Takashi Tadokoro

Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes

The prokaryotic genomes, for which complete nucleotide sequences are available, always contain at least one RNase H gene, indicating that RNase H is ubiquitous in all prokaryotic cells. Coupled with its unique substrate specificity, the enzyme has been expected to play crucial roles in the biochemical processes associated with DNA replication, gene expression and DNA repair. The physiological role of prokaryotic RNases H, especially of type 1 RNases H, has been extensively studied using Escherichia coli strains that are defective in RNase HI activity or overproduce RNase HI. However, it is not fully understood yet. By contrast, significant progress has been made in this decade in identifying novel RNases H with respect to their biochemical properties and structures, and elucidating catalytic mechanism and substrate recognition mechanism of RNase H. We review the results of these studies.

Hydrophobic effect on the stability and folding of a hyperthermophilic protein.

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HII were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol(-1) as compared with the wild-type protein. The removal of each -CH(2)- group burial decreased the stability by 5.1 kJ mol(-1) on average in the mutant proteins of Tk-RNase HII examined. This is comparable with the value of 5.3 kJ mol(-1) obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HII examined exhibited faster unfolding compared with the wild-type protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HII. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.

Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins.

BackgroundThe unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI).ResultsTo clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII) and Aquifex aeolicus (Aa-RNase HII) and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI). These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins.ConclusionsThese results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

Proline Effect on the Thermostability and Slow Unfolding of a Hyperthermophilic Protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a robust monomeric protein under kinetic control, which possesses some proline residues at the N-terminal of alpha-helices. Proline residue at the N-terminal of an alpha-helix is thought to stabilize a protein. In this work, the thermostability and folding kinetics of Tk-RNase HII were measured for mutant proteins in which a proline residue is introduced (Xaa to Pro) or removed (Pro to Ala) at the N-terminal of alpha-helices. In the folding experiments, the mutant proteins examined exhibit little influence on the remarkably slow unfolding of Tk-RNase HII. In contrast, E111P and K199P exhibit some thermostabilization, whereas P46A, P70A and P174A have some thermodestabilization. E111P/K199P and P46A/P70A double mutations cause cumulative changes in stability. We conclude that the proline effect on protein thermostability is observed in a hyperthermophilic protein, but each proline residue at the N-terminal of an alpha-helix slightly contributes to the thermostability. The present results also mean that even a natural hyperthermophilic protein can acquire improved thermostability.

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

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.

Identification of the substrate binding site in the N-terminal TBP-like domain of RNase H3

Ribonuclease H3 from Bacillus stearothermophilus (Bst‐RNase H3) has the N‐terminal TBP‐like substrate‐binding domain. To identify the substrate binding site in this domain, the mutant proteins of the intact protein and isolated N‐domain, in which six of the seventeen residues corresponding to those involved in DNA binding of TBP are individually mutated to Ala, were constructed. All of them exhibited decreased enzymatic activities and/or substrate‐binding affinities when compared to those of the parent proteins, suggesting that the N‐terminal domain of RNase H3 uses the flat surface of the β‐sheet for substrate binding as TBP to bind DNA. This domain may greatly change conformation upon substrate binding.

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.

Identification of the gene encoding a type 1 RNase H with an N‐terminal double‐stranded RNA binding domain from a psychrotrophic bacterium

The gene encoding a bacterial type 1 RNase H, termed RBD‐RNase HI, was cloned from the psychrotrophic bacterium Shewanella sp. SIB1, overproduced in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RBD‐RNase HI consists of 262 amino acid residues and shows amino acid sequence identities of 26% to SIB1 RNase HI, 17% to E. coli RNase HI, and 32% to human RNase H1. SIB1 RBD‐RNase HI has a double‐stranded RNA binding domain (RBD) at the N‐terminus, which is commonly present at the N‐termini of eukaryotic type 1 RNases H. Gel mobility shift assay indicated that this domain binds to an RNA/DNA hybrid in an isolated form, suggesting that this domain is involved in substrate binding. SIB1 RBD‐RNase HI exhibited the enzymatic activity both in vitro and in vivo. Its optimum pH and metal ion requirement were similar to those of SIB1 RNase HI, E. coli RNase HI, and human RNase H1. The specific activity of SIB1 RBD‐RNase HI was comparable to that of E. coli RNase HI and was much higher than those of SIB1 RNase HI and human RNase H1. SIB1 RBD‐RNase HI showed poor cleavage‐site specificity for oligomeric substrates. SIB1 RBD‐RNase HI was less stable than E. coli RNase HI but was as stable as human RNase H1. Database searches indicate that several bacteria and archaea contain an RBD‐RNase HI. This is the first report on the biochemical characterization of RBD‐RNase HI.

Identification of RNase HII from psychrotrophic bacterium, Shewanella sp. SIB1 as a high-activity type RNase H

The gene encoding RNase HII from the psychrotrophic bacterium, Shewanella sp. SIB1 was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RNase HII is a monomeric protein with 212 amino acid residues and shows an amino acid sequence identity of 64% to E. coli RNase HII. The enzymatic properties of SIB1 RNase HII, such as metal ion preference, pH optimum, and cleavage mode of substrate, were similar to those of E. coli RNase HII. SIB1 RNase HII was less stable than E. coli RNase HII, but the difference was marginal. The half‐lives of SIB1 and E. coli RNases HII at 30 °C were ∼ 30 and 45 min, respectively. The midpoint of the urea denaturation curve and optimum temperature of SIB1 RNase HII were lower than those of E. coli RNase HII by ∼ 0.2 m and ∼ 5 °C, respectively. However, SIB1 RNase HII was much more active than E. coli RNase HII at all temperatures studied. The specific activity of SIB1 RNase HII at 30 °C was 20 times that of E. coli RNase HII. Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII represent low‐ and high‐activity type RNases H, respectively, in SIB1. In contrast, RNases HI and HII represent high‐ and low‐activity type RNases H, respectively, in E. coli. We propose that bacterial cells usually contain low‐ and high‐activity type RNases H, but these types are not correlated with RNase H families.

Role of polar and nonpolar residues at the active site for PPIase activity of FKBP22 from Shewanella sp. SIB1

FKBP22 from the psychotropic bacterium Shewanella sp. SIB1 is a homodimeric protein with peptidyl prolyl cis–trans isomerase (PPIase) activity. According to a tertiary model, several nonpolar residues including Trp157 and Phe197 form a substrate‐binding cavity, and Asp137 and Arg142, which form a salt bridge, are located at the edge of this cavity. To analyze the role of these residues, nine single (D137A, R142A, W157A/F/Y, F197A/L/Y/W) and one double (D137A/R142A) mutant protein of SIB1 FKBP22 were constructed. The far‐ and near‐UV CD spectra of these mutant proteins suggest that the mutations at Asp137 and Arg142 do not seriously affect the protein structure, while those at Trp157 and Phe197 cause a local conformational change around the mutation site. Each mutation decreased the PPIase activities of SIB1 FKBP22 for peptide and protein substrates similarly without seriously affecting chaperone function. This result indicates that SIB1 FKBP22 does not require PPIase activity for chaperone function. The PPIase activities of R142A, D137A and D137A/R142A decreased in this order, suggesting that Asp137 and Arg142 play a principal and auxiliary role in catalytic function, respectively, but Arg142 can function as a substitute of Asp137. Because the PPIase activity of SIB1 FKBP22 was not fully lost by the removal of all polar residues around the active site, the desolvation effect may also contribute to the enzymatic activity. However, the mutations of Trp157 to Phe or Phe197 to Leu greatly decrease the enzymatic activity, suggesting that the shape of the substrate‐binding cavity is also important for enzymatic activity.