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