1c3x
|
PURINE NUCLEOSIDE PHOSPHORYLASE FROM CELLULOMONAS SP. IN COMPLEX WITH 8-IODO-GUANINE
OverviewOverview
The three-dimensional structure of the trimeric purine nucleoside, phosphorylase (PNP) from Cellulomonas sp. has been determined by X-ray, crystallography. The binary complex of the enzyme with orthophosphate was, crystallized in the orthorhombic space group P212121 with unit cell, dimensions a=64.1 A, b=108.9 A, c=119.3 A and an enzymatically active, trimer in the asymmetric unit. X-ray data were collected at 4 degrees C, using synchrotron radiation (EMBL/DESY, Hamburg). The structure was solved, by molecular replacement, with the calf spleen PNP structure as a model, and refined at 2.2 A resolution. The ternary "dead-end" complex of the, enzyme with orthophosphate and 8-iodoguanine was obtained by soaking, crystals of the binary orthophosphate complex with the very weak substrate, 8-iodoguanosine. Data were collected at 100 K with CuKalpha radiation, and, the three-dimensional structure refined at 2.4 A resolution. Although the, sequence of the Cellulomonas PNP shares only 33 % identity with the calf, spleen enzyme, and almost no identity with the hexameric Escherichia coli, PNP, all three enzymes have many common structural features, viz. the, nine-stranded central beta-sheet, the positions of the active centres, and, the geometrical arrangement of the ligands in the active centres. Some, similarities of the surrounding helices also prevail. In Cellulomonas PNP, each of the three active centres per trimer is occupied by orthophosphate, and by orthophosphate and base, respectively, and small structural, differences between monomers A, B and C are observed. This supports, cooperativity between subunits (non-identity of binding sites) rather than, existence of more than one binding site per monomer, as previously, suggested for binding of phosphate by mammalian PNPs. The phosphate, binding site is located between two conserved beta- and gamma-turns and, consists of Ser46, Arg103, His105, Gly135 and Ser223, and one or two water, molecules. The guanine base is recognized by a zig-zag pattern of possible, hydrogen bonds, as follows: guanine N-1...Glu204 O(epsilon1)...guanine, NH2...Glu204 O(epsilon2). The exocyclic O6 of the base is bridged via a, water molecule to Asn246 N(delta), which accounts for the inhibitory, but, lack of substrate, activity of adenosine. An alternative molecular, mechanism for catalysis by trimeric PNPs is proposed, in which the key, catalytic role is played by Glu204 (Glu201 in the calf and human enzymes), while Asn246 (Asn243 in the mammalian enzymes) supports binding of, 6-oxopurines rather than catalysis. This mechanism, in contrast to that, previously suggested, is consistent with the excellent substrate, properties of N-7 substituted nucleosides, the specificity of trimeric, PNPs versus 6-oxopurine nucleosides and the reported kinetic properties of, Glu201/Ala and Asn243/Ala point variants of human PNP.
About this StructureAbout this Structure
1C3X is a Single protein structure of sequence from Cellulomonas sp. with PO4, CA and 8IG as ligands. Full crystallographic information is available from OCA.
ReferenceReference
Crystal structure of the purine nucleoside phosphorylase (PNP) from Cellulomonas sp. and its implication for the mechanism of trimeric PNPs., Tebbe J, Bzowska A, Wielgus-Kutrowska B, Schroder W, Kazimierczuk Z, Shugar D, Saenger W, Koellner G, J Mol Biol. 1999 Dec 17;294(5):1239-55. PMID:10600382
Page seeded by OCA on Tue Nov 20 12:08:27 2007