title: Activity, stability, and structure of metagenome-derived LC11-RNase H1, a homolog of Sulfolobus tokodaii RNase H1.
abstract: Metagenome-derived LC11-RNase H1 is a homolog of Sulfolobus tokodaii RNase H1 (Sto-RNase H1). It lacks a C-terminal tail, which is responsible for hyperstabilization of Sto-RNase H1. Sto-RNase H1 is characterized by its ability to cleave not only an RNA/DNA hybrid but also a double-stranded RNA (dsRNA). To examine whether LC11-RNase H1 also exhibits both RNase H and dsRNase activities, LC11-RNase H1 was overproduced in Escherichia coli, purified, and characterized. LC11-RNase H1 exhibited RNase H activity with similar metal ion preference, optimum pH, and cleavage mode of substrate with those of Sto-RNase H1. However, LC11-RNase H1 did not exhibit dsRNase activity at any condition examined. LC11-RNase H1 was less stable than Sto-RNases H1 and its derivative lacking the C-terminal tail (Sto-RNase H1ΔC6) by 37 and 13 °C in T(m) , respectively. To understand the structural bases for these differences, the crystal structure of LC11-RNase H1 was determined at 1.4 Å resolution. The LC11-RNase H1 structure is highly similar to the Sto-RNase H1 structure. However, LC11-RNase H1 has two grooves on protein surface, one containing the active site and the other containing DNA-phosphate binding pocket, while Sto-RNase H1 has one groove containing the active site. In addition, LC11-RNase H1 contains more cavities and buried charged residues than Sto-RNase H1. We propose that LC11-RNase H1 does not exhibit dsRNase activity because dsRNA cannot fit to the two grooves on protein surface and that LC11-RNase H1 is less stable than Sto-RNase H1ΔC6 because of the increase in cavity volume and number of buried charged residues.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Tri-Nhan
authors.initials: TN
authors.lastname: Nguyen
authors.forename: Clement
authors.initials: C
authors.lastname: Angkawidjaja
authors.forename: Eiko
authors.initials: E
authors.lastname: Kanaya
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Koga
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Takano
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D016680:Genome, Bacterial; D006358:Hot Temperature; D054892:Metagenome; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D055672:Static Electricity; D013379:Substrate Specificity; D016944:Sulfolobus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D016914:Ribonuclease H; C078856:ribonuclease HI