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

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 cob

encodes a bifunctional enzyme consisting of an RNase H domain and an acid phosphatase domain

Naoto Ohtani1, Natsumi Saito1, Masaru Tomita1, Mitsuhiro Itaya1,2and Aya Itoh1

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and only those from Halobacterium sp NRC-1 [19], Sulfolobus toko-daii [20] and Pyrobaculum aerophilum (N Ohtani, unpublished data) were recently shown to encode active enzymes Interestingly, in another archaeon,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.

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

B12 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

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).

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-terN-ter-minal 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 RNAÆDNA 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 Mg2+, Mn2+, Co2+, and Ni2+, and preferred Mg2+or Mn2+to Co2+or Ni2+ 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

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.

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

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 m M MgCl2, 10 m M NaCl, 1 m M 2-mercaptoethanol and 50 lgÆmL)1BSA 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.

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 m M 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 m M

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)

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

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.

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 CTCATATGGCTGACCAGGCGCCCCGCCCCGCGC-3¢ (5¢-F primer) as 5¢-primer and 5¢-GGTGGTGGTAGAT CTTTATCAGCGCAGGTGGGACGTCTCGTTG-3¢ (3¢-F-primer) as 3¢-primer for the full length gene; the 5¢-F pri-mer as 5¢-pripri-mer and 5¢-GGCGCGAGATCTTTATTACGC GTCGAGCTCCGCCGTCGAGTC-3¢ as 3¢-primer for the RNase H domain; and 5¢-GGGCCGCCCCATATGGG CGCCCCCGCGACCTTC-3¢ as 5¢-primer and the 3¢-F pri-mer as 3¢-pripri-mer 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

MIC2067(DE3) was transformed with each constructed

plasmid, spread on Luria agar plates containing 50 lgÆmL)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 Ni2+-affinity column (4 mL), in

which the Chelating Sepharose Fast Flow (Amersham,

Pis-cataway, NJ, USA) had been charged with a NiSO4

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 mm Tris⁄ HCl

(pH 8.0) containing 1 mm EDTA, 150 mm NaCl, and

1 mm dithiothreitol, and used for further analyses

The concentration of the purified protein was determined

from the extent of UV absorption with A2800:1% 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)1Æcm)1 for Tyr and

5225 m)1Æcm)1for Trp at 280 nm [36]

Cleavage reaction of oligomeric RNAÆDNA

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 riboDeoxyribo-nucleotides 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 MgCl2, 10 mm NaCl, 1 mm 2-mercaptoethanol and

50 lgÆmL)1bovine 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 lL of water, 25 lL of 2.5% ammo-nium molybdate in 2.5 m H2SO4and 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 phosphos-phate 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

5 mm HEPES⁄ NaOH (pH 7.5) or 5 mm acetate ⁄ NaOH (pH 5.0) containing 10 lg CobT, 1 mm MgCl2, 10 mm KCl, 1 mm nicotinate nucleotide, 1 mm dimethylbenzimi-dazole, 200 lm PIPES as an internal standard, and 2 lg of 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

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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