(Received for publication, September 26, 1994; and in revised form, November 28, 1994)
From the
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.
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) . ()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)()(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). ()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.
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.
Figure 3:
Chromatography of bacterial extracts on
Superdex-75. Fractions were collected at the beginning of the void
volume and analyzed for protein () and activase activity
(
) 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.
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 () or pREH
(
).
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.
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.