No abstract

Background: PfEMP1 proteins cause Plasmodium falciparum-infected erythrocytes to bind human tissues during malaria.Results: The IT4VAR13 ectodomain is rigid, elongated, and monomeric, presenting a binding site for its ligand, ICAM-1.Conclusion: The IT4VAR13 ectodomain is unlike that of VAR2CSA, a PfEMP1 that adopts a compact structure with multiple domains contributing to ligand binding.Significance: PfEMP1 proteins have evolved diverse architectures to facilitate ligand recognition.

The use of nucleoside phosphorylases (NPs; EC 2.4.2.n) represents a convenient alternative to the chemical route for the synthesis of natural and modified nucleosides. We purified four recombinantly expressed nucleoside phosphorylases from the bacterial pathogens Citrobacter koseri, Clostridium perfringens, and Streptococcus pyogenes (CkPNPI, CkPNPII, CpUP, SpUP) and their substrate specificity was investigated towards either natural pyrimidine or purine nucleosides and some analogues, namely, arabinosyladenine (araA) and 2′,3′‐dideoxyinosine (ddI). A 2–3 % activity towards these latter compounds (compared to the natural substrates) was observed. Enzyme activities were compared to the specificities obtained for the enzymes pyrimidine nucleoside phosphorylase from Bacillus subtilis (BsPyNP) and purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNPII) previously reported by some of the authors. The enzymes displaying the suitable specificity for the synthesis of araA and ddI were immobilized on aldehyde–agarose. The immobilized preparations were highly stable at alkaline pH and in the presence of methanol or acetonitrile as cosolvent. They were used in the synthesis of araA and ddI by a one‐pot, bienzymatic transglycosylation achieving 74 and 44 % conversion, respectively.

Two open reading frames in the genome of Sulfolobus solfataricus (SSO2342 [corrected] and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2342 [corrected] was annotated as a gene (gpT-1) encoding a 6-oxopurine phosphoribosyltransferase (PRTase), the protein product turned out to be a PRTase highly specific for adenine and we suggest that the reading frame should be renamed apT. The other reading frame SSO2424 (gpT-2) proved to be a true 6-oxopurine PRTase active with hypoxanthine, xanthine and guanine as substrates, and we suggest that the gene should be renamed gpT. Both enzymes exhibited unusual profiles of activity versus pH. The adenine PRTase showed the highest activity at pH 7.5-8.5, but had a distinct peak of activity also at pH 4.5. The 6-oxo PRTase showed maximal activity with hypoxanthine and guanine around pH 4.5, while maximal activity with xanthine was observed at pH 7.5. We discuss likely reasons why SSO2342 [corrected] in S. solfataricus and similar open reading frames in other Crenarchaeota could not be identified as genes encoding APRTase.

Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2′-deoxyuridine to uracil and ribose 1-phosphate or 2′-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 Å resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild type SpUP showed that substrate specificity is similar to that of EcUP, while EcUP is about sevenfold more efficient than SpUP. Biochemical studies on active site mutant SpUP showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.