Tài liệu Báo cáo khoa học: The SCO2299 gene from Streptomyces coelicolor A3(2) encodes a bifunctional enzyme consisting of an RNase H domain and an acid phosphatase domain pdf

The SCO2299 gene from Streptomyces coelicolor A3(2) encodes a bifunctional enzyme consisting of an RNase H domain and an acid phosphatase domain Naoto Ohtani 1 , Natsumi Saito 1 , Masaru Tomita 1 , Mitsuhiro Itaya 1,2 and Aya Itoh 1 1 Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan 2 Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo, Japan It is generally accepted that ribonuclease H (RNase H; EC 3.1.26.4) specifically cleaves an RNA strand of RNAÆDNA hybrid endonucleolytically [1]. Various studies suggest that RNase H is involved in import- ant cellular functions such as DNA replication [2–7], DNA repair [7–9], transcription [10–12], and develop- ment [13,14]. RNase H is classified into two major families, Type 1 and Type 2, based on amino acid sequence similarities with Escherichia coli RNase HI [15] and HII [16], respectively [17,18]. Although both enzymes have been found in various organisms, Type 2 RNase H is more universal because the encoding genes exist in almost all genomes whose sequences have been determined [17,18]. On the other hand, the Type 1 gene is lacking in a large number of prokaryotic genomes, and distribution of the gene in prokaryotic genomes is not apparently correlated with the prokaryotic evolutionary relationship based on rRNA sequences [18]. For example, the Type 1 gene is rare in archaeal genomes, and only those from Halobacterium sp. NRC-1 [19], Sulfolobus toko- daii [20] and Pyrobaculum aerophilum (N. Ohtani, unpublished data) were recently shown to encode active enzymes. Interestingly, in another archaeon, Correspondence N. Ohtani, Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan Tel ⁄ Fax: +81 6 6608 3777 E-mail: nao10_oh@ybb.ne.jp (Received 12 February 2005, revised 29 March 2005, accepted 5 April 2005) doi:10.1111/j.1742-4658.2005.04704.x The SCO2299 gene from Streptomyces coelicolor encodes a single peptide consisting of 497 amino acid residues. Its N-terminal region shows high amino acid sequence similarity to RNase HI, whereas its C-terminal region bears similarity to the CobC protein, which is involved in the synthesis of cobalamin. The SCO2299 gene suppressed a temperature-sensitive growth defect of an Escherichia coli RNase H-deficient strain, and the recombinant SCO2299 protein cleaved an RNA strand of RNAÆDNA hybrid in vitro. The N-terminal domain of the SCO2299 protein, when overproduced inde- pendently, exhibited RNase H activity at a similar level to the full length protein. On the other hand, the C-terminal domain showed no CobC-like activity but an acid phosphatase activity. The full length protein also exhib- ited acid phosphatase activity at almost the same level as the C-terminal domain alone. These results indicate that RNase H and acid phosphatase activities of the full length SCO2299 protein depend on its N-terminal and C-terminal domains, respectively. The physiological functions of the SCO2299 gene and the relation between RNase H and acid phosphatase remain to be determined. However, the bifunctional enzyme examined here is a novel style in the Type 1 RNase H family. Additionally, S. coelicolor is the first example of an organism whose genome contains three active RNase H genes. Abbreviations APase, acid phosphatase; CE-ESI MS, capillary electrophoresis mass spectrometry; pNPP, p-nitrophenyl phosphate; RNase H, ribonuclease H; RT, reverse transcriptase; ts, temperature-sensitive. 2828 FEBS Journal 272 (2005) 2828–2837 ª 2005 FEBS Haloarcula marismortui, unlike any other known RNase H gene, one of the two Type 1 RNase H genes is encoded on a plasmid [21]. Reverse transcrip- tases (RTs) of retroelements, which contain amino acid sequences and structures showing high homology with E. coli RNase HI as a domain, are also included in the Type 1 RNase H family [18,22,23]. As des- cribed above, the natural distribution and style of the Type 1 RNase H are more complicated than those of the Type 2 variety. Previous work has shown that Corynebacterium glu- tamicum RNase HI (Type 1 RNase H), whose addi- tional C-terminal region showed a high amino acid sequence similarity to the CobC protein, exhibited RNase H activity in vivo in a complementation assay with an E. coli RNase H-deficient strain [24]. Although the RNase HI with the extra CobC-like region is a novel style in the Type 1 RNase H family, the C. glutamicum enzyme itself has not been charac- terized and a function of its C-terminal region remains unknown [24]. The C-terminal region of the enzyme certainly shows a similarity to the CobC pro- tein, which has been reported to be involved in syn- thesis of cobalamin, one of the precursors of vitamin B 12 synthesis [25]. However, a blast search reveals that the C-terminal region is also similar to phospho- glycerate mutase, fructose-2,6-bisphosphatase or other acid phosphatases. This suggests that its function might not be the same as the CobC generating a-ribazole from a-ribazole-5¢-phosphate but might be some other phosphatase. Therefore, we decided to characterize the RNase H activity of the enzyme and examine phosphatase activities of the C-terminal region. The C. glutamicum RNase HI-like genes are also found from genomes of bacteria classified as Actin- omycetales, i.e., Mycobacterium, Thermobifida, Nocar- dia, Corynebacterium and Streptomyces. Among them, the SCO2299 gene from Streptomyces coelicolor A3(2) was selected for our analyses, because S. coelicolor can be genetically engineered and its genome con- tains three RNase H-like genes [26]. Beacuse no organism whose genome contains three active RN- ase H genes has been reported before, it is also important to note whether the three genes of S. coe- licolor are active or not. Here, we show that the SCO2299 gene from S. coelicolor encodes a bifunc- tional enzyme consisting of the RNase H domain and the acid phosphatase (APase) domain, and pro- pose that the enzyme is a novel style in the Type 1 RNase H family. Moreover, we also announce that S. coelicolor is the first example of a genome with three active RNase H genes. Results Amino acid sequence The N-terminal region (amino acid residues 1–159) of the SCO2299 protein shows significant amino acid sequence similarity to RNase HI (Fig. 1). For example, it shows sequence identities of 27% to E. coli RNase HI, 52% to C. glutamicum RNase HI, 31% to S. toko- daii RNase HI, and 38% to Halobacterium RNase HI. A previous phylogenetic analysis [20] confirmed that the SCO2299 protein is more similar to archaeal Type 1 RNases H such as S. tokodaii and Halobac- terium enzymes than to the bacterial enzymes except for C. glutamicum RNase HI. Among the five active site residues (Asp10, Glu48, Asp70, His124 and Asp134) identified in E. coli RNase HI [27], only four acidic residues are conserved in the SCO2299 protein. As the His residue is not important for catalysis of RNase HI from Halobacterium [19] and S. tokodaii [20], the SCO2299 protein may operate in a similar manner to them. Furthermore, the SCO2299 protein lacks a basic protrusion region, which is present in other bacterial and eukaryotic Type 1 RNase H [18] and has been reported to be important for substrate binding for E. coli RNase HI [28], as in Halobacterium and S. tokodaii enzymes. On the other hand, the C-terminal region (amino acid residues 290–497) of the SCO2299 protein shows 38% sequence identity to that of C. glutamicum RNase HI. The regions of both proteins show sequence simi- larity to CobC generating a-ribazole from a-ribazole- 5¢-phosphate. For example, the SCO2299 protein shows a sequence identity of 27% to Salmonella typhimurium CobC. However, the CobC protein, phos- phoglycerate mutase, fructose-2,6-bisphosphatase or other acid phosphatases have been found to be similar to each other in their sequences and three-dimensional structures [29]. Because of this, we considered it 1 497 159 290 RNase H domain APase domain complementation of MIC2067(DE3) − + + Fig. 1. Diagram of the SCO2299 constructs. The shaded regions represent an RNase H domain or an APase domain of the SCO2299 protein. Numbers represent the positions of amino acid residues that start from the initiator Met residue. Plus or minus signs indicate temperature-sensitive complementation of the E. coli RNase H-deficient mutant MIC2067(DE3). N. Ohtani et al. A fusion protein consisting of RNase H and APase FEBS Journal 272 (2005) 2828–2837 ª 2005 FEBS 2829 probable that the C-terminal region of the SCO2299 protein might exhibit some phosphatase activity. Overproduction and purification To obtain the full length SCO2299 protein in an amount sufficient for biochemical characterization, an overproducing strain was constructed as described in Experimental procedures. Although the strain was used for complementation assays, the production level of the tag-free recombinant protein was very low. There- fore, to facilitate the purification, an overproducing strain for the N-terminal His-tagged protein was con- structed. Fortunately, in this strain, the production level was improved (data not shown). Upon induction at 18 °C, about 70% of the recombinant protein accu- mulated intracellularly in a soluble form. On the other hand, when induced at 37 °C, almost all of the protein accumulated in an insoluble form. Recombinant pro- teins of the N-terminal (amino acid residues 1–159) and C-terminal (residues 290–497) regions as shown in Fig. 1 were also overproduced in a similar manner to that of the full length protein (data not shown). The purified recombinant SCO2299 proteins are shown in Fig. 2. RNase H activity of the SCO2299 proteins E. coli rnhA rnhB double mutant strains MIC2067 [19,30] and MIC2067(DE3) [20,31] show a tempera- ture-sensitive (ts) growth defect, which can be rescued by the introduction of a gene encoding an active RNase H enzyme. For example, the C. glutamicum RNase HI can suppress the phenotype of MIC2067 [24]. Therefore, to examine whether the SCO2299 gene also encodes the active RNase H enzyme, the MIC2067(DE3) cells were transformed with a pET vec- tor containing the gene. As expected, the SCO2299 gene suppressed the ts growth defect, suggesting that the SCO2299 protein was an RNase H enzyme. Its N-ter- minal and C-terminal regions were cloned independ- ently (Fig. 1), and similar assays were performed. The results showed that the N-terminal region suppressed the ts phenotype but the C-terminal region did not. The RNase H activities of the three recombinant SCO2299 proteins were examined in vitro employing a 12-bp oligomeric RNAÆ DNA hybrid as a substrate. As shown in Fig. 3, the full length protein and the N-ter- minal domain of SCO2299 could cleave the RNA strand of the RNAÆDNA hybrid but the C-terminal region could not. This result agreed with that of the in vivo complementation assay. The cleavage efficiency of the 12-bp RNAÆDNA hybrid per mole of protein was almost the same between the full length protein and the N-terminal domain (Fig. 3). As shown in Fig. 3, addition of the C-terminal region at an equiva- lent mole level had no effect on the activity of the N-terminal domain. Characteristics of RNase H acti- vities, i.e., the divalent metal ion preference, pH dependency, and cleavage patterns of oligomeric sub- strate, were almost the same between the two proteins. These results suggested that the RNase H activity of the full length SCO2299 protein depended only on the N-terminal RNase H-like domain. The SCO2299 protein exhibited an RNase H activity in the presence of Mg 2+ ,Mn 2+ ,Co 2+ , and Ni 2+ , and preferred Mg 2+ or Mn 2+ to Co 2+ or Ni 2+ . Its activity increased as the pH increased (data not shown). These enzymatic characteristics containing the cleavage pat- tern of the 12-bp RNAÆDNA hybrid were similar to those of archaeal Type 1 RNase H [19,20]. Archaeal Type 1 RNase H can cleave an RNA–DNA junction (a junction between the 3¢ side of RNA and 5¢ side of DNA) of an Okazaki fragment-like substrate (RNA9– DNAÆDNA), unlike other cellular Type 1 RNase H [19,20]. To check whether the SCO2299 protein can also cleave the RNA–DNA junction, the RNA9– DNAÆDNA substrate was examined for the SCO2299 protein. As shown in Fig. 4, both the full length 97 66 45 30 20 14 M 1 2 3 kDa Fig. 2. SDS ⁄ PAGE of purified SCO2299 proteins. All recombinant proteins were purified as described in Experimental procedures, subjected to SDS ⁄ PAGE (15% gel), and stained with Coomassie Brilliant Blue. M, low molecular mass standards kit (Amersham); 1, the full length protein; 2, the N-terminal RNase H domain; 3, the C-terminal APase domain. Molecular masses are indicated on the left side of the gel. A fusion protein consisting of RNase H and APase N. Ohtani et al. 2830 FEBS Journal 272 (2005) 2828–2837 ª 2005 FEBS protein and the RNase H domain could cleave the RNA–DNA junction of this substrate, in a similar manner to archaeal enzymes. Function of the C-terminal domain The amino acid sequence suggested that the C-terminal region of the SCO2299 protein might function as a phosphatase. Therefore, the phosphatase activity of the SCO2299 protein was examined by using p-nitro- phenyl phosphate (pNPP) as a substrate. When activity was assayed at various pH values as described in Experimental procedures, both the full length protein and the C-terminal domain of SCO2299 showed maxi- mal activity at pH 5.0 (Fig. 5). On the other hand, the N-terminal RNase H domain showed no phosphatase full-length N-domain C-domain N-domain & C-domain MM g12 g11 c10 a9 g8 u7 a6 g5 a4 g3 g2 3' g12 g11 c10 a9 g8 u7 a6 g5 a4 g3 g2 3' 0 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Fig. 3. Cleavages of 12-bp oligomeric RNAÆDNA substrate. A 12-bp RNAÆDNA hybrid was incubated at 37 °C for 15 min with the purified pro- teins in 10 m M Tris ⁄ HCl (pH 8.5) containing 10 mM MgCl 2 ,10mM NaCl, 1 mM 2-mercaptoethanol and 50 lgÆmL )1 BSA. The concentration of the substrate was 0.5 l M. Products were separated on a 20% polyacrylamide gel containing 7 M urea as described in Experimental proce- dures. M represents products resulting from partial digestion of the 12-b RNA with snake venom phosphodiesterase. Lanes 0–4 represent samples incubated with each protein (0, 0.012, 0.12, 1.2 and 12 pmolÆmL )1 , respectively). In the lanes for ‘N-domain and C-domain’, both N-terminal and C-terminal domains of each amount were added to reaction mixtures. full-length RNase H domain M M 0 1 2 3 4 5 0 1 2 3 4 5 Fig. 4. Cleavage of Okazaki fragment-like substrates. RNA9–DNAÆDNA hybrids were incubated at 37 °C for 15 min with SCO2299 proteins. Cleavage reactions and product separation were carried out as described in Fig. 3. Lanes 0–5 represent samples incubated with each protein of amount of 0, 0.12, 1.2, 12, 120 and 1200 pmolÆmL )1 , respectively. M represents the 3¢ end-labeled RNA1–DNA (5¢-cTGCAGGTCG-3¢), which was chemically synthesized by Proligo. Cleavage at the RNA–DNA junction of the RNA9–DNAÆDNA substrate gives a product that is one base shorter than M. Products are shown schematically on the right. Deoxyribonucleotides and ribonucleotides are shown by uppercase and lowercase letters, respectively. The asterisk and the black arrowhead indicate the fluorescent-labeled site and the RNA–DNA junction, respectively. N. Ohtani et al. A fusion protein consisting of RNase H and APase FEBS Journal 272 (2005) 2828–2837 ª 2005 FEBS 2831 activity at any pH value (data not shown). As shown in Fig. 5, the specific activity of the full length protein was approximately twofold lower than that of the C-terminal domain alone. However, as the calculated molecular mass (52 438 Da) of the His-tagged full length protein is two-fold larger than that (24 044 Da) of the His-tagged C-terminal domain, the phosphatase activity per mole of protein was almost the same in the two proteins. This finding suggested that the phospha- tase activity detected in the full length SCO2299 protein depended only on its C-terminal phosphatase domain and was independent on its N-terminal RNase H domain. The phosphatase activity was examined with var- ious phosphorylated substrates at pH 5.0 as shown in Table 1. No remarkable difference in the phosphatase activity between the full length protein and the C-ter- minal domain was observed. It is noteworthy that fructose 2,6-bisphosphatase activity was not detected in the preparation of the SCO2299 proteins. Although phosphoglycerate mutase activity and CobC activity generating a-ribazole from a-ribazole-5¢-phosphate were also examined as described previously [32] or as described in Experimental procedures, neither activi- ties were detected (data not shown). The CobC activ- ity was examined using capillary electrophoresis mass spectrometry (CE-ESI MS) and coupling with E. coli CobT, because the a-ribazole-5¢-phosphate is gener- ated from nicotinate nucleotide and dimethylbenz- imidazole by a phosphoribosyltransferase enzyme (CobT). When purified E. coli CobC was used as a positive control, the resultant a-ribazole was selec- tively detected in its deprotonated ion form (data not shown), suggesting that this method was suitable for the CobC assay. These results indicated that the C-terminal phosphatase domain of SCO2299 was not equivalent to fructose 2,6-bisphosphatase, phosphogly- cerate mutase, or the CobC protein. Therefore, it was concluded that the C-terminal domain of the SCO2299 protein functioned as an APase. The SCO2299 protein exhibited APase activity in the absence of divalent metal ions, suggesting that it required no divalent metal ions for catalysis. This characteristic of the SCO2299 protein agrees with that of other APases [29,33]. Discussion The SCO2299 protein from S. coelicolor The SCO2299 gene from S. coelicolor was shown to encode a bifunctional enzyme consisting of an RNase H domain and an APase domain. The RNase H and APase activities of the full length SCO2299 protein depend on its N-terminal RNase H domain and C-ter- minal APase domain, respectively, and do not interfere or overlap with each other. Although C. glutamicum Fig. 5. The pH profile of phosphatase activity of the SCO2299 pro- teins. The full length SCO2299 protein (circle) and the C-terminal APase domain (triangle) were incubated for 10 min at 37 °C with 10 m M of p-nitrophenyl phosphate in 100 mM acecate ⁄ NaOH (closed symbol) or HEPES ⁄ NaOH (open symbol). The specific activi- ties shown were determined from the average of triplicate experi- ments and were reproducible within 10% of the mean values. Table 1. Phosphatase activity with various substrates. The full length SCO2299 protein and the C-terminal APase domain were incubated with 10 m M of substrate for 10 min at 37 °C in 100 mM acecate ⁄ NaOH (pH 5.0). The specific activities shown were deter- mined from the average of triplicate experiments and were repro- ducible within 10% of the mean values. N.A., no activity (< 0.01). Substrate Specific activity (UÆmg )1 ) APase-domain SCO2299 pNPP 3.72 5.60 Phytic acid N.A. N.A. p-Ser N.A. N.A. p-Tyr 0.17 0.28 ATP N.A. N.A. ADP N.A. N.A. Glucose 1-phosphate N.A. N.A. Fructose 1-phosphate N.A. 0.20 Fructose 1,6-bisphosphate N.A. N.A. Fructose 2,6-bisphosphate N.A. N.A. Ribose 5-phosphate 0.13 0.28 Ribulose 5-phosphate 0.76 1.10 Ribulose 1,5-bisphosphate 0.95 2.21 A fusion protein consisting of RNase H and APase N. Ohtani et al. 2832 FEBS Journal 272 (2005) 2828–2837 ª 2005 FEBS RNase HI, the SCO2299-like protein, was shown to be active as an RNase H, its phosphatase activity had not previously been examined [24]. Therefore, the SCO2299 protein is the first reported example of this bifunctional RNase HI. Genes similar to the SCO2299 gene are distributed among several bacteria classified as Actinomycetales, i.e., Streptomyces , Corynebacterium, Mycobacterium, Nocardia and Thermobifida (Fig. 6). The distribution of the gene among several bacteria implies that it might be involved in important functions for living cells. However, in-frame deletion mutants of the RN- ase H domain only (D13–155; the deletion of amino acid residues 13–155) or APase domain only (D284– 467) of the SCO2299 gene in S. coelicolor grew as well as the parental strain (N. Saito, unpublished data), suggesting that the SCO2299 gene would not be essen- tial for cell viability. The deletion strains are under analysis, and the physiological functions of the SCO2299 gene remain to be determined. It is also unclear exactly what the fusion between RNase H and APase means for living cells. APase domain The C-terminal domain of the SCO2299 protein exhib- its phosphatase activity at an acidic pH. It requires no divalent metal ion for catalytic reaction. Generally, the APases do not utilize divalent metal ions in their cata- lysis [29,33]. They instead utilize histidine to form an enzyme–phosphohistidine intermediate, which is essen- tial for their catalysis [33,34]. A His residue in an RHGXRXP motif that is highly conserved among APases has been proposed to form this intermediate [34]. His301 in the SCO2299 protein corresponds to the conserved His residue (Fig. 6). The SCO2299 Fig. 6. Intermediate regions between RNase H and APase domains in the SCO2299 orthologs. Numbers represent the positions of amino acid residues, starting from the ini- tiator Met for each protein. An asterisk indi- cates a conserved His residue proposed to form a phophohistidine–enzyme intermedi- ate. The abbreviations are as follows: Sco, SCO2299 of Streptomyces coelicolor; Sav, SAV5877 of Streptomyces avermitilis; Cgl, RNase HI (or Cg2455) of Corynebacterium glutamicum; Cef, CE2133 of Coryne- bacterium efficiens; Cdi, DIP1678 of Coryne- bacterium diphtheriae; Mtu, Rv2228c (or MT2287) of Mycobacterium tuberculosis; Mle, ML1637 of Mycobacterium leprae; Mav, MAP1980c of Mycobacterium avium; Nfa, nfa16400 of Nocardia farcinica, and Tfu, Tfus02000308 of Thermobifida fusca.The Rv2228c of M. tuberculosis is identical to Mb2253c of Mycobacterium bovis. N. Ohtani et al. A fusion protein consisting of RNase H and APase FEBS Journal 272 (2005) 2828–2837 ª 2005 FEBS 2833 protein does not share the conserved RHGXRXP motif strictly, suggesting that it may not be a true APase and may exhibit specificity to an unexamined substrate. However, this substrate has not yet been identified. Intermediate region between two domains Amino acid sequences of RNase H and APase domains in the SCO2299 orthologs are highly con- served, whereas sequences of the intermediate regions between two domains are quite different in sequence and size (Fig. 6). This fact suggests that the function of each of the two domains is strictly important for cells, whereas the intermediate region might not be. The results from truncated SCO2299 proteins indicate that the activities of the two domains do not interfere or overlap. The intermediate region of the SCO2299 protein is the longest among similar genes and con- tains many Gly residues (Fig. 6). This increased flexi- bility of the intermediate region might contribute to the independence of the two domains. Analyses of other SCO2299-like proteins and comparisons with the SCO2299 protein will provide some information on the role of the intermediate region and further information on the relation between the two domains. Multiple RNase H genes in the S. coelicolor genome The S. coeicolor genome contains two additional RNase H homologous genes besides the SCO2299 gene [26]. One is the SCO5812 gene, encoding an RNase HII-like amino acid sequence, and the other is the SCO7284 gene, encoding an RNase HI-like sequence. The result of the complementation assay with MIC2067 showed that both SCO5812 and SCO7284 were active (N. Ohtani, unpublished data). As the SCO7284 protein has no APase domain, it is more similar to E. coli RNase HI (identity of 34% in 117 amino acid residues) than the SCO2299 protein. There- fore, we refer to the SCO7284 protein as S. coelicolor RNase HI. S. coelicolor is the first example of an organism whose genome contains three active RNase H genes. This multiplicity might be a reason why dele- tion of the SCO2299 gene is not lethal for cells. The bacterium also contains many phosphatase-like genes in its genome. A novel style Type 1 RNase H Enzymatic properties (the divalent metal ion prefer- ence, pH profile, and RNA–DNA junction cleavage) of the RNase H activity of the SCO2299 protein from S. coelicolor were more similar to those of archaeal RNase HI than to other bacterial RNase HI [19,20]. A previous phylogenetic analysis based on amino acid sequences strongly supports this similarity [20]. Because the archaeal RNase HI exhibits a similar RNase H activity to the RNase H domain of RT, it has been hypothesized that the enzyme might be derived via horizontal gene transfer from RT [19,20]. Although properties of the RNase H domain of the SCO2299 protein are also similar to those of RT, it is not known whether the RNase H domain of RT fused with one APase or not. Nevertheless, the SCO2299 protein examined here is a bifunctional enzyme con- sisting of an RNase H domain and an APase domain, and it is a novel style in the Type 1 RNase H family. Experimental procedures Cells, plasmids, and materials The genomic DNA of S. coelicolor A3(2) was prepared by the salting out procedure [35]. E. coli MIC2067 is an rnhA and rnhB double mutant strain [30], and E. coli MIC2067(DE3) was previously constructed for overexpres- sion of a recombinant protein using the pET system [31]. Plasmids pET-11a and pET-28a, and E. coli Rosetta(DE3) were purchased from Novagen (Madison, WI, USA). Restriction enzymes, modifying enzymes, and PCR enzymes were from TaKaRa Bio (Kyoto, Japan). Crotalus atrox phosphodiesterase I was purchased from Sigma (St. Louis, MO, USA). The other chemical reagents were purchased from Wako (Osaka, Japan) or Sigma. In vivo complementation assay for RNase H activity Plasmids for complementation assay were constructed by ligating the DNA fragment containing the full length, the RNase H domain, or the APase domain of the SCO2299 gene to the NdeI–BamHI site of pET-11a. The DNA frag- ments were amplified by PCR using S. coelicolor genomic DNA as a template. The PCR primers were 5¢-CCTCCTC CT CATATGGCTGACCAGGCGCCCCGCCCCGCGC-3¢ (5¢-F primer) as 5¢-primer and 5¢-GGTGGTGGT AGAT CTTTATCAGCGCAGGTGGGACGTCTCGTTG-3¢ (3¢- F-primer) as 3¢-primer for the full length gene; the 5¢-F pri- meras5¢-primer and 5¢-GGCGCG AGATCTTTAT TACGC GTCGAGCTCCGCCGTCGAGTC-3¢ as 3¢-primer for the RNase H domain; and 5¢-GGGCCGCCC CATATGGG CGCCCCCGCGACCTTC-3¢ as 5¢-primer and the 3¢-F pri- mer as 3¢-primer for the APase domain. The underlined bases show the positions of the NdeI(5¢-primer) and the BglII (3¢-primer) sites. E. coli RNase H mutant strain A fusion protein consisting of RNase H and APase N. Ohtani et al. 2834 FEBS Journal 272 (2005) 2828–2837 ª 2005 FEBS MIC2067(DE3) was transformed with each constructed plasmid, spread on Luria agar plates containing 50 lgÆ mL )1 ampicillin and 30 lgÆmL )1 chloramphenicol, and incubated at 30 °C and 42 °C. Plasmid constructions, overproductions and purifications Plasmids for overexpression of His-tagged recombinant proteins were constructed by ligating the NdeI–EcoRI DNA fragment from the plasmid used for the complemen- tation assay, to the NdeI–EcoRI site of pET-28a. For over- production, E. coli Rosetta(DE3) was transformed with each constructed plasmid and grown in Luria broth con- taining 0.1% (w ⁄ v) glucose, 30 lgÆmL )1 kanamycin, and 30 lgÆmL )1 chloramphenicol at 37 °C. When the absorb- ance at 600 nm of the culture reached around 0.5, isopropyl thio b-d-galactoside was added to the culture medium (final concentration: 0.3 mm) and cultivation was continued at 37 °C for 30 min. Then, the temperature of the growth medium was shifted to 18 °C and cultivation was continued at 18 °C for an additional 15 h. Cells were harvested by centrifugation at 6000 g for 5 min. The following protein purification was carried out at 4 °C. Cells were suspended in 20 mm Tris ⁄ HCl (pH 8.0) containing 0.5 m NaCl and 50 mm imidazole (buffer A), disrupted by sonication with an ultrasonic disruptor UD-201 from TOMY Corp (Tokyo, Japan), and centrifuged at 30 000 g for 30 min. The super- natant was applied to a Ni 2+ -affinity column (4 mL), in which the Chelating Sepharose Fast Flow (Amersham, Pis- cataway, NJ, USA) had been charged with a NiSO 4 solu- tion and equilibrated with buffer A. The protein was eluted from the column using a linear gradient of imidazole from 50 to 500 mm in buffer A. The protein fractions were com- bined, concentrated, dialyzed against 20 m m Tris ⁄ HCl (pH 8.0) containing 1 mm EDTA, 150 mm NaCl, and 1mm dithiothreitol, and used for further analyses. The concentration of the purified protein was determined from the extent of UV absorption with A 280 0:1% values of 0.83 for the full length protein, 1.1 for the RNase HI domain, and 0.63 for the APase domain, which were cal- culated using e-values of 1576 m )1 Æcm )1 for Tyr and 5225 m )1 Æcm )1 for Trp at 280 nm [36]. Cleavage reaction of oligomeric RNA Æ DNA substrates The 5¢ end labeled 12-b RNA (5¢-cggagaugacgg-3¢) and the 3¢ end labeled 18-b RNA9–DNA (5¢-uugcaugccTGCA GGTCG-3¢), and their complementary DNAs were chemic- ally synthesized by Proligo (Paris, France). Deoxyribo- nucleotides and ribonucleotides are shown by uppercase and lowercase letters, respectively. 6-FAM was used for the end labeling. The RNAÆDNA hybrid (0.5 lm) was prepared by hybridizing the end-labeled RNA-containing oligonu- cleotide with 2.0 molar equivalent of its complementary DNA. Hydrolysis of the substrate was carried out at 37 °C for 15 min in 10 mm Tris ⁄ HCl (pH 8.5) containing 10 mm MgCl 2 ,10mm NaCl, 1 mm 2-mercaptoethanol and 50 lgÆmL )1 bovine serum albumin (BSA). Product analysis was carried out as described previously [19,20]. Measurement of phosphatase activity The phosphatase activity was measured according to Fiske & Subbarow [37]. For routine measurements, samples were incubated in a 96-well microtitre plate in a final volume of 100 lL. Each assay contained 100 mm acetate ⁄ NaOH (pH 5.0) and 10 mm of substrate. Assays were initiated by the addition of substrate, progressed for 10 min at 37 °C, and terminated by 5 lL of 100% (v ⁄ v) trichloroacetic acid. After dilution with 100 l L of water, 25 lL of 2.5% ammo- nium molybdate in 2.5 m H 2 SO 4 and 10 lL of Fiske–Subba- row reagent were added. The mixtures were incubated for 20 min at 37 °C and absorbance determined at 820 nm using a Spectromax 250 Microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The standard curve of phos- phate was obtained with 0–200 nmol sodium phosphate. One unit (U) of phosphatase activity is defined as the amount of enzyme resulting in the production of 1 lmol phosphate per min at 37 °C. The specific activity is defined as the enzymatic activity per milligram of protein. The opti- mal pH on pNPP was determined in 100 mm acetate ⁄ NaOH (pH 3.0–6.0) or 100 mm HEPES ⁄ NaOH (pH 6.0–8.0). Determination of a CobC activity by CE-ESI MS Overproducing strains for N-terminal His-tagged recombin- ant proteins of E. coli CobT and E. coli CobC (PhpB) were kindly donated by H Mori and T Baba (Institute for Advanced Biosciences, Keio University, Yamagata, Japan). Details on these strains in ASKA (a complete set of E. coli K-12 ORF archive) library are available at http://ecoli. aist-nara.ac.jp/index.html. They were grown in Luria broth containing 0.1% (w ⁄ v) glucose and 30 lgÆmL )1 chloram- phenicol. Induction, sonication, and purification were per- formed as described for the SCO2299 proteins. The purified proteins were dialyzed against 20 mm Tris ⁄ HCl (pH 8.0) containing 1 mm EDTA, 0.5 m NaCl, and 1 mm dithio- threitol, concentrated, and used for analyses. The assay of CobC activity was performed either in 5mm HEPES ⁄ NaOH (pH 7.5) or 5 mm acetate ⁄ NaOH (pH 5.0) containing 10 lg CobT, 1 mm MgCl 2 ,10mm KCl, 1 mm nicotinate nucleotide, 1 mm dimethylbenzimi- dazole, 200 lm PIPES as an internal standard, and 2 lgof protein samples in a final volume of 100 lL. The reaction mixture was incubated for 10 min at 37 °C and transferred to 400 lL cold methanol. The contents were stored on ice N. Ohtani et al. A fusion protein consisting of RNase H and APase FEBS Journal 272 (2005) 2828–2837 ª 2005 FEBS 2835 for 20 min and diluted four times with water. The inacti- vated enzymes were removed by filtration in a centrifugal filter (5000 molecular mass cut) according to the manufac- ture’s instructions. The filtrate was immediately freeze-dried and drawn into 40 lL of water prior to injection. As a posi- tive control, purified E. coli CobC was incubated with E. coli CobT under the described condition and the prod- ucts were determined. The analysis was performed using an Agilent CE system, Agilent 1100 series MSD mass spectrometer, an Agilent 1100 series isocratic HPLC pump, a G1603A Agilent CE ⁄ MS adapter kit, and a G1607A Agilent CE ⁄ MS sprayer kit (Agilent Technologies, Waldbronn, Germany). CE-ESI MS separations employed a SMILE (+), cationic polymer (polybrene) coated capillary column (1 m · 50 lm internal diameter) (Nakalai Tesque, Kyoto, Japan) and 50 mm ammonium acetate (pH 8.5) as the electrolyte. The other analytical conditions were as described previously [38]. In this method, niacine and a-ribazole-5¢-phosphate generated by CobT, and a-ribazole by CobC were selec- tively detected in their deprotonated ion forms (m ⁄ z: 122, 357 and 277, respectively) by MS. 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J Biol Chem 66, 375–400. 38 Soga T, Ueno Y, Naraoka H, Ohashi Y, Tomita M & Nishioka T (2002) Anal Chem 74, 2233–2239. N. Ohtani et al. A fusion protein consisting of RNase H and APase FEBS Journal 272 (2005) 2828–2837 ª 2005 FEBS 2837 . show that the SCO2299 gene from S. coelicolor encodes a bifunc- tional enzyme consisting of the RNase H domain and the acid phosphatase (APase) domain, and. exhib- ited acid phosphatase activity at almost the same level as the C-terminal domain alone. These results indicate that RNase H and acid phosphatase activities