EzCatDB: S00288

DB codeS00288
RLCP classification2.40.48000.390
CATH domainDomain 13.40.50.2020Catalytic domain
E.C.2.4.2.10
CSA1oro

CATH domainRelated DB codes (homologues)
3.40.50.2020S00289,S00287,D00131

Enzyme Name
Swiss-protKEGG

P08870P0A7E3
Protein nameOrotate phosphoribosyltransferaseOrotate phosphoribosyltransferaseorotate phosphoribosyltransferase
orotidylic acid phosphorylase
orotidine-5'-phosphate pyrophosphorylase
OPRTase
orotate phosphoribosyl pyrophosphate transferase
orotic acid phosphoribosyltransferase
orotidine 5'-monophosphate pyrophosphorylase
orotidine monophosphate pyrophosphorylase
orotidine phosphoribosyltransferase
orotidylate phosphoribosyltransferase
orotidylate pyrophosphorylase
orotidylic acid pyrophosphorylase
orotidylic phosphorylase
orotidylic pyrophosphorylase
SynonymsOPRT
OPRTase
EC 2.4.2.10
OPRT
OPRTase
EC 2.4.2.10

KEGG pathways
MAP codePathways
MAP00240Pyrimidine metabolism
MAP00983Drug metabolism - other enzymes

Swiss-prot:Accession NumberP08870P0A7E3
Entry namePYRE_SALTYPYRE_ECOLI
ActivityOrotidine 5''-phosphate + diphosphate = orotate + 5-phospho-alpha-D-ribose 1-diphosphate.Orotidine 5''-phosphate + diphosphate = orotate + 5-phospho-alpha-D-ribose 1-diphosphate.
SubunitHomodimer.Homodimer.
Subcellular location

CofactorMagnesium. Manganese can replace magnesium as the divalent metal. The role of metal is to bind PRPP and form a MgPRPP complex which then serves as substrate for OPRTase.Magnesium (By similarity).


CofactorsSubstratesProducts
KEGG-idC00305C01103C00013C00295C00119C00105C00011
CompoundMagnesiumOrotidine 5'-phosphatePyrophosphateOrotate5-Phospho-alpha-D-ribose 1-diphosphateUMPCO2
Typedivalent metal (Ca2+, Mg2+)amide group,carbohydrate,nucleotidephosphate group/phosphate ionamide group,aromatic ring (with nitrogen atoms),carboxyl groupcarbohydrate,phosphate group/phosphate ionamide group,nucleotideothers
1oprABound:_MGUnboundUnboundBound:OROBound:PRPUnboundUnbound
1oroAUnboundUnboundAnalogue:SO4UnboundUnboundUnboundUnbound
1oroBUnboundUnboundAnalogue:SO4UnboundUnboundUnboundUnbound
1stoAUnboundBound:OMPUnboundUnboundUnboundUnboundUnbound
1lh0AUnboundUnboundUnboundBound:OROUnboundUnboundUnbound
1lh0BBound:_MGUnboundUnboundBound:OROBound:PRPUnboundUnbound

Active-site residues
resource
Swiss-prot
pdbCatalytic residuesCofactor-binding residuescomment
1oprALYS  103
LYS   73;ASP  124;ASP  125(Mg2+ binding)

1oroALYS  103
LYS   73;ASP  124;ASP  125(Mg2+ binding)

1oroB        
LYS   73;ASP  124;ASP  125(Mg2+ binding)
invisible 102-108
1stoA        
LYS   73;ASP  124;ASP  125(Mg2+ binding)
invisible 103-107
1lh0ALYS 1103
LYS 1073;ASP 1124;ASP 1125(Mg2+ binding)

1lh0B        
LYS 2073;ASP 2124;ASP 2125(Mg2+ binding)
invisible 2102-2108

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[7]Fig.3, p.19-202

references
[1]
PubMed ID2271660
JournalBiochemistry
Year1990
Volume29
Pages10480-7
AuthorsBhatia MB, Vinitsky A, Grubmeyer C
TitleKinetic mechanism of orotate phosphoribosyltransferase from Salmonella typhimurium.
Related Swiss-protP08870
[2]
PubMed ID8376388
JournalJ Biol Chem
Year1993
Volume268
Pages20299-304
AuthorsGrubmeyer C, Segura E, Dorfman R
TitleActive site lysines in orotate phosphoribosyltransferase.
Related Swiss-protP08870
[3]
PubMed ID8312245
JournalBiochemistry
Year1994
Volume33
Pages1287-94
AuthorsScapin G, Grubmeyer C, Sacchettini JC
TitleCrystal structure of orotate phosphoribosyltransferase.
Related PDB1sto
Related Swiss-protP08870
[4]
PubMed ID7545004
JournalBiochemistry
Year1995
Volume34
Pages10744-54
AuthorsScapin G, Ozturk DH, Grubmeyer C, Sacchettini JC
TitleThe crystal structure of the orotate phosphoribosyltransferase complexed with orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate.
Related PDB1opr
Related Swiss-protP08870
[5]
PubMed ID7545005
JournalBiochemistry
Year1995
Volume34
Pages10755-63
AuthorsOzturk DH, Dorfman RH, Scapin G, Sacchettini JC, Grubmeyer C
TitleLocations and functional roles of conserved lysine residues in Salmonella typhimurium orotate phosphoribosyltransferase.
Related Swiss-protP08870
[6]
PubMed ID7545006
JournalBiochemistry
Year1995
Volume34
Pages10764-70
AuthorsOzturk DH, Dorfman RH, Scapin G, Sacchettini JC, Grubmeyer C
TitleStructure and function of Salmonella typhimurium orotate phosphoribosyltransferase: protein complementation reveals shared active sites.
Related Swiss-protP08870
[7]
PubMed ID8555167
JournalBiochemistry
Year1996
Volume35
Pages14-21
AuthorsTao W, Grubmeyer C, Blanchard JS
TitleTransition state structure of Salmonella typhimurium orotate phosphoribosyltransferase.
[8]
PubMed ID8620002
JournalBiochemistry
Year1996
Volume35
Pages3803-9
AuthorsHenriksen A, Aghajari N, Jensen KF, Gajhede M
TitleA flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase.
Related PDB1oro
Related Swiss-protP0A7E3

comments
The literature [4] indicated that the ribose of substrate will move in spite of the slight movement of the overall strucuture of the OPRTase itself (see Figs. 6 and 7). The literature [5] indicated that Lys103 plays an essential role in catalysis, although this residue is away from the active site. One possible role for Lys103 is to serve as an acid to protonate the leaving pyrophosphate group or as a base to deprotonate the incoming orotate. Another possible role is to shield the active site from solvent, as the oxocarbonium transition state is unstable with solvent [5]. Moreover, this literature indicated that Lys73 extends into the active site, and a conformational change allows it to interact with either the 5'-phosphate of OMP or the 2-hydroxyl and alpha-phosphoryl oxgen of PRPP in the respective complexes. The literature [6] indicated that the active site of OPRTase requires Asp125 from one subunit and Lys103 from the adjacent subunit of heterodimer.
According to [7], the partial C1'-O4' bond cleavage and double bond formation in C1'-O4' argue that the reaction undergoes an SN1-like mechanism, with a posiively-charged oxocarbonium ion in the transition state. As the oxocarbonium ion is reactive and unstable, it needs to be stablized by some negative charge in the enzyme active site. The paired Asp125 and Asp124 are highly conserved, but they are away from the oxocarbonium ion, which is difficult to stabilize.
The interaction of Lys73 with 5'-phosphate is disrupted by pyrophosphate binding, and this could initiates catalysis, thus preventing nonproductive hydrolysis via solvent capture of the oxocarbonium ion [7]. This loss of the 5'-phosphate-Lys73 interaction also may allow for the interaction between the 5'-phosphate and phosphate binding loop interactions to become stronger and initiate the movement of the ribose 5'-phosphate ring away from the orotate ring. The O4' ring oxygen initiates oxocarbonium ion formation by electron donation into the C1'-O4' bond, with subsequent lengthening of the C1'-N1 bond. Both orotate keto oxygen bond lengths have lengthened, dispersing the buildup of negative charge on the orotate ring [7]. The bipolar tansition state, with the negative charge of orotate ring and the positive charge of the ribose ring, is energetically stabilized via both "intra-molecular" and enzyme-transition state interactions [7].
As the intra-molecular stabilization decreases, and as the 5'-phosphate is pulled further into the phosphate binding loop, the swinging movement of the ribosyl 5'-phosphate caation is likely intiated by the long-range electrostatic attraction between the oxocarbonium ion and Asp125 [7]. The approach og the face of the oxocarbonium ion to bound pyrophoshate results in the formation of the covalent C1-O-PPi bond in the alpha-anomeric configuration [7].
The litarture [8] indicated that the flexible loop region (residues 102-108) is important for catalysis. The movement of this loop in association with the movement of OMP is vital to catalysis.

createdupdated
2002-05-022009-02-26
Copyright: Nozomi Nagano, JST & CBRC-AIST
Funded by PRESTO/Japan Science and Technology Corporation (JST) (December 2001 - November 2004)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2006)
Funded by Grant-in-Aid for Scientific Research (B)/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (September 2005 - September 2010)
Funded by BIRD/Japan Science and Technology Corporation (JST) (October 2007 - September 2010)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2011 - March 2012)

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