PDBsum entry 2v6i

Hydrolase

PDB id: 2v6i

Contents

Protein chain

421 a.a. *

Ligands

POP

Waters ×171

* Residue conservation analysis

PDB id:

2v6i

Name:

Hydrolase

Title:

Kokobera virus helicase

Structure:

RNA helicase. Chain: a. Fragment: helicase domain, residues 1678-2108. Engineered: yes

Source:

Kokobera virus. Organism_taxid: 44024. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Expression_system_variant: c41.

Resolution:

2.10Å R-factor: 0.234 R-free: 0.275

Authors:

S.Speroni,L.De Colibus,B.Coutard,B.Canard,A.Mattevi

Key ref:

S.Speroni et al. (2008). Structure and biochemical analysis of Kokobera virus helicase. Proteins, 70, 1120-1123. PubMed id: 18004778 DOI: 10.1002/prot.21812

Date:

18-Jul-07 Release date: 25-Mar-08

Protein chain

Q32ZD5  (POLG_KOKV) -  Genome polyprotein from Kokobera virus

Seq: 3410 a.a.

Struc: 421 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: E.C.2.1.1.56 - mRNA (guanine-N(7))-methyltransferase.
• Reaction: a 5'-end (5'-triphosphoguanosine)-ribonucleoside in mRNA + S-adenosyl-L- methionine = a 5'-end (N(7)-methyl 5'-triphosphoguanosine)-ribonucleoside in mRNA + S-adenosyl-L-homocysteine
• Enzyme class 2: E.C.2.1.1.57 - methyltransferase cap1.
• Reaction: a 5'-end (N(7)-methyl 5'-triphosphoguanosine)-ribonucleoside in mRNA + S-adenosyl-L-methionine = a 5'-end (N(7)-methyl 5'-triphosphoguanosine)- (2'-O-methyl-ribonucleoside) in mRNA + S-adenosyl-L-homocysteine + H⁺
• Enzyme class 3: E.C.2.7.7.48 - RNA-directed Rna polymerase.
• Reaction: RNA(n) + a ribonucleoside 5'-triphosphate = RNA(n+1) + diphosphate

RNA(n) + ribonucleoside 5'-triphosphate = RNA(n+1) (Bound ligand (Het Group name = POP) corresponds exactly) + diphosphate

• Enzyme class 4: E.C.3.4.21.91 - flavivirin.
• Reaction: Selective hydrolysis of Xaa-Xaa-|-Xbb bonds in which each of the Xaa can be either Arg or Lys and Xbb can be either Ser or Ala.
• Enzyme class 5: E.C.3.6.1.15 - nucleoside-triphosphate phosphatase.
• Reaction: a ribonucleoside 5'-triphosphate + H2O = a ribonucleoside 5'-diphosphate + phosphate + H⁺

ribonucleoside 5'-triphosphate + H2O = ribonucleoside 5'-diphosphate + phosphate + H(+) (Bound ligand (Het Group name = POP) matches with 55.56% similarity)

• Enzyme class 6: E.C.3.6.4.13 - Rna helicase.
• Reaction: ATP + H2O = ADP + phosphate + H⁺

ATP + H2O = ADP + phosphate + H(+) (Bound ligand (Het Group name = POP) matches with 55.56% similarity)

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1002/prot.21812

Proteins 70:1120-1123 (2008)

PubMed id: 18004778

Structure and biochemical analysis of Kokobera virus helicase.

S.Speroni, L.De Colibus, E.Mastrangelo, E.Gould, B.Coutard, N.L.Forrester, S.Blanc, B.Canard, A.Mattevi.

ABSTRACT

No abstract given.

Selected figure(s)

Figure 1.
Figure 1. (A) Helicase activity assay for wild-type (top) and Met47Thr mutant (bottom). The substrate of the partially double-stranded helicase assay[18] was obtained by annealing RNA synthetic oligonucleotide whose sequence was designed to produce a 16-base-pair duplex with a 14-nucleotide 3 overhand in the longer strand (Primm, Milan-Italy). To form a double-stranded substrate, the 5 -CACCUCUCUAGAGUCGACCUGCAGGCAUCG-3 strand was labelled with [ -^32P]ATP at its 5 end by using T4 polynucleotide kinase, and annealed with the complementary primer 5 -CGACUCUAGAGAGGUG-3 . The annealed duplex was purified with Sephadex G25 columns (GE-Healthcare). The assay was performed with 20 L of the reaction buffer containing 25 mM Hepes pH 7.5, 1 mM MgCl[2], 2 mM DTT, 2 mM ATP, 5% glycerol, 5U RNAsin for the RNA substrate and 10 fmol of RNA substrate. The reaction was started by adding the recombinant proteins at various concentrations (50-1000 nM), or an equivalent volume of the buffer, and stopped after 30 min at 37°C by adding 6 L of loading dye (50% EDTA, 0.5% SDS, 50% glycerol, 0.01% bromophenol blue). The helicase assay mixtures were resolved by electrophoresis through nondenaturing 17% polyacrylamide gels that were dried and analyzed by phosphoimage (Typhoon, GE-Healthcare). The percentage of duplex unwinding was calculated using the ImageQuant software (Amersham Bioscience) by comparing the intensities of the two bands. (B) Ribbon representation of Kokobera virus helicase. Domain 1 (residues 2-138), domain 2 (139-297), and domain 3 (297-431) are colored orange, blue, and green, respectively. The bound pyrophosphate ion is shown in ball-and-stick representation (oxygen in red and phosphorous in green). Residues 60-68 are disordered and not present in the refined model. (C) Close-up view of the ATPase site and of Met47. The orientation and domain colors are as in (B). Carbons are in yellow, nitrogen in blue, oxygen in red, and phosphorous in green. Figures for (B) and (C) were generated with Pymol (www.pymol.org).

The above figure is reprinted by permission from John Wiley & Sons, Inc.: Proteins (2008, 70, 1120-1123) copyright 2008.