Structural analyses reveal two distinct families of nucleoside phosphorylases

Abstract

The reversible phosphorolysis of purine and pyrimidine nucleosides is an important biochemical reaction in the salvage pathway, which provides an alternative to the de novo purine and pyrimidine biosynthetic pathways. Structural studies in our laboratory and by others have revealed that only two folds exist that catalyse the phosphorolysis of all nucleosides, and provide the basis for defining two families of nucleoside phosphorylases. The first family (nucleoside phosphorylase-I) includes enzymes that share a common single-domain subunit, with either a trimeric or a hexameric quaternary structure, and accept a range of both purine and pyrimidine nucleoside substrates. Despite differences in substrate specificity, amino acid sequence and quaternary structure, all members of this family share a characteristic subunit topology. We have also carried out a sequence motif study that identified regions of the common subunit fold that are functionally significant in differentiating the various members of the nucleoside phosphorylase-I family. Although the substrate-binding sites are arranged similarly for all members of the nucleoside phosphorylase-I family, a comparison of the active sites from the known structures of this family indicates significant differences between the trimeric and hexameric family members. Sequence comparisons also suggest structural identity between the nucleoside phosphorylase-I family and both 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase and AMP nucleosidase. Members of the second family of nucleoside phosphorylases (nucleoside phosphorylase-II) share a common two-domain subunit fold and a dimeric quaternary structure, share a significant level of sequence identity (>30%) and are specific for pyrimidine nucleosides. Members of this second family accept both thymidine and uridine substrates in lower organisms, but are specific for thymidine in mammals and other higher organisms. A possible relationship between nucleoside phosphorylase-II and anthranilate phosphoribosyltransferase has been identified through sequence comparisons. Initial studies in our laboratory suggested that members of the nucleoside phosphorylase-II family require significant domain movements in order for catalysis to proceed. A series of recent structures has confirmed our hypothesis and provided details of these conformational changes. Structural studies of the nucleoside phosphorylases have resulted in a wealth of information that begins to address fundamental biological questions, such as how Nature makes use of the intricate relationships between structure and function, and how biological processes have evolved over time. In addition, the therapeutic potential of suppressing the nucleoside phosphorylase activity in either family of enzymes has motivated efforts to design potent inhibitors. Several research groups have synthesized a variety of nucleoside phosphorylase inhibitors that are at various stages of preclinical and clinical evaluation.

Enzymatic synthesis and phosphorolysis of 4(2)-thioxo- and 6(5)-azapyrimidine nucleosides by E. coli nucleoside phosphorylases

“…1). It appears to be reasonable that TP manifests strict requirements to dimension and electronic structure of the C-4 substituent [4], as well as the TP catalyzed transformations depend in a greater extent on the experimental condition [51]. …”

Strained Conformations of Nucleosides in Active Sites of Nucleoside Phosphorylases

“…The equilibrium of the reaction is shifted towards nucleosides, the shift being much more significant for PNPs as compared with UPs. Uridine phosphorylases catalyze phosphorolysis of both thymidine and uridine [1]. Purine nucleoside phosphorylases cleave the N-glycosidic bond in purine ribonucleosides and 2'-deoxynucleosides.…”

“…Numerous reviews on nucleoside phosphorylases are available. The structural organization of these enzymes [1,2], the substrate specificity and methods for the synthesis of valuable nucleosides [3][4][5][6], and metabolism in biological systems [7][8][9][10] have been considered.…”

“…All nucleoside phosphorylases were classified into two families based on the sequence homology analysis of NPs and a comparison of their three-dimensional structures-NP-I and NP-II [1]. The NP-I family comprises hexameric and trimeric PNPs (including the MTAP subfamily) and hexameric UPs.…”

“…Hexameric PNPs are enzymes from lower organisms, such as bacteria and some eukaryotic protozoa, in particular, intracellular parasites. Generally, these enzymes have a very broad spectrum of substrate specificity and are active towards both 6-amino and 6-keto derivatives of purine nucleosides [1]. Hexameric MTAPs were only isolated from the thermostable archaea Sulfolobus solfataricus [24], Pyrococcus furiosus, and Aeropyrum pernix.…”