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.