DB code: S00510

RLCP classification	2.40.18000.65 : Phosphorolysis
CATH domain	3.40.50.1580 : Rossmann fold	Catalytic domain
E.C.	2.4.2.1
CSA	1a69
M-CSA	1a69
MACiE

CATH domain	Related DB codes (homologues)
3.40.50.1580 : Rossmann fold	S00375 S00376

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	RefSeq	Pfam
P0ABP8	Purine nucleoside phosphorylase deoD-type	PNP EC 2.4.2.1 Inosine phosphorylase	NP_418801.1 (Protein) NC_000913.2 (DNA/RNA sequence) YP_492514.1 (Protein) NC_007779.1 (DNA/RNA sequence)	PF01048 (PNP_UDP_1) [Graphical View]

KEGG enzyme name
purine-nucleoside phosphorylase inosine phosphorylase PNPase PUNPI PUNPII inosine-guanosine phosphorylase nucleotide phosphatase purine deoxynucleoside phosphorylase purine deoxyribonucleoside phosphorylase purine nucleoside phosphorylase purine ribonucleoside phosphorylase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P0ABP8	DEOD_ECOLI	Purine nucleoside + phosphate = purine + alpha-D-ribose 1-phosphate.	Homohexamer.

KEGG Pathways
Map code	Pathways	E.C.
MAP00230	Purine metabolism
MAP00240	Pyrimidine metabolism
MAP00760	Nicotinate and nicotinamide metabolism

Compound table
	Substrates		Products		Intermediates
KEGG-id	C15586	C00009	C15587	C00620
E.C.
Compound	Purine nucleoside	Orthophosphate	Purine	alpha-D-Ribose 1-phosphate
Type	nucleoside	phosphate group/phosphate ion	aromatic ring (with nitrogen atoms)	carbohydrate,phosphate group/phosphate ion
ChEBI	18255 18255	26078 26078	17258 35586 35588 35589 17258 35586 35588 35589	16300 16300
PubChem	68368 68368	1004 22486802 1004 22486802	1044 1044	439236 439236

1a69A	Analogue:FMB	Analogue:SO4	Unbound	Unbound
1a69B	Analogue:FMB	Analogue:SO4	Unbound	Unbound
1a69C	Analogue:FMB	Analogue:SO4	Unbound	Unbound
1ecpA	Unbound	Unbound	Unbound	Unbound
1ecpB	Unbound	Unbound	Unbound	Unbound
1ecpC	Unbound	Unbound	Unbound	Unbound
1ecpD	Unbound	Unbound	Unbound	Unbound
1ecpE	Unbound	Unbound	Unbound	Unbound
1ecpF	Unbound	Unbound	Unbound	Unbound
1k9sA	Analogue:FM2	Bound:PO4	Unbound	Unbound
1k9sB	Analogue:FM2	Bound:PO4	Unbound	Unbound
1k9sC	Analogue:FM2	Bound:PO4	Unbound	Unbound
1k9sD	Analogue:FM1	Bound:PO4	Unbound	Unbound
1k9sE	Analogue:FM1	Bound:PO4	Unbound	Unbound
1k9sF	Analogue:FM1	Bound:PO4	Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
literature [5]

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

1a69A	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1a69B	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1a69C	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1ecpA	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1ecpB	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1ecpC	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1ecpD	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1ecpE	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1ecpF	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1k9sA	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1k9sB	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1k9sC	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1k9sD	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1k9sE	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)
1k9sF	ASP 204(base binding);ARG 24(phosphate binding);ARG 217(Switch)

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[1]	p.1380
[3]	Fig.1, Fig.8	2
[5]	Scheme 6, p.363-365	2
[6]	Fig.5, Fig.7, p.14	2

References
[1]
Resource
Comments
Medline ID
PubMed ID	9351810
Journal	Structure
Year	1997
Volume	5
Pages	1373-83
Authors	Mao C, Cook WJ, Zhou M, Koszalka GW, Krenitsky TA, Ealick SE
Title	The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology.
Related PDB	1ecp
Related UniProtKB	P0ABP8
[2]
Resource
Comments
Medline ID
PubMed ID	9653038
Journal	J Mol Biol
Year	1998
Volume	280
Pages	153-66
Authors	Koellner G, Luic M, Shugar D, Saenger W, Bzowska A
Title	Crystal structure of the ternary complex of E. coli purine nucleoside phosphorylase with formycin B, a structural analogue of the substrate inosine, and phosphate (Sulphate) at 2.1 A resolution.
Related PDB	1a69
Related UniProtKB	P0ABP8
[3]
Resource
Comments
Medline ID
PubMed ID	11444966
Journal	Biochemistry
Year	2001
Volume	40
Pages	8204-15
Authors	Shi W, Basso LA, Santos DS, Tyler PC, Furneaux RH, Blanchard JS, Almo SC, Schramm VL
Title	Structures of purine nucleoside phosphorylase from Mycobacterium tuberculosis in complexes with immucillin-H and its pieces.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID	11591349
Journal	Structure (Camb)
Year	2001
Volume	9
Pages	941-53
Authors	Lee JE, Cornell KA, Riscoe MK, Howell PL
Title	Structure of E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase reveals similarity to the purine nucleoside phosphorylases.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID	11786017
Journal	J Mol Biol
Year	2002
Volume	315
Pages	351-71
Authors	Koellner G, Bzowska A, Wielgus-Kutrowska B, Luic M, Steiner T, Saenger W, Stepinski J
Title	Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID	11743878
Journal	Biochem J
Year	2002
Volume	361
Pages	1-25
Authors	Pugmire MJ, Ealick SE
Title	Structural analyses reveal two distinct families of nucleoside phosphorylases.
Related PDB
Related UniProtKB

Comments
The enzymes of this entry corresponds to the hexameric subunit members of nucleoside phosphorylase family-I [6]. FMB molecules (analogues of purine nucleoside) in 1a69 interact with two chains (A & C). The literature [6] summarized the proposed catalytic mechanism of the enzyme. Beta-nucleoside binds in a high-energy (anticlinal torsion angle of the glycosidic bond, with the ribose moiety in the uncommon C-4'-endo sugar pucker), according to the literature. This high-energy conformation produces steric strain, which induces glycosidic cleavage. The glycosidic bond is weakened further as electrons flow from O-4' of the ribose to the purine ring, resulting in an oxocarbenium ion that is stabilized by the negative charges of the phosphate ion. The phosphate ion binds on the alpha-side of the ribose ring, where it is postioned to participate in an SN1 nucleophilic attack at the C-1' position. The flow of electrons from the glycosidic bond to the purine ring is probably stabilized by active site residues (Asp) interactions at O-4 position of the purine base. According to the literature [5], the hexamer of this enzymes is found as a trimer of unsymmetric dimers, which are formed by pairs of monomers with active sites in different conformations. The conformational differences results from a flexible helix (H8: residues 214-236), which is continuous in one conformer, and segmented in the other. With the continuous helix, the entry in to the active site pocket is wide open, and the ligands are bound only loosely ("open" conformation). In contrast, by segmentation of the helix (H8: residues 214-219 and H8': residues 223-236, separated), the entry in to the active site is partially closed, the pocket is narrowed and the ligands are bound much more tightly ("closed" conformation) [5]. The possible catalytic mechanism could be described as follows [5]: (1) In the open conformation, the helix H8 is continuous and Arg217 on the helix is far away from Asp204 which can be in the acid form. The sidechain of Arg24 is floppy. Phosphate and the nucleoside both can bind to this conformation of the active site. The neutral sidechain of Asp204 donates a hydrogen bond to the purine nitrogen N7. (2) Phosphate binding stabilizes Arg24 and, in turn, favors breaking of H8 into two segments. The active site is now in the closed conformation. (3) The conformation change brings the guanidinium group of Arg217 in contact with the acid group of Asp204. This triggers proton transfer to the base, followed by formation of a salt bridge between Arg217 and Asp204. The positively charged purine base leads to the ribo-oxocarbenium ion character of the ribose, representing the transition state of bond cleavage in acid hydorolysis which can then proceed further.

Comments

The enzymes of this entry corresponds to the hexameric subunit members of nucleoside phosphorylase family-I [6].
FMB molecules (analogues of purine nucleoside) in 1a69 interact with two chains (A & C).
The literature [6] summarized the proposed catalytic mechanism of the enzyme. Beta-nucleoside binds in a high-energy (anticlinal torsion angle of the glycosidic bond, with the ribose moiety in the uncommon C-4'-endo sugar pucker), according to the literature. This high-energy conformation produces steric strain, which induces glycosidic cleavage. The glycosidic bond is weakened further as electrons flow from O-4' of the ribose to the purine ring, resulting in an oxocarbenium ion that is stabilized by the negative charges of the phosphate ion. The phosphate ion binds on the alpha-side of the ribose ring, where it is postioned to participate in an SN1 nucleophilic attack at the C-1' position. The flow of electrons from the glycosidic bond to the purine ring is probably stabilized by active site residues (Asp) interactions at O-4 position of the purine base.
According to the literature [5], the hexamer of this enzymes is found as a trimer of unsymmetric dimers, which are formed by pairs of monomers with active sites in different conformations. The conformational differences results from a flexible helix (H8: residues 214-236), which is continuous in one conformer, and segmented in the other. With the continuous helix, the entry in to the active site pocket is wide open, and the ligands are bound only loosely ("open" conformation). In contrast, by segmentation of the helix (H8: residues 214-219 and H8': residues 223-236, separated), the entry in to the active site is partially closed, the pocket is narrowed and the ligands are bound much more tightly ("closed" conformation) [5].
The possible catalytic mechanism could be described as follows [5]:
(1) In the open conformation, the helix H8 is continuous and Arg217 on the helix is far away from Asp204 which can be in the acid form. The sidechain of Arg24 is floppy. Phosphate and the nucleoside both can bind to this conformation of the active site. The neutral sidechain of Asp204 donates a hydrogen bond to the purine nitrogen N7.
(2) Phosphate binding stabilizes Arg24 and, in turn, favors breaking of H8 into two segments. The active site is now in the closed conformation.
(3) The conformation change brings the guanidinium group of Arg217 in contact with the acid group of Asp204. This triggers proton transfer to the base, followed by formation of a salt bridge between Arg217 and Asp204. The positively charged purine base leads to the ribo-oxocarbenium ion character of the ribose, representing the transition state of bond cleavage in acid hydorolysis which can then proceed further.

Created	Updated
2002-07-12	2011-09-27