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Orotidine 5'-phosphate decarboxylase

Orotidine 5'-phosphate decarboxylase (OMP decarboxylase) or orotidylate decarboxylase is an enzyme involved in pyrimidine biosynthesis. It catalyzes the decarboxylation of orotidine monophosphate (OMP) to form uridine monophosphate (UMP). The function of this enzyme is essential to the de novo biosynthesis of the pyrimidine nucleotides uridine triphosphate, cytidine triphosphate, and thymidine triphosphate. OMP decarboxylase has been a frequent target for scientific investigation because of its demonstrated extreme catalytic efficiency and its usefulness as a selection marker for yeast strain engineering.

Orotidine-5'-phosphate decarboxylase
E. coli OMP decarboxylase.[1]
Identifiers
EC no.4.1.1.23
CAS no.9024-62-8
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
Search
PMCarticles
PubMedarticles
NCBIproteins
Schematic of reaction catalyzed by OMP decarboxylase

Catalysis edit

OMP decarboxylase is known for being an extraordinarily efficient catalyst capable of accelerating the uncatalyzed reaction rate by a factor of 1017. To put this in perspective, the uncatalysed reaction which would take 78 million years to convert half the reactants into products is accelerated to 18 milliseconds when catalyzed by OMP decarboxylase.[2] This extreme enzymatic efficiency is especially interesting because OMP decarboxylases uses no cofactor and contains no metal sites[3] or prosthetic groups.[4] The catalysis relies on a handful of charged amino acid residues positioned within the active site of the enzyme.

 
Image representing the structure of the active site of OMP decarboxylase when bound to the inhibitor BMP. Note the Lys and Asp residues surrounding the 6-hydroxyl of the substrate. (Image captured from pymol viewer snapshot of 1LOR crystal structure)[5]

The exact mechanism by which OMP decarboxylase catalyzes its reaction has been a subject of rigorous scientific investigation. The driving force for the loss of the carboxyl linked to the C6 of the pyrimidine ring comes from the close proximity of an aspartate residue carboxyl group in the enzyme's active site, which destabilizes the ground state relative to the transition state of the uncatalyzed reaction. There have been multiple hypotheses about what form the transition state takes before protonation of the C6 carbon occurs to yield the final product. Many studies investigated the binding of a potent inhibitor of OMP decarboxylase, 6-hydroxy uridine monophosphate (BMP, a barbituric acid derivative), within the active site, to identify which essential amino acid residues are directly involved with stabilization of the transition state. (See figure of enzyme bound to BMP) Several mechanisms for enzymatic decarboxylation of OMP have been proposed, including protonation at O2 to form a zwitterionic species as an intermediate,[6] anion stabilization of O4,[7] or nucleophilic attack at C5.[8] Current consensus suggests that the mechanism proceeds through a stabilized carbanion at the C6 after loss of carbon dioxide. This mechanism was suggested from studies investigating kinetic isotope effects in conjunction with competitive inhibition and active site mutagenesis.[9][10][11][12] In this mechanism the short-lived carbanion species is stabilized by a nearby lysine residue, before it is quenched by a proton. (See schematic of catalytic mechanism) The intermediacy of a highly basic vinyl carbanion not benefiting from electronic stabilization is rare in an enzymatic system and in biological systems in general. Remarkably, the enzyme microenvironment helps stabilize the carbanion considerably. The pKaH of the enzyme-bound carbanionic intermediate was measured to be less than or equal to 22 based on deuterium exchange studies. While still highly basic, the corresponding pKaH of the free carbanionic intermediate is estimated to be much higher, around 30-34 (based on measurements on the analogous 1,3-dimethyluracil), leading to the conclusion that the enzyme stabilizes the carbanion by at least 14 kcal/mol.[12]

 
Reaction schematic showing the mechanism of OMP decarboxylase catalysis through a putative vinyl carbanion at the C6 position. This carbanion is likely stabilized by the nearby protonated lysine residue. The Lys93 and Asp91 residue numbering corresponds to the sequence for OMP decarboxylase from S cerevisiae.[13]

Relation to UMP synthase edit

In yeast and bacteria, OMP decarboxylase is a single-function enzyme. However, in mammals, OMP decarboxylase is part of a single protein with two catalytic activities. This bifunctional enzyme is named UMP synthase and it also catalyzes the preceding reaction in pyrimidine nucleotide biosynthesis, the transfer of ribose 5-phosphate from 5-phosphoribosyl-1-pyrophosphate to orotate to form OMP. In organisms utilizing OMP decarboxylase, this reaction is catalyzed by orotate phosphoribosyltransferase.[14]

Importance in yeast genetics edit

Mutations in the gene encoding OMP decarboxylase in yeast (URA3) leads to auxotrophy in uracil. In addition, a function OMP decarboxylase renders yeast strains sensitive to the molecule 5-fluoroorotic acid (5-FOA).[15] The establishment of the URA3 gene as a selection marker with both positive and negative selection strategies has made the controlled expression of OMP decarboxylase a significant laboratory tool for the investigation of yeast genetics.

See also edit

References edit

  1. ^ PDB: 1EIX​; Harris P, Navarro Poulsen JC, Jensen KF, Larsen S (April 2000). "Structural basis for the catalytic mechanism of a proficient enzyme: orotidine 5'-monophosphate decarboxylase". Biochemistry. 39 (15): 4217–24. doi:10.1021/bi992952r. PMID 10757968.
  2. ^ Radzicka A, Wolfenden R (January 1995). "A proficient enzyme". Science. 267 (5194): 90–3. doi:10.1126/science.7809611. PMID 7809611.
  3. ^ Miller BG, Smiley JA, Short SA, Wolfenden R (August 1999). "Activity of yeast orotidine-5'-phosphate decarboxylase in the absence of metals". J. Biol. Chem. 274 (34): 23841–3. doi:10.1074/jbc.274.34.23841. PMID 10446147.
  4. ^ Miller BG, Wolfenden R (2002). "Catalytic proficiency: the unusual case of OMP decarboxylase". Annu. Rev. Biochem. 71: 847–85. doi:10.1146/annurev.biochem.71.110601.135446. PMID 12045113.
  5. ^ Wu N, Pai EF (August 2002). "Crystal structures of inhibitor complexes reveal an alternate binding mode in orotidine-5'-monophosphate decarboxylase". J. Biol. Chem. 277 (31): 28080–7. doi:10.1074/jbc.M202362200. PMID 12011084.
  6. ^ Beak P, Siegel B (1976). "Mechanism of decarboxylation of 1,3-dimethylorotic acid. A model for orotidine 5'-phosphate decarboxylase". J Am Chem Soc. 98 (12): 3601–6. doi:10.1021/ja00428a035. PMID 1270703.
  7. ^ Lee JK, Houk KN (May 1997). "A proficient enzyme revisited: the predicted mechanism for orotidine monophosphate decarboxylase". Science. 276 (5314): 942–5. doi:10.1126/science.276.5314.942. PMID 9139656.
  8. ^ Silverman, R.B.; Groziak, M.P. (1982). "Model Chemistry for a Covalent Mechanism of Action of Orotidine 5'-Phosphate Decarboxylase". J. Am. Chem. Soc. 104 (23): 6434–6439. doi:10.1021/ja00387a047.
  9. ^ Lee, Jeehiun K; Tantillo, Dean J (2004-06-25). Orotidine Monophosphate Decarboxylase: A Mechanistic Dialogue. ISBN 9783540205661.
  10. ^ Richavy MA, Cleland WW (2000). "Determination of the Mechanism of Orotidine 5'-Monophosphate Decarboxylase by Isotope Effects". Biochemistry. 39 (16): 4569–4574. doi:10.1021/bi000376p. PMID 10769111.
  11. ^ Toth K, Amyes TL, Wood BM, Chan K, Gerlt JA, Richard JP (October 2007). "Product Deuterium Isotope Effect for Orotidine 5'-Monophosphate Decarboxylase: Evidence for the Existence of a Short-Lived Carbanion Intermediate". J. Am. Chem. Soc. 129 (43): 12946–7. doi:10.1021/ja076222f. PMC 2483675. PMID 17918849.
  12. ^ a b Amyes TL, Wood BM, Chan K, Gerlt JA, Richard JP (February 2008). "Formation and Stability of a Vinyl Carbanion at the Active Site of Orotidine 5′-Monophosphate Decarboxylase: pKa of the C-6 Proton of Enzyme-Bound UMP". J. Am. Chem. Soc. 130 (5): 1574–5. doi:10.1021/ja710384t. PMC 2652670. PMID 18186641.
  13. ^ Van Vleet JL, Reinhardt LA, Miller BG, Sievers A, Cleland WW (January 2008). "Carbon isotope effect study on orotidine 5'-monophosphate decarboxylase: support for an anionic intermediate". Biochemistry. 47 (2): 798–803. doi:10.1021/bi701664n. PMID 18081312.
  14. ^ Yablonski MJ, Pasek DA, Han BD, Jones ME, Traut TW (1996). "Intrinsic activity and stability of bifunctional human UMP synthase and its two separate catalytic domains, orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase". J Biol Chem. 271 (18): 10704–10708. doi:10.1074/jbc.271.18.10704. PMID 8631878.
  15. ^ Boeke JD, LaCroute F, Fink GR (1984). "A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance". Mol Gen Genet. 197 (2): 345–346. doi:10.1007/BF00330984. PMID 6394957.

orotidine, phosphate, decarboxylase, decarboxylase, orotidylate, decarboxylase, enzyme, involved, pyrimidine, biosynthesis, catalyzes, decarboxylation, orotidine, monophosphate, form, uridine, monophosphate, function, this, enzyme, essential, novo, biosynthesi. Orotidine 5 phosphate decarboxylase OMP decarboxylase or orotidylate decarboxylase is an enzyme involved in pyrimidine biosynthesis It catalyzes the decarboxylation of orotidine monophosphate OMP to form uridine monophosphate UMP The function of this enzyme is essential to the de novo biosynthesis of the pyrimidine nucleotides uridine triphosphate cytidine triphosphate and thymidine triphosphate OMP decarboxylase has been a frequent target for scientific investigation because of its demonstrated extreme catalytic efficiency and its usefulness as a selection marker for yeast strain engineering Orotidine 5 phosphate decarboxylaseE coli OMP decarboxylase 1 IdentifiersEC no 4 1 1 23CAS no 9024 62 8DatabasesIntEnzIntEnz viewBRENDABRENDA entryExPASyNiceZyme viewKEGGKEGG entryMetaCycmetabolic pathwayPRIAMprofilePDB structuresRCSB PDB PDBe PDBsumGene OntologyAmiGO QuickGOSearchPMCarticlesPubMedarticlesNCBIproteins Schematic of reaction catalyzed by OMP decarboxylaseContents 1 Catalysis 2 Relation to UMP synthase 3 Importance in yeast genetics 4 See also 5 ReferencesCatalysis editOMP decarboxylase is known for being an extraordinarily efficient catalyst capable of accelerating the uncatalyzed reaction rate by a factor of 1017 To put this in perspective the uncatalysed reaction which would take 78 million years to convert half the reactants into products is accelerated to 18 milliseconds when catalyzed by OMP decarboxylase 2 This extreme enzymatic efficiency is especially interesting because OMP decarboxylases uses no cofactor and contains no metal sites 3 or prosthetic groups 4 The catalysis relies on a handful of charged amino acid residues positioned within the active site of the enzyme nbsp Image representing the structure of the active site of OMP decarboxylase when bound to the inhibitor BMP Note the Lys and Asp residues surrounding the 6 hydroxyl of the substrate Image captured from pymol viewer snapshot of 1LOR crystal structure 5 The exact mechanism by which OMP decarboxylase catalyzes its reaction has been a subject of rigorous scientific investigation The driving force for the loss of the carboxyl linked to the C6 of the pyrimidine ring comes from the close proximity of an aspartate residue carboxyl group in the enzyme s active site which destabilizes the ground state relative to the transition state of the uncatalyzed reaction There have been multiple hypotheses about what form the transition state takes before protonation of the C6 carbon occurs to yield the final product Many studies investigated the binding of a potent inhibitor of OMP decarboxylase 6 hydroxy uridine monophosphate BMP a barbituric acid derivative within the active site to identify which essential amino acid residues are directly involved with stabilization of the transition state See figure of enzyme bound to BMP Several mechanisms for enzymatic decarboxylation of OMP have been proposed including protonation at O2 to form a zwitterionic species as an intermediate 6 anion stabilization of O4 7 or nucleophilic attack at C5 8 Current consensus suggests that the mechanism proceeds through a stabilized carbanion at the C6 after loss of carbon dioxide This mechanism was suggested from studies investigating kinetic isotope effects in conjunction with competitive inhibition and active site mutagenesis 9 10 11 12 In this mechanism the short lived carbanion species is stabilized by a nearby lysine residue before it is quenched by a proton See schematic of catalytic mechanism The intermediacy of a highly basic vinyl carbanion not benefiting from electronic stabilization is rare in an enzymatic system and in biological systems in general Remarkably the enzyme microenvironment helps stabilize the carbanion considerably The pKaH of the enzyme bound carbanionic intermediate was measured to be less than or equal to 22 based on deuterium exchange studies While still highly basic the corresponding pKaH of the free carbanionic intermediate is estimated to be much higher around 30 34 based on measurements on the analogous 1 3 dimethyluracil leading to the conclusion that the enzyme stabilizes the carbanion by at least 14 kcal mol 12 nbsp Reaction schematic showing the mechanism of OMP decarboxylase catalysis through a putative vinyl carbanion at the C6 position This carbanion is likely stabilized by the nearby protonated lysine residue The Lys93 and Asp91 residue numbering corresponds to the sequence for OMP decarboxylase from S cerevisiae 13 Relation to UMP synthase editIn yeast and bacteria OMP decarboxylase is a single function enzyme However in mammals OMP decarboxylase is part of a single protein with two catalytic activities This bifunctional enzyme is named UMP synthase and it also catalyzes the preceding reaction in pyrimidine nucleotide biosynthesis the transfer of ribose 5 phosphate from 5 phosphoribosyl 1 pyrophosphate to orotate to form OMP In organisms utilizing OMP decarboxylase this reaction is catalyzed by orotate phosphoribosyltransferase 14 Importance in yeast genetics editMutations in the gene encoding OMP decarboxylase in yeast URA3 leads to auxotrophy in uracil In addition a function OMP decarboxylase renders yeast strains sensitive to the molecule 5 fluoroorotic acid 5 FOA 15 The establishment of the URA3 gene as a selection marker with both positive and negative selection strategies has made the controlled expression of OMP decarboxylase a significant laboratory tool for the investigation of yeast genetics See also editCirce effectReferences edit PDB 1EIX Harris P Navarro Poulsen JC Jensen KF Larsen S April 2000 Structural basis for the catalytic mechanism of a proficient enzyme orotidine 5 monophosphate decarboxylase Biochemistry 39 15 4217 24 doi 10 1021 bi992952r PMID 10757968 Radzicka A Wolfenden R January 1995 A proficient enzyme Science 267 5194 90 3 doi 10 1126 science 7809611 PMID 7809611 Miller BG Smiley JA Short SA Wolfenden R August 1999 Activity of yeast orotidine 5 phosphate decarboxylase in the absence of metals J Biol Chem 274 34 23841 3 doi 10 1074 jbc 274 34 23841 PMID 10446147 Miller BG Wolfenden R 2002 Catalytic proficiency the unusual case of OMP decarboxylase Annu Rev Biochem 71 847 85 doi 10 1146 annurev biochem 71 110601 135446 PMID 12045113 Wu N Pai EF August 2002 Crystal structures of inhibitor complexes reveal an alternate binding mode in orotidine 5 monophosphate decarboxylase J Biol Chem 277 31 28080 7 doi 10 1074 jbc M202362200 PMID 12011084 Beak P Siegel B 1976 Mechanism of decarboxylation of 1 3 dimethylorotic acid A model for orotidine 5 phosphate decarboxylase J Am Chem Soc 98 12 3601 6 doi 10 1021 ja00428a035 PMID 1270703 Lee JK Houk KN May 1997 A proficient enzyme revisited the predicted mechanism for orotidine monophosphate decarboxylase Science 276 5314 942 5 doi 10 1126 science 276 5314 942 PMID 9139656 Silverman R B Groziak M P 1982 Model Chemistry for a Covalent Mechanism of Action of Orotidine 5 Phosphate Decarboxylase J Am Chem Soc 104 23 6434 6439 doi 10 1021 ja00387a047 Lee Jeehiun K Tantillo Dean J 2004 06 25 Orotidine Monophosphate Decarboxylase A Mechanistic Dialogue ISBN 9783540205661 Richavy MA Cleland WW 2000 Determination of the Mechanism of Orotidine 5 Monophosphate Decarboxylase by Isotope Effects Biochemistry 39 16 4569 4574 doi 10 1021 bi000376p PMID 10769111 Toth K Amyes TL Wood BM Chan K Gerlt JA Richard JP October 2007 Product Deuterium Isotope Effect for Orotidine 5 Monophosphate Decarboxylase Evidence for the Existence of a Short Lived Carbanion Intermediate J Am Chem Soc 129 43 12946 7 doi 10 1021 ja076222f PMC 2483675 PMID 17918849 a b Amyes TL Wood BM Chan K Gerlt JA Richard JP February 2008 Formation and Stability of a Vinyl Carbanion at the Active Site of Orotidine 5 Monophosphate Decarboxylase pKa of the C 6 Proton of Enzyme Bound UMP J Am Chem Soc 130 5 1574 5 doi 10 1021 ja710384t PMC 2652670 PMID 18186641 Van Vleet JL Reinhardt LA Miller BG Sievers A Cleland WW January 2008 Carbon isotope effect study on orotidine 5 monophosphate decarboxylase support for an anionic intermediate Biochemistry 47 2 798 803 doi 10 1021 bi701664n PMID 18081312 Yablonski MJ Pasek DA Han BD Jones ME Traut TW 1996 Intrinsic activity and stability of bifunctional human UMP synthase and its two separate catalytic domains orotate phosphoribosyltransferase and orotidine 5 phosphate decarboxylase J Biol Chem 271 18 10704 10708 doi 10 1074 jbc 271 18 10704 PMID 8631878 Boeke JD LaCroute F Fink GR 1984 A positive selection for mutants lacking orotidine 5 phosphate decarboxylase activity in yeast 5 fluoro orotic acid resistance Mol Gen Genet 197 2 345 346 doi 10 1007 BF00330984 PMID 6394957 Portal nbsp Biology Retrieved from https en wikipedia org w index php title Orotidine 5 27 phosphate decarboxylase amp oldid 1172356007, wikipedia, wiki, book, books, library,

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