title: Structural, thermodynamic, and mutational analyses of a psychrotrophic RNase HI.
abstract: Ribonuclease (RNase) HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 was overproduced in Escherichia coli, purified, and structurally and biochemically characterized. The amino acid sequence of MR-1 RNase HI is 67% identical to that of E. coli RNase HI. The crystal structure of MR-1 RNase HI determined at 2.0 A resolution was highly similar to that of E. coli RNase HI, except that the number of intramolecular ion pairs and the fraction of polar surface area of MR-1 RNase HI were reduced compared to those of E. coli RNase HI. The enzymatic properties of MR-1 RNase HI were similar to those of E. coli RNase HI. However, MR-1 RNase HI was much less stable than E. coli RNase HI. The stability of MR-1 RNase HI against heat inactivation was lower than that of E. coli RNase HI by 19 degrees C. The conformational stability of MR-1 RNase HI was thermodynamically analyzed by monitoring the CD values at 220 nm. MR-1 RNase HI was less stable than E. coli RNase HI by 22.4 degrees C in Tm and 12.5 kJ/mol in DeltaG(H2O). The thermodynamic stability curve of MR-1 RNase HI was characterized by a downward shift and increased curvature, which results in an increased DeltaCp value, compared to that of E. coli RNase HI. Site-directed mutagenesis studies suggest that the difference in the number of intramolecular ion pairs partly accounts for the difference in stability between MR-1 and E. coli RNases HI.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tadokoro
authors.forename: Dong-Ju
authors.initials: DJ
authors.lastname: You
authors.forename: Yumi
authors.initials: Y
authors.lastname: Abe
authors.forename: Hyongi
authors.initials: H
authors.lastname: Chon
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Matsumura
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; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017124:Conserved Sequence; D003460:Crystallization; D004795:Enzyme Stability; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000201:enzymology*; D012995:Solubility; D013696:Temperature; D013816:Thermodynamics; D014462:Ultrafiltration; D014508:Urea / Q000494:pharmacology; D014961:X-Ray Diffraction
chemical_list: D014508:Urea; D016914:Ribonuclease H; C078856:ribonuclease HI