PDBsum entry 2yv0

Hydrolase

PDB id: 2yv0

Contents

Protein chain

155 a.a. *

Waters ×217

* Residue conservation analysis

PDB id:

2yv0

Name:

Hydrolase

Title:

Structural and thermodynamic analyses of e. Coli ribonuclease hi variant with quintuple thermostabilizing mutations

Structure:

Ribonuclease hi. Chain: x. Synonym: rnase hi, ribonuclease h, rnase h. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: rnha. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.40Å R-factor: 0.182

Authors:

M.Haruki,T.Motegi,T.Tadokoro,Y.Koga,K.Takano,S.Kanaya

Key ref:

M.Haruki et al. (2007). Structural and thermodynamic analyses of Escherichia coli RNase HI variant with quintuple thermostabilizing mutations. Febs J, 274, 5815-5825. PubMed id: 17944939

Date:

06-Apr-07 Release date: 18-Mar-08

Protein chain

P0A7Y4  (RNH_ECOLI) -  Ribonuclease HI from Escherichia coli (strain K12)

Seq: 155 a.a.

Struc: 155 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.26.4 - ribonuclease H.
• Reaction: Endonucleolytic cleavage to 5'-phosphomonoester.

Febs J 274:5815-5825 (2007)

PubMed id: 17944939

Structural and thermodynamic analyses of Escherichia coli RNase HI variant with quintuple thermostabilizing mutations.

M.Haruki, M.Tanaka, T.Motegi, T.Tadokoro, Y.Koga, K.Takano, S.Kanaya.

ABSTRACT

A combination of five thermostabilizing mutations, Gly23-->Ala, His62-->Pro, Val74-->Leu, Lys95-->Gly, and Asp134-->His, has been shown to additively enhance the thermostability of Escherichia coli RNase HI [Akasako A, Haruki M, Oobatake M & Kanaya S (1995) Biochemistry34, 8115-8122]. In this study, we determined the crystal structure of the protein with these mutations (5H-RNase HI) to analyze the effects of the mutations on the structure in detail. The structures of the mutation sites were almost identical to those of the mutant proteins to which the mutations were individually introduced, except for G23A, for which the structure of the single mutant protein is not available. Moreover, only slight changes in the backbone conformation of the protein were observed, and the interactions of the side chains were almost conserved. These results indicate that these mutations almost independently affect the protein structure, and are consistent with the fact that the thermostabiling effects of the mutations are cumulative. We also determined the protein stability curve describing the temperature dependence of the free energy of unfolding of 5H-RNase HI to elucidate the thermostabilization mechanism. The maximal stability for 5H-RNase HI was as high as that for the cysteine-free variant of Thermus thermophilus RNase HI. In contrast, the heat capacity of unfolding for 5H-RNase H was similar to that for E. coli RNase HI, which is considerably higher than that for T. thermophilus RNase HI. These results suggest that 5H-RNase HI is stabilized, in part, by the thermostabilization mechanism adopted by T. thermophilus RNase HI.