PDBsum entry 2zz1

Lyase

PDB id: 2zz1

Contents

Protein chains

215 a.a. *

Ligands

BMP ×2

6CN ×2

GOL ×4

Waters ×287

* Residue conservation analysis

PDB id:

2zz1

Name:

Lyase

Title:

Snapshot of the reaction from 6-cn-ump to bmp catalyzed by orotidine monophosphate deacarboxylase from m. Thermoautotrophicum

Structure:

Orotidine 5'-phosphate decarboxylase. Chain: a, b. Synonym: omp decarboxylase, ompdcase, ompdecase. Engineered: yes. Mutation: yes

Source:

Methanothermobacter thermautotrophicus. Organism_taxid: 145262. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.57Å R-factor: 0.157 R-free: 0.182

Authors:

M.Fujihashi,E.F.Pai

Key ref:

M.Fujihashi et al. (2009). Structural characterization of the molecular events during a slow substrate-product transition in orotidine 5'-monophosphate decarboxylase. J Mol Biol, 387, 1199-1210. PubMed id: 19236876 DOI: 10.1016/j.jmb.2009.02.037

Date:

05-Feb-09 Release date: 24-Mar-09

Protein chains

O26232  (PYRF_METTH) -  Orotidine 5'-phosphate decarboxylase from Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H)

Seq: 228 a.a.

Struc: 215 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.4.1.1.23 - orotidine-5'-phosphate decarboxylase.
• Pathway: Pyrimidine Biosynthesis
• Reaction: orotidine 5'-phosphate + H⁺ = UMP + CO2

orotidine 5'-phosphate + H(+) (Bound ligand (Het Group name = 6CN) matches with 88.00% similarity) = UMP + CO2 (Bound ligand (Het Group name = BMP) matches with 95.45% similarity)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2009.02.037

J Mol Biol 387:1199-1210 (2009)

PubMed id: 19236876

Structural characterization of the molecular events during a slow substrate-product transition in orotidine 5'-monophosphate decarboxylase.

M.Fujihashi, L.Wei, L.P.Kotra, E.F.Pai.

ABSTRACT

Crystal structures of substrate-product complexes of Methanobacterium thermoautotrophicum orotidine 5'-monophosphate decarboxylase, obtained at various steps in its catalysis of the unusual transformation of 6-cyano-uridine 5'-monophosphate (UMP) into barbituric acid ribosyl monophosphate, show that the cyano substituent of the substrate, when bound to the active site, is first bent significantly from the plane of the pyrimidine ring and then replaced by an oxygen atom. Although the K72A and D70A/K72A mutants are either catalytically impaired or even completely inactive, they still display bending of the C6 substituent. Interestingly, high-resolution structures of the D70A and D75N mutants revealed a covalent bond between C6 of UMP and the Lys72 side chain after the -CN moiety's release. The same covalent bond was observed when the native enzyme was incubated with 6-azido-UMP and 6-iodo-UMP; in contrast, the K72A mutant transformed 6-iodo-UMP to barbituric acid ribosyl 5'-monophosphate. These results demonstrate that, given a suitable environment, native orotidine 5'-monophosphate decarboxylase and several of its mutants are not restricted to the physiologically relevant decarboxylation; they are able to catalyze even nucleophilic substitution reactions but consistently maintain distortion on the C6 substituent as an important feature of catalysis.

Selected figure(s)

Figure 2.
Fig. 2. Close-ups of the active sites of native and mutant MtODCase complexes with 6-CN-UMP. (a–d) F[o] − F[c] omit electron density maps superimposed on the atomic model. The pyrimidine ring and residues Lys72, Asp70, Lys42, and Asp75^B (or the corresponding mutant residues) are omitted for the map calculation. (a) K72A mutant. Map contoured at 2.5σ. (b) Native MtODCase incubated with 6-CN-UMP for 1 day at RT and 1 day at 4 °C. (c) Native MtODCase incubated with 6-CN-UMP for 2 months at RT. (d) D70A/K72A double mutant. Maps (b)–(d) are contoured at 3.0σ. (e) View approximately perpendicular to that in (b). The F[o] − F[c] omit electron density map was calculated with the pyrimidine ring and the surrounding water molecules excluded from the phasing model. (f) F[o](2 days) − F[o](2 months) difference electron density map superimposed on the model of the native MtODCase–6-CN-UMP complex. The ligand molecule was excluded from the model used in calculating the phases for this map. Green (positive difference density) and red (negative difference density) meshes are contoured at + 6.5σ and − 6.5σ, respectively. (g–j) Stereo views of the side-chain arrangements in the reaction center of MtODCase. Numbers colored in both blue and red indicate the distances between atoms in Ångstrom. (g) The K72A–6-CN-UMP complex. Lys72 of native MtODCase is superimposed for comparison and shown in transparent gray. (h) Native MtODCase–6-CN-UMP incubated for 1 day at RT and 1 day at 4 °C. The hydroxyl group of the product BMP is shown in transparent gray and red. (i) D70A/K72A–6-CN-UMP. Asp70 and Lys72 of native ODCase are superimposed for comparison and shown in transparent gray. (j) D70A/K72A–OMP (PDB ID 1KM6). The D70A/K72A–6-CN-UMP complex was superimposed, and 6-CN-UMP is shown in transparent gray for comparison. Water molecules bound in the D70A/K72A–6-CN-UMP complex are shown in green. These panels were prepared with PyMOL (http://pymol.sourceforge.net).

Figure 6.
Fig. 6. Cartoon of the proposed reaction schemes. Distortions of the carboxyl, cyano, iodo, and azido groups play an important role in the reaction catalyzed by ODCase. “P-Rib” indicates ribose 5′-monophosphate. (a) The reaction from OMP to UMP catalyzed by native ODCase. (b) Covalent-bond formation between the mutant ODCase and 6-CN-UMP. (c) The conversion of 6-CN-UMP to BMP catalyzed by native ODCase. (d) Covalent-bond formation between the native ODCase and 6-I-UMP or 6-N[3]-UMP. (e) The conversion of 6-I-UMP to BMP by K72A MtODCase. Distortion of the bond linking the pyrimidine ring to the C6 substituent is conserved in all these reactions, the physiologically relevant decarboxylation and the nucleophilic mechanisms.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 387, 1199-1210) copyright 2009.

Figures were selected by the author.

Author's comment

The results of our "slow motion" time-resolved crystallography (enabled by the very slow reaction rates of the 6-CN-UMP substrate analogue) on native orotidine 5'-monophosphate decarboxylase (ODCase) show that the enzyme employs distortion on the C6 substituent as an important feature in its catalysis. Additional structures of complexes of ODCase mutants with a variety of C6-substituted substrate analogues demonstrate that, given a suitable environment, the enzyme is not restricted to the physiologically relevant decarboxylation but is able to catalyze even nucleophilic substitutions.
Masahiro Fujihashi, Emil F. Pai