THREE DIMENSIONAL STRUCTURES OF E.COLI PNP COMPLEXES. Chenglong Li and Steven Ealick, Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853

We present here several complex structures of E.Coli PNP with its substratesand substrate analogs. Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribo or 2'-deoxyribonucleosides to the purine and ribose or 2-deoxyribose-1-phosphate. The enzyme has been isolated from both eukaryotic and prokaryotic organisms and functions in the purine salvage pathways. The human and bovine PNPs are specific for the 6-oxo-purines and many of their analogs,and both are trimers with identical subunits. E.Coli PNP represents another class of PNP identified from various sources,which is hexameric,has no sequence similarity with human and bovine PNPs and accepts both 6-amino and 6-oxopurines as substrates. E.Coli PNP has a strikingly different active site and binding features to its substrates compared with that of mammalian PNPs. The phosphate binding site consists of backbone Gly20 and three arginine residues Arg43 and Arg87 from one subunit and Arg24 from the neighboring subunit. The three arginine residue hold the phosphate to form a strong binding net and yet are flexible enough to allow phosphate to initiate the nucleophilic attack due to the long arms of arginine residues. E.Coli PNP does not undergo a large conformational change during the enzymatic reaction like in the case of mammalian PNP. The base binding site consists of residues Phe159,Phe167,Ile206,Val178 and Asp204. It is mainly made of hydrophobic residues and is more open (therefore maybe less specific) than its mammalian counterpart. Another remarkable feature is that the nucleoside binding conformations in the E.Coli and mammalian PNP are totally different. Although the ribosyl and phosphate groups bind to E.Coli PNP and mammalian PNP in similar ways,the purine base is rotated 180 degree about the glycosidic bond. E.Coli PNP has only one H-bonding residue Asp204 and mammalian PNPs have two H-bonding residues Asn243 and Glu 201 near the purine base.