Biochemical and structural characterization of mammalian‐like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus

Abstract

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolytic cleavage of the glycosidic bond of purine nucleosides to produce ribose-1-phosphate and a free purine base [1][2][3]. PNPs have been characterized in a variety of species and may be grouped into two main groups, PNP-1 and PNP-2. PNP-1 are found in prokaryotes, are homohexamers with a subunit of 26 kDa and recognize both 6-oxo and 6-amino purine nucleosides as substrates. PNP-2 are homotrimers, with a subunit molecular mass of We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5¢-deoxy-5¢-methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family members. Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50% identity with MTAP from P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfM-TAP indicates that only four residue changes are able to switch the specificity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120°C and is characterized by extreme thermodynamic stability (T m , 110°C that increases to 120°C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100°C), and remarkable SDS-resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasubunit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S ⁄ C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability.Abbreviations hMTAP, human 5¢-deoxy-5¢-methylthioadenosine phosphorylase; MTA, 5¢-deoxy-5¢-methylthioadenosine; MTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase; PfMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Pyrococcus furiosus; PfPNP, purine nucleoside phosphorylase from P. furiosus; PNP, purine nucleoside phosphorylase; SsMTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase from Sulfolobus solfataricus; SsMTAPII, 5¢-deoxy-5¢-methylthioadenosine phosphorylase II from S. solfataricus.

Humanized ADEPT comprised of an engineered human purine nucleoside phosphorylase and a tumor targeting peptide for treatment of cancer

“…Guanosine is a natural substrate of hPNP (21). The wild-type enzyme catalyzes the reversible phosphorolysis of guanosine to guanine and ribose-1-phosphate with high efficiency.…”

“…Crystal structures of hPNP in complex with either guanosine or inosine indicate that the N1-C2 side of these nucleotides faces Glu201 of hPNP (8, 16, 22). In fact, the carboxyl side chain of Glu201 forms hydrogen bonds with the amino group at C2 in guanosine and with the N1-hydrogen of both guanosine and inosine (16, 18, 21). Since the prodrugs contain highly electronegative fluorine or chlorine at the C2 position, replacing Glu201 with glutamine should allow the amide group in Gln to form hydrogen bonds with the C2-fluorine (or C2-Chlorine) and N1 of the prodrugs, which may enable the enzyme to accept the prodrugs with higher efficiency (Supplementary Fig.…”

Expression and Association of Group IV Nitrogenase NifD and NifH Homologs in the Non-Nitrogen-Fixing ArchaeonMethanocaldococcus jannaschii

“…Since the samples were treated with 20 percent ␤-mercaptoethanol and boiled for 20 min (conditions that would normally break protein complexes into their corresponding monomers), the appearance of multimeric forms was a surprise. However, it is often observed that multimeric protein complexes from hyperthermophilic organisms such as M. jannaschii persist under SDS-PAGE analysis in even the harshest of conditions (6).…”

Purine nucleoside phosphorylases from hyperthermophilic Archaea require a CXC motif for stability and folding

“…By contrast, in intracellular proteins from well‐known organisms, and as a result of the reductive chemical environment inside the cells [3], the presence of these covalent links is limited to proteins involved in the mechanism of the response to redox stress [4] or to proteins catalyzing oxidation–reduction processes [1,5]. Despite this classical view, recent computational, structural and biochemical studies have highlighted the critical role of these covalent links in the structural stabilization of intracellular proteins in some hyperthermophilic Archaea and Bacteria [6–12].…”

“…PfPNP displays a much higher similarity with MTAP than with PNP family members [12]. Similar to human PNP [18], PfPNP shows an absolute specificity for inosine and guanosine [12]. SsMTAPII and PfPNP show features of exceptional thermophilicity and thermostability and are characterized by stabilizing disulfide bonds.…”