ppnP lack the divalent metal-binding site
We then analyze the homolog proteins with the Dali lite server, the
search result with monomeric Ec ppnP revealed many structures with
high similarity but with low sequence identity (Figure 4A )15. However, many of these proteins are unknown
functions. Therefore, based on the DALI results, we selected two
published structures, which are complexed with their substrates, to
further dissect the substrate-binding properties of Ec ppnP. The
HppE from Streptomyces wedmorensis (4J1X) and BacB fromBacillus subtilis are selected to compare with Ec ppnP16,17. HppE is a non-heme-dependent dioxygenase that
catalyzes the oxidative epoxidation of (S)-2-hydroxypropylphosphonate
into (1R,2S)-epoxypropylphosphonate and is shown as a homotetramer17. BacB is indicated to be responsible for the
biosynthesis of dipeptide antibiotic bacilysin with double cupin fold16. Superposition of Ec ppnP with HppE and BacB
showed the r.m.s.d. about 0.996 Å and 1.105 Å with the whole chain ofEc ppnP (Figure 4B,4C ). We then detailly compared the
substrate-binding pocket region, both the HppE and BacB contains a metal
chelated by histidine and other residues to promote the catalysis
process, however, no metal can be modeled in the counterpart inEc ppnP, furthermore, the EcppnP lack the corresponding amino
acids that can be used to chelate the metal ion (Figure
4D,4E,4F ).
The members of the NP-I family include PNPs (EC 2.4.2.1), UP (EC
2.4.2.3), and MTAP (EC 2.4.2.28). PNPs from a variety of species
revealed either the trimeric form specific for guanine and hypoxanthine
(2’-deoxy) ribonucleosides, or the hexameric form accepts adenine as
well as guanine and hypoxanthine (2’-deoxy) ribonucleosides1,18-20. UP functions as a hexamer specific for
uridine nucleosides in bacteria and also accepts 2’-deoxypyrimidine
nucleosides in higher organisms 21. MTAP is reported
to function as a trimer in all species except Sulfolobus
solfataricus 22, in which it functions as a hexamer
and catalyzes the phosphorolysis of inosine, guanosine, and adenosine23. Compared with the NP-I family, TP (EC 2.4.2.4) and
pyrimidine nucleoside phosphorylases (PyNPs; EC 2.4.2.2) belong to the
NP-II family and function as a dimer in all cases1,24,25. Our structure studies revealed that the ppnP
family proteins showed dimeric quaternary conformations like NP-II
class, but with a different dimerization mode belonging to the cupin
fold.
Trimeric PNPs (e.g. bPNP and hPNP) are specific for 6-oxo purine
nucleosides, whereas the hexameric PNPs accept adenosine in addition to
6-oxo purine nucleosides. MTAP is specific for 5’-methylthioadenosine
but will accept various substrate analogs 26,27. UP
accepts pyrimidine nucleosides and lacks specificity at the 2’-ribose
position in higher organisms. TP is the high specificity of TP for the
2’-deoxyribose moieties in these studies, interestingly, UP will not
cleave cytidine, which is not cleaved by any known nucleoside
phosphorylase 21. The ppnP family proteins
are validated to catalyze the reaction involving both purine and
pyrimidine, indicating the broadest substrate selectivity. Although we
tried our best to obtain the complex structure to illustrate the binding
properties for these nucleotides, we cannot capture the substrates in
our structures. Therefore, further studies are needed to illustrate the
mechanism for ppnP in catalyzing various ribonucleosides. On the other
hand, the main feature of the ppnP family proteins are members of the
cupin fold superfamily, the structural comparison of ppnP withStreptomyces wedmorensis HppE and Bacillus subtili BacB
revealed a conserved pocket that has the potential to bind the
substrate, however, in ppnP, it lacks the metal-binding residues. How
the catalytic scheme is achieved needs further investigation.