Literature summary for 2.7.7.8 extracted from

Hardwick, S.W.; Gubbey, T.; Hug, I.; Jenal, U.; Luisi, B.F.
Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly (2012), Open biology, 2, 120028.

Cloned(Commentary)

Cloned (Comment)	Organism
gene pnp, cloned in a plasmid with rne gene, encoding endoribonuclease RNase E, fragments, expression of N-terminally GST- or His6-tagged enzyme fragments in Escherichia coli	Caulobacter vibrioides

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant RNA-free and RNA-bound PNPase, as protein-peptide and protein-RNA complexes, mixing of PNPase recognition peptide from RNase E, KPRRGWWRR, with apo-PNPase in a 2:1 ratio, sitting drop vapour diffusion method, mixing of equal volumes of protein solution and cyrstallization solution, containing w/v PEG 3350, 0.1 M Bis-Tris, pH 5.5, 0.1 M ammonium acetate for RNA-bound crystals, and with the apo-enzyme containing w/v PEG3000, 0.1 M trisodium citrate, pH 5.5, for hexagonal crystals and w/v PEG 3350, 0.15 M DL-malic acid for rhombohedral crystals, 18°C, 1 week, X-ray diffraction structure determination aand analysis at 2.6-3.3 A resolution, molecular replacement	Caulobacter vibrioides

Crystallization (Comment)

Organism

purified recombinant RNA-free and RNA-bound PNPase, as protein-peptide and protein-RNA complexes, mixing of PNPase recognition peptide from RNase E, KPRRGWWRR, with apo-PNPase in a 2:1 ratio, sitting drop vapour diffusion method, mixing of equal volumes of protein solution and cyrstallization solution, containing w/v PEG 3350, 0.1 M Bis-Tris, pH 5.5, 0.1 M ammonium acetate for RNA-bound crystals, and with the apo-enzyme containing w/v PEG3000, 0.1 M trisodium citrate, pH 5.5, for hexagonal crystals and w/v PEG 3350, 0.15 M DL-malic acid for rhombohedral crystals, 18°C, 1 week, X-ray diffraction structure determination aand analysis at 2.6-3.3 A resolution, molecular replacement

Caulobacter vibrioides

Organism

Organism	UniProt	Comment	Textmining
Caulobacter vibrioides	-	gene pnp	-
Caulobacter vibrioides NA1000	-	gene pnp	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally GST-tagged enzyme fragments from Escherichia coli by glutathione affinity chromatography, of His6-tagged enzyme fragments by nickel affinity chromatography, followed by gel filtration in both procedures	Caulobacter vibrioides

Subunits

Subunits	Comment	Organism
trimer	the enzyme has a ring-like, trimeric architecture that creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, domain organization with modular organization of conserved structural domains, overview	Caulobacter vibrioides

Synonyms

Synonyms	Comment	Organism
PNPase	-	Caulobacter vibrioides
polynucleotide phosphorylase	-	Caulobacter vibrioides

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Caulobacter vibrioides

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Caulobacter vibrioides

General Information

General Information	Comment	Organism
evolution	two domains, both resembling closely the phosphorolytic exoribonuclease RNase PH, EC 27.7.56, almost certainly have originated from duplication and fusion of an ancestral gene. While the C-terminal RNase PH-like domain catalyses phosphorolytic attack of RNA, the N-terminal domain has lost this capacity. Instead, it contributes to the ring-like quaternary structure of the trimeric PNPase assembly	Caulobacter vibrioides
additional information	the enzyme has a ring-like, trimeric architecture that creates a central channel where phosphorolytic active sites reside, with asymmetry within the catalytic core of the enzyme. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains. In the RNA-free form, the S1 domains adopt a splayed conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. Structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Access to the PNPase active sites is through the central channel, which can accommodate single-stranded RNA with some structural adjustment of a constricted aperture at the channel entrance, residues and motifs involved in RNA directionality, recognition, and quarternary changes in the core, structure-function-relationship, detailed overview	Caulobacter vibrioides
physiological function	polynucleotide phosphorylase is an exoribonuclease that cleaves single-stranded RNA substrates with 3' -5' directionality and processive behaviour	Caulobacter vibrioides