Remarkable stabilization of a psychrotrophic RNase HI by a combination of thermostabilizing mutations identified by the suppressor mutation method.
Ribonuclease HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 (So-RNase HI) is much less stable than Escherichia coli RNase HI (Ec-RNase HI) by 22.4 degrees C in T m and 12.5 kJ mol (-1) in Delta G(H 2O), despite their high degrees of structural and functional similarity. To examine whether the stability of So-RNase HI increases to a level similar to that of Ec-RNase HI via introduction of several mutations, the mutations that stabilize So-RNase HI were identified by the suppressor mutation method and combined. So-RNase HI and its variant with a C-terminal four-residue truncation (154-RNase HI) complemented the RNase H-dependent temperature-sensitive (ts) growth phenotype of E. coli strain MIC3001, while 153-RNase HI with a five-residue truncation could not. Analyses of the activity and stability of these truncated proteins suggest that 153-RNase HI is nonfunctional in vivo because of a great decrease in stability. Random mutagenesis of 153-RNase HI using error-prone PCR, followed by screening for the revertants, allowed us to identify six single suppressor mutations that make 153-RNase HI functional in vivo. Four of them markedly increased the stability of the wild-type protein by 3.6-6.7 degrees C in T m and 1.7-5.2 kJ mol (-1) in Delta G(H 2O). The effects of these mutations were nearly additive, and combination of these mutations increased protein stability by 18.7 degrees C in T m and 12.2 kJ mol (-1) in Delta G(H 2O). These results suggest that several residues are not optimal for the stability of So-RNase HI, and their replacement with other residues strikingly increases it to a level similar to that of the mesophilic counterpart.
Tadokoro
Takashi
T
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Matsushita
Kyoko
K
Abe
Yumi
Y
Rohman
Muhammad Saifur
MS
Koga
Yuichi
Y
Takano
Kazufumi
K
Kanaya
Shigenori
S
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011489:Protein Denaturation / Q000187:drug effects; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020592:Shewanella / Q000201:enzymology / Q000235:genetics; D013489:Suppression, Genetic / Q000235:genetics*; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D014508:Urea; D016914:Ribonuclease H; C078856:ribonuclease HI