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unpublished data) were recently shown to encode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:
(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.
Acknowledgements
This research was partially supported by the Ministry
of Education, Culture, Sports, Science and Technol-
ogy, Grant-in-Aid for the 21st Century Center of
Excellence (COE) Program entitled ‘Understanding
and Control of Life’s Function via Systems Biology
(Keio University)’ and a grant from New Energy and
Industrial Technology Development Organization
(NEDO) of the Ministry of Economy, Trade and
Industry of Japan (Development of a Technological

Infrastructure for Industrial Bioprocesses Project).
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