5-monofosfat Dekarboksilase

Cancer is one of the leading causes of death in the world. Various cancer treatments had undergone many developments, one of which is through the process of drug discovery of cancer drugs. 5-Fluorouridine Monophosphate is a potential active metabolite of 5-fluorouracil and the ligand for the enzyme Orotidine-5-monophosphate decarboxylase (OMPD), so it deserves further development through structural modification. This study aims to predict the cytotoxic activity and toxicity of the parent compound 5-Fluorouridine Monophosphate and twenty derivative compounds as candidates for anticancer drugs. The mechanism of action of 5-Fluorouridine Monophosphate derivatives as anticancer by inhibiting OMPD, a carboxylation enzyme that is important in nucleotide biosynthesis in the process of cellular replication. Biological activity was predicted through molecular modeling called in silico assay using the Molegro Virtual Docker (MVD) program, while compound toxicity was predicted using the pkCSM online tools program. The in silico test is carried out by docking the compound whose activity will be predicted against the OMPD receptor target with the code PDB. 2QCF. The results of docking in the form of bond energy are indicated by the value of the Rerank Score (RS). Compounds that have a small RS value are predicted to have more stable ligand-receptor binding and have high activity. Based on the results of the in silico test, it can be concluded that all derivatives of 5-Fluorouridine Monophosphate meet the requirements of Lipinski's law. Then the results of molecular docking showed that the RS value of the derivative compound number 16 was lower than the parent compound 5-Fluorouridine Monophosphate, but had more hydrogen bonds with amino acid residues than the parent compound. Therefore, it can be concluded that the 16th derivative with the formula C16H25F2N2O7P is the most feasible to be synthesized and continued by testing its activity in vitro and in vivo.

Protein-Ribofuranosyl Interactions Activate Orotidine 5'-Monophosphate Decarboxylase for Catalysis.

The role of a global, substrate-driven, enzyme conformational change in enabling the extraordinarily large rate acceleration for orotidine 5'-monophosphate decarboxylase (OMPDC)-catalyzed decarboxylation of orotidine 5'-monophosphate (OMP) is examined in experiments that focus on the interactions between OMPDC and the ribosyl hydroxyl groups of OMP. The D37 and T100' side chains of OMPDC interact, respectively, with the C-3' and C-2' hydroxyl groups of enzyme-bound OMP. D37G and T100'A substitutions result in 1.4 kcal/mol increases in the activation barrier ΔG⧧ for catalysis of decarboxylation of the phosphodianion-truncated substrate 1-(β-d-erythrofuranosyl)orotic acid (EO) but result in larger 2.1-2.9 kcal/mol increases in ΔG⧧ for decarboxylation of OMP and for phosphite dianion-activated decarboxylation of EO. This shows that these substitutions reduce transition-state stabilization by the Q215, Y217, and R235 side chains at the dianion binding site. The D37G and T100'A substitutions result in <1.0 kcal/mol increases in ΔG⧧ for activation of OMPDC-catalyzed decarboxylation of the phosphoribofuranosyl-truncated substrate FO by phosphite dianions. Experiments to probe the effect of D37 and T100' substitutions on the kinetic parameters for d-glycerol 3-phosphate and d-erythritol 4-phosphate activators of OMPDC-catalyzed decarboxylation of FO show that ΔG⧧ for sugar phosphate-activated reactions is increased by ca. 2.5 kcal/mol for each -OH interaction eliminated by D37G or T100'A substitutions. We conclude that the interactions between the D37 and T100' side chains and ribosyl or ribosyl-like hydroxyl groups are utilized to activate OMPDC for catalysis of decarboxylation of OMP, EO, and FO.

Linear Free Energy Relationships for Enzymatic Reactions: Fresh Insight from a Venerable Probe.

ConspectusLinear free energy relationships (LFERs) for substituent effects on reactions that proceed through similar transition states provide insight into transition state structures. A classical approach to the analysis of LFERs showed that differences in the slopes of Brønsted correlations for addition of substituted alkyl alcohols to ring-substituted 1-phenylethyl carbocations and to the β-galactopyranosyl carbocation intermediate of reactions catalyzed by β-galactosidase provide evidence that the enzyme catalyst modifies the curvature of the energy surface at the saddle point for the transition state for nucleophile addition. We have worked to generalize the use of LFERs in the determination of enzyme mechanisms. The defining property of enzyme catalysts is their specificity for binding the transition state with a much higher affinity than the substrate. Triosephosphate isomerase (TIM), orotidine 5′-monophosphate decarboxylase (OMPDC), and glycerol 3-phosphate dehydrogenase (GPDH) show effective catalysis of reactions of phosphorylated substrates and strong phosphite dianion activation of reactions of phosphodianion truncated substrates, with rate constants kcat/Km (M–1 s–1) and kcat/KdKHPi (M–2 s–1), respectively. Good linear logarithmic correlations, with a slope of 1.1, between these kinetic parameters determined for reactions catalyzed by five or more variant forms of each catalyst are observed, where the protein substitutions are mainly at side chains which function to stabilize the cage complex between the enzyme and substrate. This shows that the enzyme-catalyzed reactions of a whole substrate and substrate pieces proceed through transition states of similar structures. It provides support for the proposal that the dianion binding energy of whole phosphodianion substrates and of phosphite dianion is used to drive the conversion of these protein catalysts from flexible and entropically rich ground states to stiff and catalytically active Michaelis complexes that show the same activity toward catalysis of the reactions of whole and phosphodianion truncated substrates. There is a good linear correlation, with a slope of 0.73, between values of the dissociation constants log Ki for release of the transition state analog phosphoglycolate (PGA) trianion and log kcat/Km for isomerization of GAP for wild-type and variants of TIM. This correlation shows that the substituted amino acid side chains act to stabilize the complex between TIM and the PGA trianion and that ca. 70% of this stabilization is observed at the transition state for substrate deprotonation. The correlation provides evidence that these side chains function to enhance the basicity of the E165 side chain of TIM, which deprotonates the bound carbon acid substrate. There is a good linear correlation, with a slope of 0.74, between the values of ΔG‡ and ΔG° determined by electron valence bond (EVB) calculations to model deprotonation of dihydroxyacetone phosphate (DHAP) in water and when bound to wild-type and variant forms of TIM to form the enediolate reaction intermediate. This correlation provides evidence that the stabilizing interactions of the transition state for TIM-catalyzed deprotonation of DHAP are optimized by placement of amino acid side chains in positions that provide for the maximum stabilization of the charged reaction intermediate, relative to the neutral substrate.

Inhibitory Effect of Active Substances of Lollyfish (Holothuria atra) Against the Development of Plasmodium falciparum Based on In Silico Study

The high level of artemisinin resistance as the antimalarial drug makes the active substances found of lollyfish (Holothuria atra) become a very useful discovery as a new antimalarial drug. The purpose of this research is to find out the inhibitory effect of the active substances of lollyfish against the development of Plasmodium falciparum with in silico method. This is a one-shot experimental study research. Based on the test of potentially active substances of lollyfish through PubChem (https://pubchem.ncbi.nlm.nih.gov/), there are pyrogallol and catechin that have potential as the antimalarial drug. Pyrogallol, chlorogenic acid, catechin dan ascorbic acid have indirect inhibition to P. falciparum Orotidine 5-Monophosphate Decarboxylase (PfOMPDC) through carbon dioxide (CO2) and it is visualized by STITCH DB Version 5.0 (http://stitch.embl.de/). The binding affinity score of catechin, obtained from molecular docking, is higher than other substances and artemisinin. The Physicochemical and pharmacokinetic activity of the substance was predicted through SWISS ADME (http://www.swissadme.ch/index.php), while the toxicity was predicted through Pro-Tox (http://tox.charite.de/protox_II/). Catechin is a substance in lollyfish that is the safest because its lowest toxicity and very effective to be used as the antimalarial drug because of its high lethal dose 50 (LD50). Therefore, active substances in lollyfish have inhibitory effects against the development of P. falciparum based on in silico study.

An efficient transformation system for Trichoderma atroviride using the pyr4 gene as a selectable marker.

The development of an efficient transformation system is essential to enrich the genetic understanding of Trichoderma atroviride. To acquire an additional homologous selectable marker, uracil auxotrophic mutants were generated. First, the pyr4 gene encoding OMP decarboxylase was replaced by the hph marker gene, encoding a hygromycin phosphotransferase. Then, uracil auxotrophs were employed to determine that 5mM uracil restores their growth and conidia production, and 1mgml-1 is the lethal dose of 5-fluoroorotic acid in T. atroviride. Subsequently, uracil auxotrophic strains, free of a drug-selectable marker, were selected by 5-fluoroorotic acid resistance. Two different deletions in pyr4 were mapped in four auxotrophs, encoding a protein with frameshifts at the 310 and 335 amino acids in their COOH-terminal. Six auxotrophs did not have changes in the pyr4 ORF even though a specific cassette to delete the pyr4 was used, suggesting that 5-FOA could have mutagenic activity. The Ura-1 strain was selected as a genetic background to knock out the MAPKK Pbs2, MAPK Tmk3, and the blue light receptors Blr1/Blr2, using a short version of pyr4 as a homologous marker. The ∆tmk3 and ∆pbs2 mutants selected with pyr4 or hph marker were phenotypically identical, highly sensitive to different stressors, and affected in photoconidiation. The ∆blr1 and ∆blr2 mutants were not responsive to light, and complementation of uracil biosynthesis did not interfere in the expression of blu1, grg2, phr1, and env1 genes upregulated by blue light. Overall, uracil metabolism can be used as a tool for genetic manipulation in T. atroviride.

Orotidine 5'-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits.

The D37 and T100′ side chains of orotidine 5′-monophosphate decarboxylase (OMPDC) interact with the C-3′ and C-2′ ribosyl hydroxyl groups, respectively, of the bound substrate. We compare the intra-subunit interactions of D37 with the inter-subunit interactions of T100′ by determining the effects of the D37G, D37A, T100′G, and T100′A substitutions on the following: (a) kcat and kcat/Km values for the OMPDC-catalyzed decarboxylations of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP) and (b) the stability of dimeric OMPDC relative to the monomer. The D37G and T100′A substitutions resulted in 2 kcal mol–1 increases in ΔG† for kcat/Km for the decarboxylation of OMP, while the D37A and T100′G substitutions resulted in larger 4 and 5 kcal mol–1 increases, respectively, in ΔG†. The D37G and T100′A substitutions both resulted in smaller 2 kcal mol–1 decreases in ΔG† for the decarboxylation of FOMP compared to that of OMP. These results show that the D37G and T100′A substitutions affect the barrier to the chemical decarboxylation step while the D37A and T100′G substitutions also affect the barrier to a slow, ligand-driven enzyme conformational change. Substrate binding induces the movement of an α-helix (G′98–S′106) toward the substrate C-2′ ribosyl hydroxy bound at the main subunit. The T100′G substitution destabilizes the enzyme dimer by 3.5 kcal mol–1 compared to the monomer, which is consistent with the known destabilization of α-helices by the internal Gly side chains [Serrano, L., et al. (1992) Nature, 356, 453–455]. We propose that the T100′G substitution weakens the α-helical contacts at the dimer interface, which results in a decrease in the dimer stability and an increase in the barrier to the ligand-driven conformational change.

Orotidine 5'-Monophosphate Decarboxylase: Probing the Limits of the Possible for Enzyme Catalysis.

ConspectusThe mystery associated with catalysis by what were once regarded as protein black boxes, diminished with the X-ray crystallographic determination of the three-dimensional structures of enzyme–substrate complexes. The report that several high-resolution X-ray crystal structures of orotidine 5′-monophosphate decarboxylase (OMPDC) failed to provide a consensus mechanism for enzyme-catalyzed decarboxylation of OMP to form uridine 5′-monophosphate, therefore, provoked a flurry of controversy. This controversy was fueled by the enormous 1023-fold rate acceleration for this enzyme, which had “jolted many biochemists’ assumptions about the catalytic potential of enzymes.” Our studies on the mechanism of action of OMPDC provide strong evidence that catalysis by this enzyme is not fundamentally different from less proficient catalysts, while highlighting important architectural elements that enable a peak level of performance. Many enzymes undergo substrate-induced protein conformational changes that trap their substrates in solvent occluded protein cages, but the conformational change induced by ligand binding to OMPDC is incredibly complex, as required to enable the development of 22 kcal/mol of stabilizing binding interactions with the phosphodianion and ribosyl substrate fragments of OMP. The binding energy from these fragments is utilized to activate OMPDC for catalysis of decarboxylation at the orotate fragment of OMP, through the creation of a tight, catalytically active, protein cage from the floppy, open, unliganded form of OMPDC. Such utilization of binding energy for ligand-driven conformational changes provides a general mechanism to obtain specificity in transition state binding. The rate enhancement that results from the binding of carbon acid substrates to enzymes is partly due to a reduction in the carbon acid pKa that is associated with ligand binding. The binding of UMP to OMPDC results in an unusually large >12 unit decrease in the pKa = 29 for abstraction of the C-6 substrate hydrogen, due to stabilization of an enzyme-bound vinyl carbanion, which is also an intermediate of OMPDC-catalyzed decarboxylation. The protein–ligand interactions operate to stabilize the vinyl carbanion at the enzyme active site compared to aqueous solution, rather than to stabilize the transition state for the concerted electrophilic displacement of CO2 by H+ that avoids formation of this reaction intermediate. There is evidence that OMPDC induces strain into the bound substrate. The interaction between the amide side chain of Gln-215 from the phosphodianion gripper loop and the hydroxymethylene side chain of Ser-154 from the pyrimidine umbrella of ScOMPDC position the amide side chain to interact with the phosphodianion of OMP. There are no direct stabilizing interactions between dianion gripper protein side chains Gln-215, Tyr-217, and Arg-235 and the pyrimidine ring at the decarboxylation transition state. Rather these side chains function solely to hold OMPDC in the catalytically active closed conformation. The hydrophobic side chains that line the active site of OMPDC in the region of the departing CO2 product may function to stabilize the decarboxylation transition state by providing hydrophobic solvation of this product.

Enzyme Architecture: Erection of Active Orotidine 5′-Monophosphate Decarboxylase by Substrate-Induced Conformational Changes

Orotidine 5′-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with kcat/Km = 1.4 × 10–7 M–1 s–1. Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induced enzyme conformational change.

Bifunctional activity of fused Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5′-monophosphate decarboxylase

Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having a COOH-terminal orotate phosphoribosyltransferase (OPRT) and an NH2-terminal orotidine 5′-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT and expressed the bifunctional protein in Escherichia coli. The enzyme was purified to homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The activities, although unstable, were stabilized by its substrate and product during purification and long-term storage. Furthermore, the enzyme expressed a perfect catalytic efficiency (kcat/Km). The kcat was selectively enhanced up to three orders of magnitude, while the Km was not much affected and remained at low μM levels when compared to the monofunctional enzymes. The fusion of the two enzymes, creating a “super-enzyme” with perfect catalytic power and more flexibility, reflects cryptic relationship of enzymatic reactivities and metabolic functions on molecular evolution.

Primary Deuterium Kinetic Isotope Effects From Product Yields: Rationale, Implementation, and Interpretation.

A simple and convenient method is described to determine primary deuterium kinetic isotope effects (1°DKIEs) on reactions where the hydron incorporated into the reaction product is derived from solvent water. The 1°DKIE may be obtained by 1H NMR analyses as the ratio of the yields of H- and D-labeled products from a reaction in 50:50 (v/v) HOH/DOD. The procedures for these 1H NMR analyses are reviewed. This product deuterium isotope effect (PDIE) is defined as 1/ϕEL for fractionation of hydrons between solvent and the transition state for the reaction examined. When the solvent is not the direct hydron donor, it is necessary to correct the PDIE for the fractionation factor ϕEL for partitioning of the hydron between the solvent and the direct donor EL. This method was used to determine the 1°DKIE on decarboxylation reactions catalyzed by wild-type orotidine 5'-monophosphate decarboxylase (OMPDC) and by mutants of OMPDC, and then in the determination of the 1°DKIE on the decarboxylation reaction catalyzed by 5-carboxyvanillate decarboxylase. The experimental procedures used in studies on OMPDC and the rationale for these procedures are described.

Nonreplicating, cyst-defective type II Toxoplasma gondii vaccine strains stimulate protective immunity against acute and chronic infection.

Live attenuated vaccine strains, such as type I nonreplicating uracil auxotroph mutants, are highly effective in eliciting lifelong immunity to virulent acute infection by Toxoplasma gondii. However, it is currently unknown whether vaccine-elicited immunity can provide protection against acute infection and also prevent chronic infection. To address this problem, we developed nonreverting, nonreplicating, live attenuated uracil auxotroph vaccine strains in the type II Δku80 genetic background by targeting the deletion of the orotidine 5'-monophosphate decarboxylase (OMPDC) and uridine phosphorylase (UP) genes. Deletion of OMPDC induced a severe uracil auxotrophy with loss of replication, loss of virulence in mice, and loss of the ability to develop cysts and chronic infection. Vaccination of mice using type II Δku80 Δompdc mutants stimulated a fully protective CD8(+) T cell-dependent immunity that prevented acute infection by type I and type II strains of T. gondii, and this vaccination also severely reduced or prevented cyst formation after type II challenge infection. Nonreverting, nonreplicating, and non-cyst-forming Δompdc mutants provide new tools to examine protective immune responses elicited by vaccination with a live attenuated type II vaccine.

Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution

Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5'-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼10(17)) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate ((HO)S) was used to generate the ester backbone substitution (S127(HO)S). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼10(2)) to the 10(17) rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.

Enzyme architecture: deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5'-monophosphate decarboxylase.

The mechanism for activation of orotidine 5′-monophosphate decarboxylase (OMPDC) by interactions of side chains from Gln215 and Try217 at a gripper loop and R235, adjacent to this loop, with the phosphodianion of OMP was probed by determining the kinetic parameters kcat and Km for all combinations of single, double, and triple Q215A, Y217F, and R235A mutations. The 12 kcal/mol intrinsic binding energy of the phosphodianion is shown to be equal to the sum of the binding energies of the side chains of R235 (6 kcal/mol), Q215 (2 kcal/mol), Y217 (2 kcal/mol), and hydrogen bonds to the G234 and R235 backbone amides (2 kcal/mol). Analysis of a triple mutant cube shows small (ca. 1 kcal/mol) interactions between phosphodianion gripper side chains, which are consistent with steric crowding of the side chains around the phosphodianion at wild-type OMPDC. These mutations result in the same change in the activation barrier to the OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces (1-β-d-erythrofuranosyl)orotic acid (EO) and phosphite dianion. This shows that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The 12 kcal/mol intrinsic phosphodianion binding energy of OMP is divided between the 8 kcal/mol of binding energy, which is utilized to drive a thermodynamically unfavorable conformational change of the free enzyme, resulting in an increase in (kcat)obs for OMPDC-catalyzed decarboxylation of OMP, and the 4 kcal/mol of binding energy, which is utilized to stabilize the Michaelis complex, resulting in a decrease in (Km)obs.

Pyrimidine nucleotide synthesis in Pseudomonas nitroreducens and the regulatory role of pyrimidines

Control of pyrimidine biosynthesis in the commercially important, hydrocarbon-utilizing bacterium Pseudomonas nitroreducens ATCC 33634 was investigated. When glucose-grown wild-type cells were supplemented with uracil or orotic acid, the pyrimidine biosynthetic activities were depressed. Pyrimidine limitation of glucose-grown cells of an orotate phosphoribosyltransferase mutant caused aspartate transcarbamoylase and dihydroorotase activities to increase by about 4-fold while the other enzyme activities about doubled. In succinate-grown phosphoribosyltransferase mutant cells subjected to pyrimidine limitation, transcarbamoylase and dehydrogenase activities rose by about 5-fold while dihydroorotase activity more than tripled. In an OMP decarboxylase mutant, pyrimidine limitation of glucose-grown cells increased transcarbamoylase, dihydroorotase, dehydrogenase and phosphoribosyltransferase activities by 4-, 10-, 6- and 3.8-fold, respectively. Pyrimidine limitation of the succinate-grown decarboxylase mutant cells increased aspartate transcarbamoylase or dihydroorotase by more than 4-fold and the other activities by about 2-fold. Pyrimidine biosynthetic enzyme synthesis appeared to be regulated by pyrimidines with the regulation being influenced by the carbon source present. Aspartate transcarbamoylase activity in Ps. nitroreducens was regulated at the level of enzyme activity since the enzyme was strongly inhibited by UDP, pyrophosphate, ATP and ADP. Overall, the regulation of pyrimidine biosynthesis in Ps. nitroreducens can be used to differentiate it from other taxonomically related species of Pseudomonas.

Study of orotidine 5′-monophosphate decarboxylase in complex with the top three OMP, BMP, and PMP ligands by molecular dynamics simulation

Catalytic mechanism of orotidine 5′-monophosphate decarboxylase (OMPDC), one of the nature most proficient enzymes which provides large rate enhancement, has not been fully understood yet. A series of 30 ns molecular dynamics (MD) simulations were run on X-ray structure of the OMPDC from Saccharomyces cerevisiae in its free form as well as in complex with different ligands, namely 1-(5′-phospho-D-ribofuranosyl) barbituric acid (BMP), orotidine 5′-monophosphate (OMP), and 6-phosphonouridine 5′-monophosphate (PMP). The importance of this biological system is justified both by its high rate enhancement and its potential use as a target in chemotherapy. This work focuses on comparing two physicochemical states of the enzyme (protonated and deprotonated Asp91) and three ligands (substrate OMP, inhibitor, and transition state analog BMP and substrate analog PMP). Detailed analysis of the active site geometry and its interactions is properly put in context by extensive comparison with relevant experimental works. Our overall results show that in terms of hydrogen bond occupancy, electrostatic interactions, dihedral angles, active site configuration, and movement of loops, notable differences among different complexes are observed. Comparison of the results obtained from these simulations provides some detailed structural data for the complexes, the enzyme, and the ligands, as well as useful insights into the inhibition mechanism of the OMPDC enzyme. Furthermore, these simulations are applied to clarify the ambiguous mechanism of the OMPDC enzyme, and imply that the substrate destabilization and transition state stabilization contribute to the mechanism of action of the most proficient enzyme, OMPDC.

A unique insertion of low complexity amino acid sequence underlies protein-protein interaction in human malaria parasite orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase

ObjectiveTo investigate the multienzyme complex formation of human malaria parasite Plasmodium falciparum (P. falciparum) orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC), the fifth and sixth enzyme of the de novo pyrimidine biosynthetic pathway. Previously, we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric (OPRT)2(OMPDC)2 complex containing two subunits each of OPRT and OMPDC, and that the complex have catalytic kinetic advantages over the monofunctional enzyme. MethodsBoth enzymes were cloned and expressed as recombinant proteins. The protein-protein interaction in the enzyme complex was identified using bifunctional chemical cross-linker, liquid chromatography-mass spectrometric analysis and homology modeling. ResultsThe unique insertions of low complexity region at the α 2 and α 5 helices of the parasite OMPDC, characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms, was located on the OPRT-OMPDC interface. The structural models for the protein-protein interaction of the heterotetrameric (OPRT)2(OMPDC)2 multienzyme complex were proposed. ConclusionsBased on the proteomic data and structural modeling, it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction. The structural complex of the parasite enzymes, thus, represents an efficient functional kinetic advantage, which in line with co-localization principles of evolutional origin, and allosteric control in protein-protein-interactions.

The use of reaction timecourses to determine the level of minor contaminants in enzyme preparations

Enzyme mutagenesis is a commonly used tool to investigate the structure and activity of enzymes. However, even minute contamination of a weakly active mutant enzyme by a considerably more active wild-type enzyme can partially or completely obscure the activity of the mutant enzyme. In this work, we propose a theoretical approach using reaction timecourses and initial velocity measurements to determine the actual contamination level of an undesired wild-type enzyme. To test this method, we applied it to a batch of the Q215A/R235A double mutant of orotidine 5′-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae that was inadvertently contaminated by the more active wild-type OMPDC from Escherichia coli. The enzyme preparation showed significant deviations from the expected kinetic behavior at contamination levels as low as 0.093mol%. We then confirmed the origin of the unexpected kinetic behavior by deliberately contaminating a sample of the mutant OMPDC from yeast that was known to be pure, with 0.015% wild-type OMPDC from E. coli and reproducing the same hybrid kinetic behavior.