Literature summary for 3.1.13.3 extracted from

Lee, C.W.; Park, S.H.; Jeong, C.S.; Cha, S.S.; Park, H.; Lee, J.H.
Structural basis of small RNA hydrolysis by oligoribonuclease (CpsORN) from Colwellia psychrerythraea strain 34H (2019), Sci. Rep., 9, 2649 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene orn, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha	Colwellia psychrerythraea

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant unliganded wild-type enzyme and pNP-TMP-bound D163A mutant, and uridine-bound wild-type and mutant enzymes, sitting drop vapour diffusion method, crystals of unliganded CpsORN are obtained from 15% w/v PEG 3350 and 0.1 M magnesium formate, 20°C, for larger single crystals, by hanging-drop vapour-diffusion method, the drop volume is increased from 200 nl to 0.001 ml against 0.5 ml reservoir solution, optimised single crystals are obtained in the same condition as that of initial screening. The crystals of pNP-TMP-bound CpsORN are observed under several conditions. Among them, the most suitable crystals are obtained from the 1.4 M ammonium tartrate dibasic and 0.1 M Tris, pH 8.5, condition. The crystals of linked RNA-bound form are obtained with 0.04 M citric acid, 0.06 M Bis-Tris propane pH 6.4, and 20% w/v PEG 3350. Crystals of uridine-bound form are obtained with 0.2 M potassium thiocyanate and 20% w/v PEG 3350. X-ray diffraction structure determination and analysis at 2.0-2.7 A resolution, molecular replacement and modelling	Colwellia psychrerythraea

Crystallization (Comment)

Organism

purified recombinant unliganded wild-type enzyme and pNP-TMP-bound D163A mutant, and uridine-bound wild-type and mutant enzymes, sitting drop vapour diffusion method, crystals of unliganded CpsORN are obtained from 15% w/v PEG 3350 and 0.1 M magnesium formate, 20°C, for larger single crystals, by hanging-drop vapour-diffusion method, the drop volume is increased from 200 nl to 0.001 ml against 0.5 ml reservoir solution, optimised single crystals are obtained in the same condition as that of initial screening. The crystals of pNP-TMP-bound CpsORN are observed under several conditions. Among them, the most suitable crystals are obtained from the 1.4 M ammonium tartrate dibasic and 0.1 M Tris, pH 8.5, condition. The crystals of linked RNA-bound form are obtained with 0.04 M citric acid, 0.06 M Bis-Tris propane pH 6.4, and 20% w/v PEG 3350. Crystals of uridine-bound form are obtained with 0.2 M potassium thiocyanate and 20% w/v PEG 3350. X-ray diffraction structure determination and analysis at 2.0-2.7 A resolution, molecular replacement and modelling

Colwellia psychrerythraea

Protein Variants

Protein Variants	Comment	Organism
D163A	site-directed mutagenesis, mutation of the catalytic residue, inactive mutant	Colwellia psychrerythraea
H158A	site-directed mutagenesis, the mutant completely loses its hydrolytic cleavage ability	Colwellia psychrerythraea
H66A	site-directed mutagenesis, mutation is located on the alpha2 helix, inactive mutant	Colwellia psychrerythraea
S108A	site-directed mutagenesis, the mutant exhibits similar activity as the wild-type	Colwellia psychrerythraea
S167A	site-directed mutagenesis, mutant exhibits similar activity as the wild-type	Colwellia psychrerythraea
Y129A	site-directed mutagenesis, mutant exhibits similar activity as the wild-type	Colwellia psychrerythraea
Y129F	site-directed mutagenesis, mutant exhibits similar activity as the wild-type	Colwellia psychrerythraea

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required for catalysis	Colwellia psychrerythraea
Mn2+	activates	Colwellia psychrerythraea

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
36000	-	analytical ultracentrifugation	Colwellia psychrerythraea

Organism

Organism	UniProt	Comment	Textmining
Colwellia psychrerythraea	Q47VZ4	i.e. Vibrio psychroerythus	-
Colwellia psychrerythraea ATCC BAA-681	Q47VZ4	i.e. Vibrio psychroerythus	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Colwellia psychrerythraea

Reaction

Reaction	Comment	Organism	Reaction ID
exonucleolytic cleavage of oligonucleotides to yield nucleoside 5'-phosphates	RNA binding and hydrolysis mechanism of oligoribonuclease CpsORN, overview. The His158 residue shows inward movement toward the active site to interact with the cleaved uridine phosphate, in the two-uridine complex structure. The side chain of His158 is also near the cleavage site of the U-U RNA substrate. The His158 is an activator of the water molecule during the enzymatic reaction of enzyme CpsORN. The manganese ion forms a new interaction with the 3'-oxygen of the ribose in the U1 ribonucleotide	Colwellia psychrerythraea	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
4-nitrophenyl-TMP + H2O	artificial assay substrate	Colwellia psychrerythraea	4-nitrophenol + TMP	-	?
4-nitrophenyl-TMP + H2O	artificial assay substrate	Colwellia psychrerythraea ATCC BAA-681	4-nitrophenol + TMP	-	?
additional information	CpsORN activities on 5-mer RNA or DNA are determined using custom-made 5'-fluorescein-labelled oligonucleotides, and assay on 24-mer RNA oligonucleotide. Small RNA hydrolysis mechanism of CpsORN, mechanism, overview	Colwellia psychrerythraea	?	-	?
additional information	CpsORN activities on 5-mer RNA or DNA are determined using custom-made 5'-fluorescein-labelled oligonucleotides, and assay on 24-mer RNA oligonucleotide. Small RNA hydrolysis mechanism of CpsORN, mechanism, overview	Colwellia psychrerythraea ATCC BAA-681	?	-	?

Subunits

Subunits	Comment	Organism
dimer	2 * 20600, sequence calcualtion	Colwellia psychrerythraea
More	CpsORN is a tight dimer, with two separated active sites and one divalent metal cation ion in each active site. The dimerisation of CpsORN is mediated by hydrophobic interactions between the alpha7, alpha8, and alpha9 helices	Colwellia psychrerythraea

Synonyms

Synonyms	Comment	Organism
CpsORN	-	Colwellia psychrerythraea
oligoribonuclease	-	Colwellia psychrerythraea

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Colwellia psychrerythraea

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Colwellia psychrerythraea

General Information

General Information	Comment	Organism
additional information	structure analysis, identification of active site structure and key residues responsible for enzymatic catalysis of CpsORN, the active site of CpsORN has a negatively charged surface, created by Asp12, Glu14, Asp112, and Asp163, overview. His66 is located near the active site and potentially stabilises the negative charge of the nucleotide. His66 residue also plays a significant role in the cleavage mechanism. His66 and His158 are completely conserved among other ORNs. Residues Ser108 and Tyr129 are not essential for the RNase activity of CpsORN	Colwellia psychrerythraea
physiological function	cells regulate their intracellular mRNA levels by using specific ribonucleases. Oligoribonuclease (ORN) is a 3'-5' exoribonuclease for small RNA molecules, important in RNA degradation and re-utilisation	Colwellia psychrerythraea