Literature summary for 3.1.13.2 extracted from

Villa, J.A.; Pike, D.P.; Patel, K.B.; Lomonosova, E.; Lu, G.; Abdulqader, R.; Tavis, J.E.
Purification and enzymatic characterization of the hepatitis B virus ribonuclease H, a new target for antiviral inhibitors (2016), Antiviral Res., 132, 186-195 .

Application

Application	Comment	Organism
drug development	an active recombinant His6-tagged HBV RNaseH is suitable for low-throughput drug screening and can be used to discover enzyme inhibitors, many of which work against viral replication in cells	Hepatitis B virus

Cloned(Commentary)

Cloned (Comment)	Organism
HBV RNaseH sequences from genotype C isolate V01460 encoding residues corresponding to amino acids 688 to 844 of the HBV polymerase (reference strain ADW2, genotype A) are cloned by gene synthesis into pMAL-c5X-His with sequences encoding a His6-tag appended to the 3' end of the RNaseH gene to create pMal-HRHgtC. Sequences encoding Ala-Gly-Ala are inserted between the maltose binding protein (MBP) and HBV RNaseH sequences, and sequences encoding Gly-Ala-Gly are inserted between sequences for the RNaseH and the His6-tag. Recombinant expression of wild-type and mutant His6-tagged enzymes in Escherichia coli strain BL21(DE3)	Hepatitis B virus

Cloned (Comment)

Organism

HBV RNaseH sequences from genotype C isolate V01460 encoding residues corresponding to amino acids 688 to 844 of the HBV polymerase (reference strain ADW2, genotype A) are cloned by gene synthesis into pMAL-c5X-His with sequences encoding a His6-tag appended to the 3' end of the RNaseH gene to create pMal-HRHgtC. Sequences encoding Ala-Gly-Ala are inserted between the maltose binding protein (MBP) and HBV RNaseH sequences, and sequences encoding Gly-Ala-Gly are inserted between sequences for the RNaseH and the His6-tag. Recombinant expression of wild-type and mutant His6-tagged enzymes in Escherichia coli strain BL21(DE3)

Hepatitis B virus

Protein Variants

Protein Variants	Comment	Organism
D702A/E731A	sequences encoding HBV RNaseH residues 809-844 are deleted from pMal-HRHgtC to create pMal-HRHgtCDELTA5. Active site residues D702 and E731 are mutated to alanines to create pMAL-HRHgtC(D702A/E731A) which encodes an inactive RNaseH	Hepatitis B virus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required, four RNaseH active site conserved carboxylates (the DEDD motif) coordinate two divalent cations, usually Mg2+	Hepatitis B virus

Organism

Organism	UniProt	Comment	Textmining
Hepatitis B virus	-	HBV, genotype C isolate V01460	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant C-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) cell 54000 x g supernatant by nickel affinity chromatography and dialysis. Purification in the presence of ATP and Mg2+ greatly increases the specific activity of the RNaseH in an DNA oligonucleotide-directed RNA cleavage assay	Hepatitis B virus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	the 3'-5' exoribonuclease activity of the HBV RNaseH is determined. The enzyme also shows endolytic activity (EC 3.1.26.4) in DNA oligonucleotide (ODN)-directed RNA cleavage assays are conducted. Substrate specificity of the HBV RNaseH, overview	Hepatitis B virus	?	-	?

Synonyms

Synonyms	Comment	Organism
More	cf. EC 3.1.26.4	Hepatitis B virus
ribonuclease H	-	Hepatitis B virus
RNaseH	-	Hepatitis B virus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
42	-	assay at	Hepatitis B virus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Hepatitis B virus

General Information

General Information	Comment	Organism
evolution	RNaseH enzymes belong to the nucleotidyl transferase superfamily whose members share a similar protein fold and catalytic mechanism	Hepatitis B virus
additional information	the RNaseH active site contains four conserved carboxylates (the DEDD motif) that coordinate two divalent cations, usually Mg2+. The RNA cleavage mechanism requires both cations to promote a hydroxyl-mediated nucleophilic scission reaction	Hepatitis B virus
physiological function	the endonucleolytic RNaseH activity (EC 3.1.26.4) requires an DNA:RNA duplex 14 nt or more and cannot tolerate a stem-loop in either the RNA or DNA strands. It tolerates a nick in the DNA strand but not a gap. The RNaseH has no obvious sequence specificity or positional dependence within the RNA, and it cuts the RNA at multiple positions even within the minimal 14 nt duplex. The RNaseH also possesses a processive 3'-5' exoribonuclease activity (EC 3.1.13.2) that is slower than the endonucleolytic reaction. The HBV reverse transcription mechanism features an initial endoribonucleolytic cut, 3'-5' degradation of RNA, and a sequence-independent terminal RNA cleavage	Hepatitis B virus