KEYWORDS
Nucleotides metabolism; ppnP; Cupin
INTRODUCTION
Nucleotides metabolism is a fundamental process involving a series of chemical reactions to the nitrogenous base, pentose sugar, and phosphate. The differences of pyrimidine or purine nucleotides are determined by their nitrogenous bases, which are surveilled by several enzymes to keep the metabolic balance 1. The biosynthesis of nucleotides mainly proceeds through two pathways in the cell: 1) the de novo pathway, which uses a variety of amino acids and other precursors to produce nucleotides; and 2) the salvage pathway, which uses preformed nucleobases and nucleosides as precursors to produce nucleotides 1-4. The salvage pathway enables the cell to avoid the energy-costly de novo pathway when appropriate precursors are available. In the salvage pathway, one of the important biochemical reactions is the reversible phosphorolysis of purine and pyrimidine nucleosides. Two families of nucleoside phosphorylases that catalyze the phosphorolytic cleavage of the glycosidic bond in nucleosides have been discovered, the trimeric or hexameric nucleoside phosphorylase-I (NP-I) family enzymes share a common α/β-subunit fold and accept a range of purine nucleosides, as well as uridine nucleotide; and 2) the nucleoside phosphorylase-II (NP-II) family enzyme that display a dimeric structure and accept both thymidine and uridine in lower organisms, but are specific for thymidine in higher species 1. The proposed enzymic reaction for the purine/pyrimidine nucleosides was established previously, whereby the C-N glycosidic bond is cleaved by a phosphate ion5.
Recently, a combinative approach based on non-targeted metabolomics and activity-based enzyme discovery at the proteome scale discovered 241 potential novel enzymes in E. coli , 12 of which were experimentally validated 6. One of these enzymes is Pyrimidine/purine nucleoside phosphorylase (ppnP), which is further identified to catalyze the phosphorolysis of diverse nucleosides such as uridine, adenosine, guanosine, cytidine, thymidine, inosine, and xanthosine as substrates, and yielding D-ribose 1-phosphate and the respective free bases 6. Therefore, the reaction scheme of ppnP is indicated as p urine/p yrimidinen ucleoside Phosphorylase and showed non-nucleotides specific and reversible activity6.
Here we determine the crystal structures of ppnP in many species of bacteria, our structural studies show that the ppnP family proteins present dual-layer beta-fence cupin fold, and adopt dimeric quaternary structure through a set of hydrophobic interactions. Detailed analysis also identified the potential substrate-binding pocket. Our structures uncover the structural basis for the new class nucleoside phosphorylase and shed light on deeply understanding the nucleotides’ metabolism in bacteria and further usage of biocatalysts for industrial applications.
MATERIALS AND METHODS