©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Generation of the Glycyl Radical of the Anaerobic Escherichia coli Ribonucleotide Reductase Requires a Specific Activating Enzyme (*)

(Received for publication, September 26, 1994; and in revised form, November 28, 1994)

Xueyin Sun (1) (2) Rolf Eliasson (1) Elisabet Pontis (1) Jessica Andersson (2) Girbe Buist (3) Britt-Marie Sjöberg (2) Peter Reichard (1)(§)

From the  (1)Department of Biochemistry 1, Medical Nobel Institute, MBB, Karolinska Institute, S-17177 Stockholm, Sweden, the (2)Department of Molecular Biology, Stockholm University, S-10691 Stockholm, Sweden, and the (3)Department of Genetics, University of Groningen, 9741 NN Haren, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The anaerobic ribonucleotide reductase from Escherichia coli contains a glycyl radical as part of its polypeptide structure. The radical is generated by an enzyme system present in E. coli. The reductase is coded for by the nrdD gene located at 96 min. Immediately downstream, we now find an open reading frame with the potential to code for a 17.5-kDa protein with sequence homology to a protein required for the generation of the glycyl radical of pyruvate formate lyase. The protein corresponding to this open reading frame is required for the generation of the glycyl radical of the anaerobic reductase and binds tightly to the reductase. The ``activase'' contains iron, required for activity. The general requirements for generation of a glycyl radical are identical for the reductase and pyruvate formate lyase. For the reductase, the requirement of an iron-containing activase suggests the possibility that the iron-sulfur cluster of the enzyme is not involved in radical generation but may participate directly in the reduction of the ribonucleotide.


INTRODUCTION

Escherichia coli uses different enzymes for the reduction of ribonucleotides to deoxyribonucleotides during aerobic and anaerobic growth(1) . Both enzymes utilize radical chemistry for the reaction but employ different mechanisms for radical generation. The aerobic enzyme contains a stable tyrosyl radical deeply buried in the structure of the enzyme(2) . It initiates a second protein radical located to a cysteinyl residue (3, 4) that directly participates in the catalytic process. The anaerobic reductase contains an oxygen-sensitive glycyl radical located at Gly-681 (5) . (^1)It is not known if the glycyl radical participates directly or indirectly during catalysis. The anaerobic enzyme also contains a poorly defined iron-sulfur cluster (6) .

As isolated, the anaerobic reductase is inactive and lacks the glycyl radical. In earlier work we found an enzyme system in E. coli that generates the glycyl radical and activates the reductase. This system consists of flavodoxin and flavodoxin reductase and employs S-adenosylmethionine and NADPH in the activating reaction(7, 8, 9) . During activation, S-adenosylmethionine was cleaved reductively into methionine and 5`-deoxyadenosine(7) .

The activation reaction shows considerable similarity to the activation of E. coli pyruvate formate lyase (pfl)(^2)(10, 11, 12) . Seminal work by Knappe and co-workers (10, 11) has shown that activation of the lyase involves the generation of a glycyl radical by a specific 28-kDa iron-containing activase with the aid of S-adenosylmethionine and dihydroflavodoxin. We tested the possibility that the pfl-activase might activate also the anaerobic ribonucleotide reductase but found that an extract of a manipulated E. coli strain lacking this enzyme showed good reductase-activating ability(7) .

This situation appeared in a different light when one of us (G. B.) by chance cloned and sequenced from Lactococcus lactis subspecies cremoris MG1363 two consecutive open reading frames (ORF). (^3)The first could encode a protein of 84.2 kDa showing 49% amino acid sequence identity with the anaerobic reductase of E. coli, and the second could encode a protein of 23.3 kDa showing some homology with the pfl activase. This suggested that E. coli might contain a specific activase for its ribonucleotide reductase similar to but distinct from the pfl-activase. In the present communication we describe the isolation and characterization of such an enzyme, required for the generation of the glycyl radical of the anaerobic E. coli ribonucleotide reductase.


EXPERIMENTAL PROCEDURES

Materials

Strain JM109(DE3) was from Promega. Plasmid pet3b was from Novagen, AMS Biotechnology, and plasmids pTZ18R and pTZ19R were from Pharmacia Biotech Inc. Oligonucleotide primers were synthesized by Scandinavian Gene Synthesis, Köping, Sweden. Taq DyeDeoxy terminator cycle sequencing kit was from Applied Biosystems.

Construction of Plasmids

Plasmid pPX41 that contains both the nrdD and activase genes was constructed by insertion of a 6.5-kilobase PstI fragment from pSX25 (5) into PstI-linearized pTZ18R. This plasmid was used to determine the nucleotide sequence of the activase gene.

Plasmid pREH was made by inversion of the insertion of pEH10 (5) into a pTZ19R vector. Plasmid pRSS was made from pSS17 (5) by the same procedure.

Plasmid pN9 was constructed by introduction of the activase gene into vector pet3b. The insertion was generated by the overlap extension PCR strategy as described in (13) . The activase gene was amplified from pPX41 by PCR and then joined with a PCR fragment covering the promotor region of the vector, such that the presumed initiator codon of the activase gene was joined to the T7 promotor. The ends of the amplified overlap PCR fragment were then digested with SphI and BamHI and ligated into SphI/BamHI linearized pet3b vector DNA. On sequence analysis we found two silent mutations in the activase gene of pN9, one at position +21 (TAC instead of TAT) and the second at position +27 (GTG instead of GTC). pN9 was transformed into E. coli JM109(DE3) for the construction of an overproducing strain.

DNA Sequence Analyses

The activase gene of pPX41 and pN9 was sequenced by Taq DyeDeoxy terminator cycle sequencing with an automated laser fluorescent DNA sequencer.

Expression of Activase Gene

E. coli JM109(DE3) containing plasmid pN9 was grown anaerobically at 37 °C in Luria-Bertani medium with 0.2% glucose and 0.2 mg/ml ampicillin. When the A was 0.5, isopropyl-1-thio-beta-D-galactopyranoside (final concentration, 0.5 mM) was added. Two h after induction, cells were collected by centrifugation.

Purification of Activase

All purification was made at close to 4 °C in air. Ten g of JM109(DE3) carrying pN9 was extracted with 15 ml of a solution of 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM DTT, 0.5% Nonidet P-40 containing 15 mg of egg white lysozyme and centrifuged for 60 min at 100,000 times g. Ten ml of the supernatant solution (23 mg of protein/ml) were added to a column of Superdex 75 (preparative grade, Pharmacia Sweden) equilibrated with 30 mM Tris-HCl, pH 7.5, 1 M KCl, 5 mM DTT and chromatographed at 1.3 ml/min. After 70 min, fractions (6.5 ml) were collected and analyzed for protein and enzyme activity (see Fig. 3A under ``Results''). In a similar experiment 0.5 ml of extract from pREH bacteria were analyzed by chromatography on an analytical Superdex 75 column with the results shown in Fig. 3B.


Figure 3: Chromatography of bacterial extracts on Superdex-75. Fractions were collected at the beginning of the void volume and analyzed for protein (times) and activase activity (bullet) as described under ``Experimental Procedures.'' A, extract from JM109(DE3) carrying plasmid pN9. The inset shows results from an SDS Phast (Pharmacia) gel electrophoresis. From left to right: marker proteins, 0.2 µg of fractions 20, 19, 18, 17, and 16, and 1 µg of protein from the bacterial extract. B, extract from bacteria carrying plasmid pREH.



Enzyme Assays

The activity of the anaerobic reductase was measured as described earlier(7) . In this assay activation was obtained from the intrinsic activating ability of the reductase derived from overproducing bacteria carrying plasmid pREH. Activating enzyme was determined from its ability to activate 1 µg of reductase prepared from overproducing bacteria carrying plasmid pRSS. This reductase lacked intrinsic activating ability. For both enzymes, 1 unit of activity represents the reduction of 1 nmol of CTP during 1 min. Specific activity is defined as units per mg of protein.

Treatment of Activase with Iron

Anaerobic conditions were maintained during the whole procedure. Activase (0.20 mg of fraction 18 in Fig. 3A) was incubated in a final volume of 0.2 ml with 30 mM Tris-HCl, pH 7.5, 0.1 M KCl, 5 mM DTT for 30 min. Ten µl of an anaerobic solution of 40 mM Fe(NH(4))(2)(SO(4))(2) were added, and incubation was continued for 30 min. The tube was then transferred to an anaerobic hood, and the material was equilibrated with 30 mM Tris-HCl, pH 8.0, by passage through a 3-ml column of Sephadex G-25. The final material (0.10 mg of protein in 0.2 ml of buffer) was assayed for activase activity without exposure to air.

Other Methods

Protein was determined by the method of Bradford(14) . Iron was analyzed as described(15) . The N-terminal amino acid sequence of the activase was determined in a Applied Biosystems 491 protein sequencer. Protein homology comparisons were carried out by means of the Fasta program(16) .


RESULTS

When we sequenced the DNA downstream of the E. coli nrdD gene we found an additional ORF apparently within the same operon as the nrdD gene (Fig. 1A). This frame had the potential to code for a 17.5-kDa protein with considerable homology to the L. lactis 23.3-kDa protein, the T4 gene 55.9 protein, and to a lesser extent to the pfl activase (Fig. 1B). The T4 gene 55.9 protein was recently shown to code for an enzyme required for the activation of the T4 NrdD protein(17) . The common homology is particularly striking for the 50 N-terminal amino acids, with 26% position identity for the four proteins, among them 3 of the 4 cysteines implicated in the binding of iron of the pfl activase(10) . It therefore appeared likely that the E. coli 17.5-kDa protein is an activase required for the generation of the glycyl radical of the anaerobic reductase.


Figure 1: A, nucleotide sequence of DNA immediately downstream of the nrdD gene containing the open reading frame coding for a 17.5-kDa protein. The stop codon of nrdD is indicated as are the two complete and one truncated REP sequences(5) . A possible terminator downstream of the E. coli activase (Ecact) is underlined. The deduced amino acid sequence corresponding to the open reading frame is shown in the one-letter code. B, comparison of the deduced sequence of the 50 N-terminal amino acids of the E. coli activase with those for the pfl activase (pflact), the T4 gene 55.9 protein (T455.9), and the L. lactis activase (Llact).



The plasmid (pREH) used earlier for overproduction of the reductase contained the ORF for the activase, suggesting the possibility that bacteria carrying the plasmid overproduced also the activase, which then was carried along during the purification of the reductase. When the highly purified reductase was analyzed by denaturing gel electrophoresis we observed on heavily overloaded gels a faint band of a 17-kDa protein. We estimated the amount of protein present in this band to be between 0.5 and 1% of the total protein. Further chromatography of the reductase on MonoQ or Hypatite columns did not remove the minor protein (data not shown).

We next constructed plasmid pRSS that lacks the ORF and prepared the reductase from bacteria carrying this plasmid. This preparation of the reductase was no longer activated by the previously employed system and did not show the 17-kDa band on SDS gel electrophoresis. However, the enzyme was activated by crude extracts of bacteria carrying plasmid pREH containing the ORF coding for the putative 17.5-kDa protein (Fig. 2). Crude extracts from bacteria carrying plasmid pRSS were inactive (Fig. 2). Taken together these results strongly suggested that the 17.5-kDa protein is an activase of the reductase.


Figure 2: Activation of ribonucleotide reductase prepared from E. coli carrying plasmid pRSS with extracts from bacteria carrying pRSS (circle) or pREH (times).



The ORF sequence coding for the activase was cloned into plasmid pN9 to construct an overproducing strain of E. coli. Extracts of this strain showed an excellent ability to activate the pRSS-derived reductase. Chromatography on a Superdex-75 column separated a well defined enzymatically active protein peak, appearing at 0.63 column volume (Fig. 3A). In a parallel experiment the markers trypsin inhibitor (20.1 kDa) and lysozyme (14.4 kDa) appeared at 0.58 and 0.76 column volumes, respectively, suggesting that the activase exists as a monomer in solution. The inset to Fig. 3A shows analyses of the peak fractions and the starting material by denaturing gel electrophoresis. The starting material contains a pronounced band at 17 kDa, and this is also the major band present in the active peak from the chromatogram. We estimate that in fractions 18 and 19 the 17-kDa band accounts for 90% or more of the total protein.

Fig. 3B shows a similar Superdex-75 chromatogram of the extract from bacteria carrying plasmid pREH used for the experiment shown in Fig. 2. Now, activase activity appears in a peak in the void volume with a long trail in the remaining part of the chromatogram, again demonstrating the tight association with the reductase.

Fractions in Fig. 3A containing activase activity had a pronounced brown color. The visible spectrum of the activase shown in Fig. 4contains three poorly resolved absorption bands between 380 and 620 nm suggesting the presence of sulfur-linked iron in the protein. Iron analysis indeed demonstrated the presence of 30 nmol of iron/mg of protein, corresponding to 0.5 mol of iron/mol of protein. Total amino acid analysis was in good agreement with the results predicted from the DNA sequence of pN9 (data not shown). N-terminal amino acid analysis gave a clean sequence of the first 18 amino acids corresponding to the sequence deduced from the nucleotide sequence (Fig. 1). All analyses were made with the protein present in fraction 18 of Fig. 3A.


Figure 4: Spectrum of activase (2 mg/ml) in 30 mM Tris-HCl, pH 7.5, 1 M KCl, 5 mM DTT.



The specific enzyme activity of fraction 18 was 1510 as compared with a specific activity of 510 in the crude extract, indicating some loss of activity during purification, possibly caused by loss of iron. Free iron is a strong inhibitor of anaerobic reductase activity (18) and can therefore not be added directly during the assay of the enzyme. Instead, we treated the activase preparation with iron under anaerobic conditions, removed excess metal, and assayed for activation of the reductase. The treatment increased the specific activity of the activase to a value of 2700.


DISCUSSION

The results of this paper clearly demonstrate that generation of the glycyl radical of the anaerobic E. coli ribonucleotide reductase requires a specific activating enzyme in addition to the earlier demonstrated requirements for reduced flavodoxin and S-adenosylmethionine(7, 8, 9) . The gene for the activase forms an operon together with nrdD. In solution, the activase is tightly bound to the reductase and was overlooked in earlier experiments.

The general requirements for generation of a glycyl radical are thus identical for the reductase and for pfl. It seems very likely that the same mechanism, via an inferred deoxyadenosyl radical, occurs with both enzymes.

As isolated, the E. coli activase contains substoichiometric amounts of iron but can be further activated by anaerobic treatment with Fe. The N-terminal structure contains the three conserved cysteines, which in the pfl-activase were suggested to bind iron. The visible spectrum of the activase suggests the presence of sulfur-bonded iron in the protein(19) . Taken together these results strongly suggest that the E. coli activase in its active form contains iron. The close similarity between ribonucleotide reductase and pfl further strengthens earlier speculations (20) concerning an evolutionary relationship between the two enzymes.

The discovery of a specific iron-containing activase for the reductase indicates that the iron-sulfur cluster of the reductase may not be involved in radical generation, as was believed originally(6) . Instead, it may participate in the actual reduction of the ribotide.


FOOTNOTES

*
This work was supported by grants from the Swedish Cancer Society and the Swedish Science Research Council (to B-M. S.) and from the Swedish Medical Research Council, the European Union, and AB Astra (to P. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
X. Sun, M. Atta, R. Eliasson, P. Reichard, and B-M. Sjöberg, unpublished data.

(^2)
The abbreviations used are: pfl, pyruvate formate lyase; ORF, open reading frame; PCR, polymerase chain reaction; DTT, dithiothreitol.

(^3)
G. Buist, unpublished data.


ACKNOWLEDGEMENTS

We thank Katarina Gell for help with DNA sequencing and Stefan Björklund for help with early experiments showing tight association between reductase and activase.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.