Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis

Naoto Ohtani, Mitsuru Haruki, Masaaki Morikawa and Shigenori Kanaya,1

Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2–1 Yamadaoka, Suita, Osaka 565-0871, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The rnhA gene encoding RNase HI from a psychrotrophic bacterium, Shewanella sp. SIB1, was cloned, sequenced and overexpressed in an rnh mutant strain of Escherichia coli. SIB1 RNase HI is composed of 157 amino acid residues and shows 63% amino acid sequence identity to E.coli RNase HI. Upon induction, the recombinant protein accumulated in the cells in an insoluble form. This protein was solubilized and purified in the presence of 7 M urea and refolded by removing urea. Determination of the enzymatic activity using M13 DNA–RNA hybrid as a substrate revealed that the enzymatic properties of SIB1 RNase HI, such as divalent cation requirement, pH optimum and cleavage mode of a substrate, are similar to those of E.coli RNase HI. However, SIB1 RNase HI was much less stable than E.coli RNase HI and the temperature (T1/2) at which the enzyme loses half of its activity upon incubation for 10 min was ~25°C for SIB1 RNase HI and ~60°C for E.coli RNase HI. The optimum temperature for the SIB1 RNase HI activity was also shifted downward by 20°C compared with that of E.coli RNase HI. Nevertheless, SIB1 RNase HI was less active than E.coli RNase HI even at low temperatures. The specific activity determined at 10°C was 0.29 units/mg for SIB1 RNase HI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesis studies suggest that the amino acid substitution in the middle of the {alpha}I-helix (Pro52 for SIB1 RNase HI and Ala52 for E.coli RNase HI) partly accounts for the difference in the stability and activity between SIB1 and E.coli RNases HI.

Keywords: cold-adaptation/psychrotroph/ribonuclease H/site-directed mutagenesis/thermal stability


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Microorganisms that can grow at 0–4°C but cannot grow at temperatures higher than 30°C, such as psychrophiles and psychrotrophs, usually produce cold-adapted enzymes. A cold-adapted enzyme has been specified by the increase in the catalytic efficiency at low temperatures, the downward shift in the apparent optimum temperature for activity and the reduction in the stability at moderate temperatures compared with those of its mesophilic counterpart (Feller et al., 1996Go; Gerday et al., 1997Go,1999Go; Marshall, 1997Go; Russell, 2000Go). Because these properties are useful in various applications (Gerday et al., 2000Go), understanding of the structural determinants of the cold-adaptation is important not only to answer the questions as to how these enzymes adapt to a cold environment, but also to develop a technique to engineer cold-adapted enzymes in a rational manner. However, the molecular basis for cold-adaptation remains largely unknown. One of the promising strategies to analyze the adaptation mechanisms of enzymes to cold environment is to compare the structure and function of a given enzyme from psychrophiles or psychrotrophs with those of the mesophilic counterpart and identify the amino acid substitutions responsible for these differences.

Ribonuclease H (RNase H) (EC 3.1.26.4) specifically cleaves the RNA strand of RNA–DNA hybrids (Crouch and Dirksen, 1882). The enzyme is ubiquitously present in various organisms and thought to be involved in DNA replication and repair. Based on the amino acid sequence information, the RNase H enzymes have been classified into two major families, Type 1 and Type 2 RNases H (Ohtani et al., 1999aGo,bGo). The Type 1 RNase H family can be further divided into three subfamilies, viral RNase H, bacterial RNase HI and eukaryotic RNase H1. The Type 2 RNase H family can be further divided into four subfamilies, bacterial RNases HII and HIII, archaeal RNase HII and eukaryotic RNase H2. Of various RNase H enzymes, Escherichia coli RNase HI, which represents bacterial Type 1 RNases H, has been most extensively studied for structures and functions (Kanaya, 1998Go). This enzyme is monomeric and relatively small in size (155 amino acid residues). Its crystal structure has been determined and the amino acid residues involved in the catalytic function and substrate binding have been identified. Therefore, a system using E.coli RNase HI as one of the pair of mesophilic and psychrophilic/psychrotrophic RNases HI would have potential for exploring the molecular mechanisms of cold-adaptations in proteins. However, no information is available on the structures and functions of RNases HI from microorganisms that adapt to cold environments. Even the genes encoding these enzymes have not so far been identified.

Shewanella sp. strain SIB1 is a psychrotrophic bacterium, which grows most rapidly at 20°C (Kato et al., 2001Go). This strain can grow even at 0°C but cannot grow at temperatures higher than 30°C. In this work, we cloned the gene encoding RNase HI from this strain (SIB1 RNase HI), overexpressed it in E.coli, purified the recombinant protein and compared its enzymatic activity and stability with those of E.coli RNase HI. In addition, we showed that the amino acid substitution at position 52 contributes to the difference in the stability and activity between SIB1 and E.coli RNases HI.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cells and plasmids

The psychrotrophic bacterium Shewanella sp. SIB1 was isolated from Japanese oil reservoir water in our laboratory (Kato et al., 2001Go). E.coli MIC2067 with the rnhA and rnhB double mutation [F- {lambda}- IN(rrnD–rrnE)1 rnhA339::cat rnhB716::kam] (Itaya et al., 1999Go) was kindly donated by M.Itaya. Plasmids pBR322 and pET-25b were obtained from Takara Shuzo and Novagen, respectively. E.coli MIC2067(DE3) (Ohtani et al., 2000Go), which is a {lambda}DE3 lysogen of E.coli MIC2067, and plasmid pBR860 (Haruki et al., 1994Go), in which the transcription and translation of the rnhA gene from E.coli (Ec-rnhA) is under the control of its own promoter and SD sequence, were constructed previously. The E.coli transformants were grown in Luria–Bertani medium containing 50 mg/l ampicillin.

Materials

[{gamma}-32P]ATP (>5000 Ci/mmol) was obtained from Amersham. E.coli RNase HI was purified previously (Kanaya et al., 1993Go). All DNA oligomers for PCR were synthesized by Hokkaido System Science.

Cloning of the rnhA gene from SIB1

The genomic DNA of Shewanella sp. SIB1 was prepared as described previously (Imanaka et al., 1981Go) and used as a template to amplify a part of the rnhA gene (Sh-rnhA) by polymerase chain reaction (PCR). The sequences of the PCR primers are 5'-TCATGTTTAGGTAATCCWGG-3' for 5'-primer and 5'-CCTGCRTGGCCTTTWACCCA-3' for 3'-primer, where R represents A + G and W represents A + T. PCR was performed with a GeneAmp PCR system 2400 (Perkin-Elmer) using a KOD polymerase (Toyobo) according to the procedures recommended by the supplier. The amplified DNA fragment was used as a probe for Southern blotting and colony hybridization to clone the entire Sh-rnhA gene. Southern blotting and colony hybridization were carried out by using the AlkPhos Direct system (Amersham Pharmacia Biotech) according to the procedures recommended by the supplier. The DNA sequence was determined with a Prism 310 DNA sequencer (Applied Biosystems).

Construction of plasmid for complementation assay

Plasmid pBR1000eS, in which the transcription and translation of the Sh-rnhA gene are controlled by the promoter and the SD sequence of the Ec-rnhA gene, was constructed by performing PCR twice, as described previously for the construction of pBR800es (Ohtani et al., 1999aGo). The sequences of the PCR primers are 5'-TTCAAGAATTCTCATGTTTGAC-3' for 5'-primer, 5'-CGCGTCGACACTAACAGGGCTGATTGACGAGTC-3' for 3'-primer, 5'-TCTACCAGAGATGGCCGAACTCAAACAGCT-3' for 5'-fusion primer and 5'-GTTCGGCCATCTCTGGTAGACTTCCTGTAA-3' for 3'-fusion primer, where underlined bases show the positions of the EcoRI (5'-primer) and SalI (3'-primer) sites, boxed bases show the position of the codon for the initial methionine residue and italic bases represent those of the Sh-rnhA gene.

Overproduction and purification

Plasmid pET600S for overproduction of SIB1 RNase HI was constructed by ligating the 600 bp DNA fragment, which contains the Sh-rnhA gene, to the NdeI–SalI site of pET-25b. This DNA fragment was amplified by PCR using the cloned Sh-rnhA gene as a template. The sequences of the PCR primers are 5'-CGCTATGGTAACAACCATATGGCCGAACTC-3' for 5'-primer and 5'-CGCGTCGACACTAACAGGGCTGATTGACGAGTC-3' for 3'-primer, where underlined bases show the positions of the NdeI (5'primer) and SalI (3'-primer) sites. For overproduction, E.coli MIC2067(DE3) was transformed with pET600S and grown at 30°C. When the absorbance at 660 nm of the culture reached around 0.6, 1 mM of isopropyl ß--thiogalactopyranoside (IPTG) was added to the culture medium and cultivation was continued at 30°C for 30 min. Then, the temperature of the growth medium was shifted to 15°C and cultivation was continued at 15°C for an additional 15 h. Cells were harvested by centrifugation at 6000 g for 10 min, suspended in 20 mM Tris–HCl (pH 8.0) containing 1 mM EDTA, disrupted by sonication lysis and centrifuged at 15 000 g for 30 min. The pellet obtained after sonication lysis was dissolved in the solution containing 20 mM sodium acetate (pH 5.5), 7 M urea and 1 mM dithiothreitol (DTT) (urea buffer). The clear lysate was applied to a cation-exchange column (3 ml) of P-11 (Whatman) equilibrated with the urea buffer. After washing the column with the urea buffer, the protein was eluted from the column with a linear gradient of NaCl from 0 to 0.5 M in the urea buffer. The protein fractions at an NaCl concentration of ~0.15 M were pooled and dialyzed against 20 mM sodium acetate (pH 5.5) containing 1 mM DTT for refolding. The N-terminal amino acid sequence was determined with a Procise 491 protein sequencer (Applied Biosystems).

For the estimation of the production level and the amount of the proteins in soluble and insoluble forms, whole cell extracts and soluble and insoluble fractions obtained after sonication lysis were analyzed by SDS–PAGE on a 15% polyacrylamide gel (Laemmli, 1970Go), followed by staining with Coomassie Brilliant Blue. The protein concentration was determined from the UV absorption assuming that the A280 nm0.1% value of SIB1 RNase HI is identical with that of E.coli RNase HI, which has been determined to be 2.02 (Kanaya et al., 1990Go). The A280 nm0.1% value of SIB1 RNase HI calculated by using {varepsilon} = 1576 M-1 cm-1 for Tyr and 5225 M-1 cm-1 for Trp at 280 nm (Goodwin and Morton, 1946Go) is comparable to that of E.coli RNase HI.

Mutagenesis

The gene encoding the mutant protein Sh-P52A, in which Pro52 of SIB1 RNase HI is replaced by Ala, was constructed by site-directed mutagenesis using PCR, as described previously (Kanaya et al., 1993Go). For PCR, the Sh-rnhA gene in pET600S was used as a template. Oligonucleotides, which were used as mutagenic primers, were designed to alter the codon for Pro52 (CCA) to that for Ala (GCA). The nucleotide sequence of the gene encoding the mutant protein was confirmed by using a Prism 310 DNA sequencer (Applied Biosystems). Overproduction and purification of the mutant protein Sh-P52A were carried out as described for the wild-type protein. The concentration of this mutant protein was determined from the UV absorption with an A280 nm0.1% value of 2.02.

Enzymatic activity

The RNase H activity was determined at 20°C for 15 min by measuring the radioactivity of the acid-soluble digestion product from 3H-labeled M13 DNA–RNA hybrid, as described previously (Kanaya et al., 1991Go), unless stated otherwise. The reaction mixture contained 10 pmol of the substrate and an appropriate amount of the enzyme in 10 µl of 10 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 30 mM KCl, 1 mM 2-mercaptoethanol (2-Me) and 50 µg/ml BSA (for SIB1 RNase HI) or 10 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 50 mM NaCl, 1 mM 2-Me and 50 µg/ml BSA (for E.coli RNase HI). One unit was defined as the amount of enzyme producing 1 µmol of acid-soluble material per minute. The specific activity was defined as the enzymatic activity per milligram of protein. For the determination of the kinetic parameters, the substrate concentration was varied from 0.041 to 0.33 µM. The hydrolysis of the M13 DNA–RNA hybrid by the enzyme followed Michaelis–Menten kinetics and the kinetic parameters were determined from the Lineweaver–Burk plot.

Substrate specificity

The 12 bp RNA–DNA, RNA–RNA and DNA–DNA duplexes (1 µM) were prepared by hybridizing the 5'-end-labeled 12 b RNA or DNA with a sequence of 5'-CGGAGA(U/T)GACGG-3' with 1.5 molar equivalent of the complementary 12 b DNA or RNA, as described previously (Ohtani et al., 1999aGo). Hydrolysis of the substrate was carried out at 20°C for 15 min under the same conditions as used to determine the enzymatic activity for the hydrolysis of the M13 DNA–RNA hybrid. Products were analyzed on a 20% polyacrylamide gel containing 7 M urea and quantified using an Instant Imager (Packard), as described previously (Ohtani et al., 1999aGo).

Circular dichroism (CD) spectra

The far- and near-UV CD spectra were measured on a J-725 spectropolarimeter (Japan Spectroscopic) at 4°C in 20 mM sodium acetate (pH 5.5) containing 1 mM DTT, as described previously (Akasako et al., 1997Go). The mean residue ellipticity ({theta}, deg cm2 dmol-1) was calculated by using an average amino acid molecular weight of 110.


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Cloning

When the amino acid sequences of various bacterial RNases HI are compared with one another, the sequences SCLGNPG and WVKGHAG, which correspond to Ser12–Gly18 and Trp120–Gly126 of E.coli RNase HI, respectively, are highly conserved (Ohtani et al., 1999bGo). Therefore, we constructed the PCR primers based on these sequences and used to amplify a part of the Sh-rnhA gene. A PCR by using the genomic DNA of Shewanella sp. SIB1 as a template produced only the 344 bp DNA fragment, which encodes part of the SIB1 RNase HI sequence. Southern blotting and colony hybridization by using this DNA fragment as a probe indicated that a 4.2 kbp PstI fragment of the SIB1 genome contained the entire Sh-rnhA gene (data not shown). Determination of the nucleotide sequence of the Sh-rnhA gene revealed that SIB1 RNase HI is composed of 157 amino acid residues with a calculated molecular weight of 17 899 and an isoelectric point (pI) of 8.0. A potential Shine Dalgarno (SD) sequence (GGTAA) is located seven nucleotides upstream of the initiation codon for translation and a possible promoter sequence is located upstream of this SD sequence. The nucleotide sequence of the Sh-rnhA gene is deposited in DDBJ with accession number AB070445.

Amino acid sequence

The amino acid sequence of SIB1 RNase HI is compared with those of E.coli and Thermus thermophilus RNases HI in Figure 1Go. SIB1 RNase HI shows the amino acid sequence identities of 63% to E.coli RNase HI and 42% to T.thermophilus RNase HI. The positions of the amino acid residues of the SIB1 RNase HI sequence were numbered based upon the E.coli RNase HI sequence instead of from its N-terminus. The amino acid residues involved in divalent cation binding and catalytic function (Asp10, Glu48, Asp70, His124 and Asp134), which are fully conserved in the various RNase HI sequences, are also conserved in the SIB1 RNase HI sequence, suggesting that SIB1 RNase HI structurally and functionally resembles to E.coli and T.thermophilus RNases HI.



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Fig. 1. Alignment of the RNase HI sequences. SIB1, Eco and Tth represent SIB1, E.coli and T.thermophilus RNases HI, respectively. The amino acid residues that are conserved in at least two different proteins are highlighted in black. Gaps are denoted by dashes. The conserved residues in various RNase HI sequences, which are involved in metal binding and catalytic function (Asp10, Glu48, Asp70, His124 and Asp134), are denoted by asterisks. Numbers above the sequences indicate the positions of the amino acid residues relative to the initiator methionine of E.coli RNase HI. The ranges of the {alpha}-helices and ß-strands of E.coli RNase HI are also indicated above the sequences. The sequences of E.coli and T.thermophilus RNases HI have been deposited in EMBL with accession numbers V00337 and X60507, respectively.

 
Complementation assay

E.coli MIC2067 shows RNase H-dependent temperaturesensitive (ts) growth phenotype (Itaya et al., 1999Go). It can form colonies at 30°C but cannot at 42°C. This ts phenotype can be complemented by the functional RNase H genes. To examine whether the Sh-rnhA gene complements the ts phenotype of this strain, E.coli MIC2067 cells were transformed with pBR1000eS, in which the transcription and translation of the Sh-rnhA gene are under the control of the promoter and the SD sequence of the Ec-rnhA gene. The resultant MIC2067 transformants could grow at 42°C, suggesting that SIB1 RNase HI exhibits the enzymatic activity in vivo. However, these transformants grew very poorly, suggesting that SIB1 RNase HI exhibits a very low level of enzymatic activity at this temperature, probably owing to its high sensitivity to thermal inactivation.

Overproduction and purification

We used E.coli MIC2067(DE3) as a host for overproduction of SIB1 RNase HI to avoid contamination of host-derived RNases HI and HII. Upon induction at 15°C, most of the recombinant protein accumulated intracellularly in an insoluble form (Figure 2Go). The protein was solubilized in the presence of 7 M urea and purified to give a single band on SDS–PAGE (Figure 2Go). The production level of the recombinant SIB1 RNase HI protein was estimated to be ~70 mg/l culture and ~35 mg of the purified protein was obtained from 1 l of culture. This purified protein was refolded by removing urea with a yield of nearly 100% and used for further characterization. The N-terminal amino acid sequence of the protein was determined to be MAELKQL, which was identical with that predicted from the DNA sequence. The far- and near-UV CD spectra of SIB1 RNase HI are similar to those of E.coli RNase HI, except for the depth of a trough at 280–300 nm (Figure 3Go). These results suggest that local conformations around the aromatic residues of SIB1 RNase HI are slightly different from those of E.coli RNase HI, but its overall main chain fold is similar to that of E.coli RNase HI.



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Fig. 2. SDS–PAGE of SIB1 RNase HI overproduced in E.coli cells. Samples were subjected to 15% SDS–PAGE and stained with Coomassie Brilliant Blue. Whole cell extract (lane 2) and soluble (lane 3) and insoluble (lane 4) fractions after sonication lysis were analyzed. Lane 5, purified SIB1 RNase HI; lane 1, a low molecular weight marker kit (Pharmacia Biotech) containing phosphorylase b, albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor and {alpha}-lactalbumin. Numbers along the gel represent the molecular weights of individual standard proteins.

 


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Fig. 3. CD spectra. The far-UV (A) and near-UV (B) CD spectra of SIB1 RNase HI (thick line), E.coli RNase HI (thin line) and the Sh-P52A (broken line) are shown. All spectra were measured as described under Materials and methods.

 
Enzymatic activities

SIB1 RNase HI showed the maximum activity for the hydrolysis of the M13 DNA–RNA hybrid at pH 7.5 in the presence of 5 mM MgCl2 and 30 mM KCl. It showed ~50% of the maximum activity at pH 7.0 or 8.5 or in the presence of 10 or 100 mM KCl. The enzyme prefers KCl to NaCl and the enzymatic activity determined in the presence of NaCl was roughly half of that determined in the presence of KCl. Both SIB1 and E.coli RNases HI exhibited the activity in the presence of Mg2+ and Mn2+, but did not exhibit it in the presence of Ba2+, Ca2+, Co2+, Cu2+, Fe2+, Ni2+, Sr2+ and Zn2+. Like E.coli RNase HI, which shows Mn2+- and Mg2+-dependent RNase H activities at the divalent cation concentrations of 2–20 µM and >1 mM, respectively (Keck and Marqusee, 1996Go; Keck et al., 1998Go), SIB1 RNase HI showed the maximum Mn2+- and Mg2+-dependent RNase H activities at 1 µM and 5 mM, respectively. The specific activity determined in the presence of 1 µM MnCl2 was 5% of that determined in the presence of 5 mM MgCl2. The enzyme showed less than 10% of the maximum activity at MgCl2 concentrations below 0.5 or above 20 mM.

In order to compare the optimum temperatures for the SIB1 RNase HI and E.coli RNase HI activities, it is necessary to determine the specific activities of these enzymes at temperatures which span the optimum one. However, when the SIB1 RNase HI activity was analyzed at temperatures higher than 25°C, the amount of the acid-soluble digestion products did not increase in proportion to the reaction time for 15 min (Figure 4Go), indicating that SIB1 RNase HI is not fully stable for 15 min at these temperatures. The specific activity of the enzyme could be determined from the initial slope of the reaction at 25 and 30°C. However, it seemed difficult to determine it at temperatures higher than 35°C. Likewise, it was difficult to determine the specific activity of E.coli RNase HI at temperatures higher than 60°C. Therefore, we compared the amounts of the acid-soluble digestion products accumulated upon incubation with these enzymes at various temperatures for 15 min. As long as these amounts were compared with one another, SIB1 RNase HI most effectively hydrolyzed the substrate at 30°C, whereas E.coli RNase HI did so at 50°C (Figure 5Go). Thus, the optimum temperature for the SIB1 RNase HI activity was apparently shifted downward by ~20°C as compared with that for the E.coli RNase HI activity.



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Fig. 4. Time dependence of the SIB1 RNase HI activity. The M13 DNA–RNA hybrid (10 pmol) was hydrolyzed by 200 pg of SIB1 RNase HI at 20°C (•), 25°C ({circ}), 30°C ({blacktriangleup}) and 35°C (x) in 10 µl of the reaction mixture, as described under Materials and methods. With appropriate intervals, the reaction was terminated and the amount of the acid-soluble digestion products was plotted against the reaction time.

 


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Fig. 5. Temperature dependence of the activities of SIB1 and E.coli RNases HI. The M13 DNA–RNA hybrid (10 pmol) was hydrolyzed by 200 pg of SIB1 RNase HI (•) or 10 pg of E.coli RNase HI ({circ}) at the temperatures indicated in 10 µl of the reaction mixture for 15 min and the amount of the acid-soluble digestion products accumulated upon enzymatic reaction was plotted against the temperature. The composition of the reaction mixture for assay is described under Materials and methods.

 
From the initial slope of the reaction, the specific activity of SIB1 RNase HI was determined as 0.29 units/mg at 10°C, 0.64 units/mg at 20°C and 1.4 units/mg at 30°C. Likewise, the specific activity of E.coli RNase HI was determined as 1.3 units/mg at 10°C, 4.4 units/mg at 20°C, 9.5 units/mg at 30°C and 17.8 units/mg at 40°C. Thus, the specific activity of SIB1 RNase HI was lower than that of E.coli RNase HI by 5–7-fold even at temperatures below 20°C. Based on these specific activities, the activation energy was determined as 13.8 kcal/mol for SIB1 RNase HI in the temperature range 10–30°C and 15.2 kcal/mol for E.coli RNase HI in the temperature range 10–40°C (Figure 6Go). At 20°C, the Km value of SIB1 RNase HI was comparable to that of E.coli RNase HI, whereas the Vmax value of SIB1 RNase HI was lower than that of E.coli RNase HI by 7-fold (Table IGo). Hence these two enzymes differ mainly in the hydrolysis rate.



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Fig. 6. Plot of the specific activity (V) as a function of the reciprocal of absolute temperature (T). The specific activities of SIB1 RNase HI (•), E.coli RNase HI ({circ}) and the Sh-P52A ({blacksquare}) determined from the initial velocity of the reaction at various temperatures (in µmol/mg.min) were plotted against 1/T. The activation energies, which were determined from the slope of semilog plots of V versus 1/T, are indicated. The lines were obtained by linear regression of the data.

 

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Table I. Specific activities and kinetic parameters
 
Substrate specificity

To examine whether SIB1 RNase HI specifically cleaves the RNA strand of RNA–DNA hybrids, the 12 b RNA, 12 b DNA, 12 bp RNA–RNA duplex, 12 bp DNA–DNA duplex or 12 bp RNA–DNA hybrid was incubated with the enzyme under conditions which were optimum for the cleavage of the M13 DNA–RNA hybrid. Of these substrates, only the 12 bp RNA–DNA hybrid was cleaved, indicating that SIB1 RNase HI does not have any nuclease activity for the single-stranded RNA, single-stranded DNA, double-stranded RNA and double-stranded DNA. SIB1 RNase HI cleaved the 12 bp RNA–DNA hybrid at A6–U7, U7–G8 and A9–C10, as did E.coli RNase HI (Kanaya and Kanaya, 1995Go) (data not shown). However, unlike E.coli RNase HI, which cleaves the A9–C10 bond most preferably, SIB1 RNase HI cleaved the A6–U7 bond most preferably.

Stability

In order to compare the thermal stabilities of SIB1 and E.coli RNases HI, they were incubated in 20 mM Tris–HCl (pH 7.5) containing 5 mM MgCl2, 0.1 M KCl, 1 mM EDTA, 10% glycerol and 0.1 mg/ml BSA at various temperatures for 10 min and the residual activities were determined at 20°C. Under these conditions, both enzymes are irreversible in thermal denaturation. The protein concentrations were 0.02–0.2 µg/ml. Under these conditions, the temperature (T1/2) at which the enzyme loses half of its activity was roughly 60°C for E.coli RNase HI and 25°C for SIB1 RNase HI (Figure 7Go). Hence SIB1 RNase HI was less stable than E.coli RNase HI by ~35°C in T1/2.



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Fig. 7. Thermal stability of SIB1 RNase HI, E.coli RNase HI and the Sh-P52A. The enzyme was dissolved in 20 mM Tris–HCl (pH 7.5) containing 5 mM MgCl2, 0.1 M KCl, 1 mM EDTA, 10% glycerol and 0.1 mg/ml BSA at a concentration of 0.02–0.2 µg/ml, incubated for 10 min at the temperatures indicated and determined for residual activity at 20°C. The residual activities of SIB1 RNase HI (•), E.coli RNase HI ({circ}) and the Sh-P52A ({blacksquare}) were plotted against the temperature.

 
Pro52->Ala mutation

The amino acid residue at position 52 is Pro for SIB1 RNase HI and Ala for E.coli RNase HI (Figure 1Go). This amino acid substitution is intriguing, because it introduces a proline residue into the middle of the {alpha}I-helix of SIB1 RNase HI. Introduction of Pro into {alpha}-helices must destabilize proteins, because it breaks a hydrogen bond that stabilizes a helical structure. In addition, this substitution may affect the enzymatic activity, because one of the catalytically essential residues, Glu48, is located near this position. Therefore, we constructed the mutant protein Sh-P52A to examine whether the amino acid substitution at position 52 contributes to the difference in the stability and/or activity between SIB1 and E.coli RNases HI.

The Sh-P52A was overproduced in E.coli in an insoluble form, solubilized in the presence of 7 M urea, purified and refolded by removing urea, as was the parent enzyme. The far- and near-UV CD spectra of the Sh-P52A were similar to those of the parent enzyme, suggesting that the Pro52->Ala mutation does not seriously affect the SIB1 RNase HI structure. When the thermal stability of this mutant enzyme was analyzed under the same conditions in which the thermal stabilities of SIB1 and E.coli RNases HI were compared, the T1/2 of the mutant enzyme was shown to be higher than that of the parent enzyme by ~5°C (Figure 7Go), indicating that the Pro52->Ala mutation stabilizes SIB1 RNase HI.

The temperature dependence of the Sh-P52A activity is compared with that of the parent enzyme in Figure 8Go. The enzymatic activity of the Sh-P52A was higher than that of the wild-type protein at all temperatures examined. In addition, the optimum temperature of the Sh-P52A seemed to be shifted slightly upwards by a few degrees compared with that of the parent enzyme. The specific activity of the Sh-P52A was determined as 0.46 units/mg at 10°C, 1.3 units/mg at 20°C and 2.7 units/mg at 30°C. The activation energy of the Sh-P52A was determined as 15.6 kcal/mol in the temperature range 10–30°C (Figure 6Go). This value was higher than that of the parent enzyme, but was comparable to that of E.coli RNase HI. Comparison of the kinetic parameters of the Sh-P52A with those of the parent enzyme determined at 20°C indicated that the Vmax value of the Sh-P52A was higher than that of the parent enzyme by 2.1-fold, whereas the Km value of the Sh-P52A was comparable to that of the parent enzyme (Table IGo). These results indicate that the Pro52->Ala mutation increases both the stability and the hydrolysis rate of SIB1 RNase HI.



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Fig. 8. Temperature dependence of the activities of SIB1 RNase HI and the Sh-P52A. The M13 DNA–RNA hybrid (10 pmol) was hydrolyzed by 65 pg of SIB1 RNase HI (•) or the Sh-P52A ({blacksquare}) at the temperatures indicated in 10 µl of the reaction mixture for 15 min, as described in the caption of Figure 5Go and the amount of the acid-soluble digestion products accumulated upon enzymatic reaction was plotted against the temperature.

 

    Discussion
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 Abstract
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 Materials and methods
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 Discussion
 References
 
rnhA gene from SIB1

Many bacterial genomes contain multiple RNase H genes, such as rnhA, rnhB and rnhC (Ohtani et al., 1999bGo). The combination of these genes in bacteria varies in a non-obvious manner. The Type 2 RNase H gene (rnhB or rnhC) is universally present in various bacterial genomes, whereas the Type 1 RNase H gene (rnhA) is not. However, it has been proposed that the genomes of the proteobacteria always contain the rnhA gene (Ohtani et al., 1999bGo). Our result that the genome from Shewanella sp. SIB1, which is a proteobacterium, contains the rnhA gene agrees with this proposal.

Stability and activity of SIB1 RNase HI

The recombinant SIB1 RNase HI protein accumulated intracellularly in an insoluble form, probably owing to its striking instability. Therefore, the refolded protein was used to analyze the enzymatic properties of SIB1 RNase HI. It remained to be determined whether the refolded protein is structurally and functionally identical with the natural one. It seems difficult to isolate the enzyme from Shewanella strain, because of its poor content in the cells (unpublished data). In addition, attempts to produce soluble recombinant protein by changing a condition or system for overproduction have so far been unsuccessful. However, the enzymatic properties of this refolded protein probably reflect those of the native protein, because the far-UV CD spectrum and the enzymatic properties of this refolded protein resembled to those of E.coli RNase HI.

Despite the high sequence similarity, SIB1 RNase HI is much less stable than E.coli RNase HI. Because the optimum growth temperature of E.coli is ~40°C, the difference in the optimum growth temperatures between the SIB1 strain and E.coli (~20°C) is comparable to the difference in the optimum temperature for activity between SIB1 and E.coli RNases HI (~20°C), but smaller than the difference in T1/2 between SIB1 and E.coli RNases HI (~35°C). E.coli RNase HI has been shown to unfold reversibly in a single cooperative fashion in thermal and guanidine hydrochloride (GdnHCl) denaturation experiments under appropriate conditions (Kanaya et al., 1991Go). From the GdnHCl denaturation curve of this protein, the free energy change of unfolding in the absence of the denaturant has been determined as 8.9 kcal/mol. Therefore, it would be informative to determine the free energy change of unfolding of SIB1 RNase HI. However, SIB1 RNase HI did not give a clear urea or GdnHCl denaturation curve, because it is very unstable and starts to unfold even in the presence of 0.1 M urea at 4°C (N.Ohtani, unpublished work). It also did not give a thermal denaturation curve, because it aggregates to form precipitates upon thermal denaturation under all conditions examined.

SIB1 RNase HI is less active than E.coli RNase HI even at low temperatures. The SIB1 genome contains the rnhB gene encoding RNase HII in addition to the rnhA gene, as does the E.coli genome (N.Ohtani, unpublished work). RNases HI and HII have been shown to be structurally and functionally related with each other (Lai et al., 2000Go;Muroya et al., 2001Go). Therefore, the low stability and low activity of SIB1 RNase HI may be a result of the lack of selective pressure towards thermal stability and activity. Several enzymes from psychrophilic or psychrotrophic bacteria have been shown to exhibit lower activities than the mesophilic counterparts at low temperatures (Rentier-Delrue et al., 1993Go; Gerike et al., 1997Go; Birolo et al., 2000Go). The activation energy of SIB1 RNase HI, which reflects the catalytic efficiency of the enzyme, was slightly lower than that of E.coli RNase HI. The activation energies of the enzymes from cold-adapted microorganisms are usually lower than those of the mesophilic counterparts (Feller et al., 1996Go; Choo et al., 1998Go), with a few exceptions (Yumoto et al., 2000Go).

Effect of Pro52->Ala mutation

The Pro52->Ala mutation increased both the stability and activity of SIB1 RNase HI, suggesting that the amino acid substitution at position 52 partly accounts for the difference in both stability and activity between SIB1 and E.coli RNases HI. The activation energy was also increased by this mutation. Statistical analyses in natural proteins have suggested that {alpha}-helices are kinked by 26 ± 5° on average at the positions where Pro is introduced (Barlow and Thornton, 1988Go). A slight increase in the depth of the CD signals at 208 nm caused by the Pro52->Ala mutation (Figure 3Go) may suggest that the {alpha}I-helix of SIB1 RNase HI is kinked at Pro52 and the Pro52->Ala mutation alters the conformation of this helix, such that this kink is removed. This conformational change may account for the increase in the catalytic function of the Sh-P52A, because Glu48, which is one of the active-site residues of the enzyme, is located in the {alpha}I-helix. The conformational change of the {alpha}I-helix may shift the position of Glu48, which is favorable for the enzymatic activity. Further structural studies will be required to elucidate the effect of the Pro52->Ala mutation on the stability and activity of the enzyme.

We previously showed that the Ala52->Pro mutation decreased the stability of E.coli RNase HI by 5.4°C in Tm, without seriously affecting the enzymatic activity (Akasako et al., 1997Go). This result is consistent with the effect of the Pro52->Ala mutation on the stability of SIB1 RNase HI, but inconsistent with that on the activity of SIB1 RNase HI. Because the hydrophobic interactions in the interior of the protein molecule of E.coli RNase HI, that stabilize the {alpha}I-helix, seem to be stronger than those of SIB1 RNase HI, introduction of Pro into the {alpha}I-helix of E.coli RNase HI may not create a considerable kink and therefore does not seriously affect the enzymatic activity.

Cold-adaptation

Because of the instability and low optimum temperature for activity, SIB1 RNase HI can be defined as a cold-adapted enzyme. SIB1 and E.coli RNases HI show an amino acid sequence identity of 63%. This means that they have adapted to the different environmental temperatures through the 37% difference in the amino acid sequence (55 amino acid substitutions). Of these amino acid substitutions, 13 substitutions are located at buried sites and 16 are located at exposed sites. According to the crystal structure of E.coli RNase HI (Katayanagi et al., 1990Go,1992Go), 62 residues are almost fully buried inside the protein molecule, whereas 26 residues are almost fully exposed to the solvent. Here, the buried and exposed residues represent those for which the ratio of the aqueous surface area in the folded state to that in the unfolded state is calculated to be <20% and >70%, respectively (Ooi and Oobatake, 1988Go; Oobatake and Ooi, 1993Go). Therefore, frequency of the amino acid substitutions at exposed sites (62%) is much higher than that at buried sites (21%). These results suggest that SIB1 and E.coli RNases HI adapt to the different temperatures by changing the exposed residues, rather than the buried ones, probably because the mutations at buried sites often produce proteins incorrectly folded.

Identification of the amino acid substitution at position 52 as one of the factors that makes SIB1 RNase HI less stable and less active than E.coli RNase HI suggests that the difference in stability and activity between SIB1 and E.coli RNases HI reflects the sum of the individual local interactions that affect a protein stability or activity. It has been suggested that the Arg->Lys and Glu->Ala substitutions at exposed sites and the Val->Ala and Val->Ile substitutions at buried sites are responsible for cold-adaptation of proteins (Gianese et al., 2001Go). It has also been suggested that elimination of the proline residues from the loop region is responsible for making cold-adapted enzymes flexible and functional at low temperatures (Aghajari et al., 1998Go). However, no typical amino acid substitutions in the direction from ‘hot’ to ‘cold’ enzymes are detected in the SIB1 RNase HI sequence, except for the Arg->Lys substitution. In addition, the Pro content of SIB1 RNase HI is higher than that of E.coli RNase HI. Therefore, strategic or random replacement of the amino acid sequences of SIB1 RNases HI with those from E.coli RNase HI will be necessary to identify other amino acid substitutions that make SIB1 RNase HI less stable and less active than E.coli RNase HI.

It is noted that His62 of E.coli RNase HI, which connects the {alpha}I-helix to the ßD-strand, is replaced by Pro in both SIB1 and T.thermophilus RNases HI (Figure 1Go). We previously showed that the His62->Pro mutation stabilizes E.coli RNase HI by 4.1°C in Tm and 1.1 kcal/mol in {Delta}G, without seriously affecting the enzymatic activity (Kimura et al., 1992Go). These results suggest that SIB1 RNase HI adapts to cold environment by the combination of both stabilizing and destabilizing amino acid substitutions.


    Notes
 
1 To whom correspondence should be addressed.E-mail: kanaya{at}ap.chem.eng.osaka-u.ac.jp Back


    Acknowledgments
 
We thank Dr M.Itaya for providing rnh mutant strains of E.coli, MIC3001, MIC3037 and MIC2067 and Drs N.Esaki and M.Oobatake for helpful discussions. This work was supported by the Asahi Glass Foundation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received July 13, 2001; revised August 27, 2001; accepted September 10, 2001.