The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity.
Thermotoga maritima ribonuclease H (RNase H) I (Tma-RNase HI) contains a hybrid binding domain (HBD) at the N-terminal region. To analyze the role of this HBD, Tma-RNase HI, Tma-W22A with the single mutation at the HBD, the C-terminal RNase H domain (Tma-CD) and the N-terminal domain containing the HBD (Tma-ND) were overproduced in Escherichia coli, purified and biochemically characterized. Tma-RNase HI prefers Mg(2+) to Mn(2+) for activity, and specifically loses most of the Mg(2+)-dependent activity on removal of the HBD and 87% of it by the mutation at the HBD. Tma-CD lost the ability to suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that the HBD is responsible for in vivo RNase H activity. The cleavage-site specificities of Tma-RNase HI are not significantly changed on removal of the HBD, regardless of the metal cofactor. Binding analyses of the proteins to the substrate using surface plasmon resonance indicate that the binding affinity of Tma-RNase HI is greatly reduced on removal of the HBD or the mutation. These results indicate that there is a correlation between Mg(2+)-dependent activity and substrate binding affinity. Tma-CD was as stable as Tma-RNase HI, indicating that the HBD is not important for stability. The HBD of Tma-RNase HI is important not only for substrate binding, but also for Mg(2+)-dependent activity, probably because the HBD affects the interaction between the substrate and enzyme at the active site, such that the scissile phosphate group of the substrate and the Mg(2+) ion are arranged ideally.
Jongruja
Nujarin
N
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.
You
Dong-Ju
DJ
Kanaya
Eiko
E
Koga
Yuichi
Y
Takano
Kazufumi
K
Kanaya
Shigenori
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005816:Genetic Complementation Test; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D008345:Manganese; D012965:Sodium Chloride; D016914:Ribonuclease H; C078856:ribonuclease HI; D008274:Magnesium