DB code: S00288

RLCP classification	2.40.48000.390 : Phosphorolysis
CATH domain	3.40.50.2020 : Rossmann fold	Catalytic domain
E.C.	2.4.2.10
CSA	1oro
M-CSA	1oro
MACiE

CATH domain	Related DB codes (homologues)
3.40.50.2020 : Rossmann fold	S00289 S00287 D00131

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	RefSeq	Pfam
P08870	Orotate phosphoribosyltransferase	OPRT OPRTase EC 2.4.2.10	NP_462633.1 (Protein) NC_003197.1 (DNA/RNA sequence)	PF00156 (Pribosyltran) [Graphical View]
P0A7E3	Orotate phosphoribosyltransferase	OPRT OPRTase EC 2.4.2.10	NP_418099.1 (Protein) NC_000913.2 (DNA/RNA sequence) YP_491791.1 (Protein) NC_007779.1 (DNA/RNA sequence)	PF00156 (Pribosyltran) [Graphical View]

KEGG enzyme name
orotate 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

KEGG enzyme name

orotate 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

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P08870	PYRE_SALTY	Orotidine 5''-phosphate + diphosphate = orotate + 5-phospho-alpha-D-ribose 1-diphosphate.	Homodimer.		Magnesium. 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.
P0A7E3	PYRE_ECOLI	Orotidine 5''-phosphate + diphosphate = orotate + 5-phospho-alpha-D-ribose 1-diphosphate.	Homodimer.		Magnesium (By similarity).

KEGG Pathways
Map code	Pathways	E.C.
MAP00240	Pyrimidine metabolism
MAP00983	Drug metabolism - other enzymes

Compound table
	Cofactors	Substrates		Products				Intermediates
KEGG-id	C00305	C01103	C00013	C00295	C00119	C00105	C00011
E.C.
Compound	Magnesium	Orotidine 5'-phosphate	Pyrophosphate	Orotate	5-Phospho-alpha-D-ribose 1-diphosphate	UMP	CO2
Type	divalent metal (Ca2+, Mg2+)	amide group,carbohydrate,nucleotide	phosphate group/phosphate ion	amide group,aromatic ring (with nitrogen atoms),carboxyl group	carbohydrate,phosphate group/phosphate ion	amide group,nucleotide	others
ChEBI	18420 18420	15842 15842	29888 29888	16742 16742	17111 17111	16695 16695	16526 16526
PubChem	888 888	160617 160617	1023 21961011 1023 21961011	967 967	7339 7339	6030 6030	280 280

1oprA	Bound:_MG	Unbound	Unbound	Bound:ORO	Bound:PRP	Unbound	Unbound
1oroA	Unbound	Unbound	Analogue:SO4	Unbound	Unbound	Unbound	Unbound
1oroB	Unbound	Unbound	Analogue:SO4	Unbound	Unbound	Unbound	Unbound
1stoA	Unbound	Bound:OMP	Unbound	Unbound	Unbound	Unbound	Unbound
1lh0A	Unbound	Unbound	Unbound	Bound:ORO	Unbound	Unbound	Unbound
1lh0B	Bound:_MG	Unbound	Unbound	Bound:ORO	Bound:PRP	Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
Swiss-prot

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

1oprA	LYS 103	LYS 73;ASP 124;ASP 125(Mg2+ binding)
1oroA	LYS 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
1lh0A	LYS 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
References	Sections	No. of steps in catalysis
[7]	Fig.3, p.19-20	2

References
[1]
Resource
Comments
Medline ID
PubMed ID	2271660
Journal	Biochemistry
Year	1990
Volume	29
Pages	10480-7
Authors	Bhatia MB, Vinitsky A, Grubmeyer C
Title	Kinetic mechanism of orotate phosphoribosyltransferase from Salmonella typhimurium.
Related PDB
Related UniProtKB	P08870
[2]
Resource
Comments
Medline ID
PubMed ID	8376388
Journal	J Biol Chem
Year	1993
Volume	268
Pages	20299-304
Authors	Grubmeyer C, Segura E, Dorfman R
Title	Active site lysines in orotate phosphoribosyltransferase.
Related PDB
Related UniProtKB	P08870
[3]
Resource
Comments
Medline ID
PubMed ID	8312245
Journal	Biochemistry
Year	1994
Volume	33
Pages	1287-94
Authors	Scapin G, Grubmeyer C, Sacchettini JC
Title	Crystal structure of orotate phosphoribosyltransferase.
Related PDB	1sto
Related UniProtKB	P08870
[4]
Resource
Comments
Medline ID
PubMed ID	7545004
Journal	Biochemistry
Year	1995
Volume	34
Pages	10744-54
Authors	Scapin G, Ozturk DH, Grubmeyer C, Sacchettini JC
Title	The crystal structure of the orotate phosphoribosyltransferase complexed with orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate.
Related PDB	1opr
Related UniProtKB	P08870
[5]
Resource
Comments
Medline ID
PubMed ID	7545005
Journal	Biochemistry
Year	1995
Volume	34
Pages	10755-63
Authors	Ozturk DH, Dorfman RH, Scapin G, Sacchettini JC, Grubmeyer C
Title	Locations and functional roles of conserved lysine residues in Salmonella typhimurium orotate phosphoribosyltransferase.
Related PDB
Related UniProtKB	P08870
[6]
Resource
Comments
Medline ID
PubMed ID	7545006
Journal	Biochemistry
Year	1995
Volume	34
Pages	10764-70
Authors	Ozturk DH, Dorfman RH, Scapin G, Sacchettini JC, Grubmeyer C
Title	Structure and function of Salmonella typhimurium orotate phosphoribosyltransferase: protein complementation reveals shared active sites.
Related PDB
Related UniProtKB	P08870
[7]
Resource
Comments
Medline ID
PubMed ID	8555167
Journal	Biochemistry
Year	1996
Volume	35
Pages	14-21
Authors	Tao W, Grubmeyer C, Blanchard JS
Title	Transition state structure of Salmonella typhimurium orotate phosphoribosyltransferase.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID	8620002
Journal	Biochemistry
Year	1996
Volume	35
Pages	3803-9
Authors	Henriksen A, Aghajari N, Jensen KF, Gajhede M
Title	A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase.
Related PDB	1oro
Related UniProtKB	P0A7E3

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.

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.

Created	Updated
2002-05-02	2009-02-26