(Received for publication, December 5, 1995; and in revised form, January 19, 1996)
From the
Escherichia coli contains the genetic information for
three separate ribonucleotide reductases. Two of them (class I
enzymes), coded by the nrdAB and nrdEF genes,
respectively, contain a tyrosyl radical, whose generation requires
oxygen. The NrdAB enzyme is physiologically active. The function of the nrdEF gene is not known. The third enzyme (class III), coded
by nrdDG, operates during anaerobiosis. The DNA of Lactococcus lactis contains sequences homologous to the nrdDG genes. Surprisingly, an nrdD mutant of L. lactis grew well under standard anaerobic
growth conditions. The ribonucleotide reductase system of this mutant
was shown to consist of an enzyme of the NrdEF-type and a small
electron transport protein. The coding operon contains the nrdEF genes and two open reading frames, one of which (nrdH)
codes for the small protein. The same gene organization is present in E. coli. We propose that the aerobic class I ribonucleotide
reductases contain two subclasses, one coded by nrdAB, active
in E. coli and eukaryotes (class Ia), the other coded by nrdEF, present in various microorganisms (class Ib). The NrdEF
enzymes use NrdH proteins as electron transporter in place of
thioredoxin or glutaredoxin used by NrdAB enzymes. The two classes also
differ in their allosteric regulation by dATP.
Ribonucleotide reductases are essential enzymes that catalyze the reduction of ribonucleoside di- or triphosphates and thereby provide the building blocks required for DNA replication and repair. Three different classes of enzymes are known(1) , each with a distinct protein structure but all requiring a protein radical for catalysis and all regulated by similar allosteric effects.
Class I reductases are aerobic enzymes present in all higher organisms and certain microorganisms, among them Escherichia coli(2) . This bacterium actually has the potential to produce two separate class I enzymes. One of them, coded for by the nrdA and -B genes (3) is the functional enzyme during the growth of E. coli and has been the prototype for all class I enzymes. The second enzyme is coded for by the nrdE and -F genes(4) , first discovered in Salmonella typhimurium(5) , and is normally not fully functional. Expression of the chromosomal nrdEF genes thus is not sufficient to complement mutations in nrdAB(5) . The two enzymes show a limited sequence similarity but contain certain strategical amino acids in identical positions. They differ to some extent in their allosteric regulation and with respect to their hydrogen donors(6) . A functionally active reductase of the NrdEF-type was recently found in Mycobacterium tuberculosis(7) . Mycoplasma genitalium contains the nrdEF genes but not the nrdAB genes(8) .
All class I enzymes consist of two proteins that are named R1 and R2 for NrdAB enzymes (2) and R1E and R2F (6) for the NrdEF enzymes. Each protein has specific functions: R1 and R1E contain the binding sites for both substrates and allosteric effectors and carry out the actual reduction of the ribonucleotide. R2 and R2F contain diferric iron centers (9) and the tyrosyl radical (10) required for catalysis. Generation of the tyrosyl radical requires oxygen (9) and class I enzymes are therefore believed not to function in the absence of oxygen.
In line with this concept,
anaerobically growing E. coli contains a third and completely
different reductase (11) that has a glycyl radical (12) but no tyrosyl radical. This enzyme, coded by nrdD(12) and nrdG(13) , is the prototype for
a whole group of class III enzymes whose presence in several other
anaerobically growing organisms can be inferred from specific DNA
sequences. Such sequences have been found in the DNA from E. coli phage T4(14) , Lactococcus lactis, ()and Haemophilus influenzae(15) . The
glycyl radical of this group of enzymes is formed by a complicated
activation reaction requiring S-adenosylmethionine and a
reducing enzyme system(16) . The exquisite oxygen sensitivity
of this radical limits the function of class III enzymes to bacteria
growing in the absence of oxygen.
A third group of reductases operates with a radical that does not require oxygen for its generation and is not oxygen sensitive. These enzymes use adenosylcobalamin as radical generator and function during both aerobic and anaerobic conditions. They are found in many different microorganisms and form a class II(17, 18) .
The reduction of ribose requires a source of electrons. With class I and II enzymes, two small proteins named thioredoxin and glutaredoxin fulfill this function(19) . They contain two active cysteine thiols that reduce by dithiol interchange two cysteines in the active center of the reductase. The reduced forms of both small proteins are regenerated by separate enzyme systems: thioredoxin, by a specific thioredoxin reductase + NADPH; glutaredoxin, by glutathione, glutathione reductase, and NADPH.
L. lactis subsp. cremoris is a member of the
family of lactic acid Gram-positive bacteria that grow anaerobically
but tolerate low concentrations of oxygen. Recently two open reading
frames homologous to the nrdD and -G genes of E.
coli were discovered and sequenced in L. lactis subsp. cremoris MG1363, suggesting the activity of an
anerobic reductase in this bacterium. By marker exchange with an
internally deleted nrdD gene an nrdD
mutant was constructed that surprisingly grew normally under
standard anaerobic conditions. (
)This suggested the presence
of an additional reductase able to reduce ribonucleotides under
anaerobic conditions supporting the growth of the mutant.
In this paper we characterize the ribonucleotide reductase of the mutant and identify it as a class I enzyme of the NrdEF-type. We also identify an apparently new type of hydrogen donor for this enzyme.
All manipulations during the following protein purification were done at close to 4 °C. In a typical experiment, 5.2 g of bacterial pellet was sonicated in 8 ml of 50 mM Tris-HCl, pH 7.5, 1 mM phenylmethanesulfonyl fluoride, 1 mM EDTA, 10 mM DTT and centrifuged at 45,000 rpm in a Ty 65 Beckman rotor for 60 min. Nucleic acids were removed by centrifugation after slow addition of 0.15 volume of 10% streptomycin sulfate to the supernatant solution. Solid ammonium sulfate was added during a 1-h period to the supernatant solution to 70% saturation. The resulting precipitate was collected by centrifugation, dissolved in a small volume of buffer A (50 mM Tris-HCl, pH 7.5, 10 mM DTT), and dialyzed against buffer A overnight with one change of buffer. The dialyzed solution was then added to a 30-ml column of DE52 equilibrated with buffer A. The column was first eluted at a rate of 0.5 ml/min with a linear KCl gradient (0-0.2 M KCl in buffer A, 60 + 60 ml), followed by additional elution with 60 ml of 0.2 M KCl in buffer A. Final elution was made with 40 ml of 0.4 M KCl in buffer A. Fractions (3 ml) were collected and analyzed for protein and reductase activity. This chromatographic step separated two fractions (Fig. 1), each inactive by itself but together providing reductase activity. From here on, the two fractions (DE1 and DE2) were purified separately.
Figure 1:
Separation of two protein fractions
required for CDP reduction. The material after ammonium sulfate
precipitation was chromatographed on DEAE-cellulose as described under
``Experimental Procedures.'' Fractions were analyzed for
protein (+), DE1 activity (), and DE2 activity (
). Each
protein fraction was analyzed in the presence of an excess of the other
fraction.
Figure 2:
Dependence of CDP reduction on DE1 and
DE2. A, increasing amounts of DE1 were incubated with 15
() or 29 (
) µg of DE2; B, increasing amounts of
DE2 were incubated with 31 (
) or 92 (
) µg of DE1. The
experiment was done with fractions after the DEAE step under standard
conditions. mU, milliunits.
In enterobacteriaceae CDP reduction by the ``classical'' NrdAB enzyme requires ATP and is inhibited by dATP(1, 2) , whereas the same reaction when catalyzed by the NrdEF enzyme is strongly stimulated by dATP, with ATP giving only a marginal effect(6) . As shown in Fig. 3A, CDP reduction by the L. lactis enzyme system is strongly stimulated by dATP but not by ATP. From this point of view, the L. lactis enzyme thus behaves like an NrdEF enzyme.
Figure 3:
A, effect of ATP and dATP on CDP
reduction. Incubation was with 12 µg of DE1 and 25 µg of DE2
(DEAE fractions), replacing the standard concentration of 0.3 mM dATP by the indicated concentrations of either ATP () or dATP
(
); B, effect of DTT on CDP reduction. DE2 (77 µg,
DEAE fraction) was incubated under standard conditions except for the
concentration of DTT shown on the abscissa, with (
) or
without (
) 1.5 µg of DE1 (Superdex-75 fraction). mU,
milliunits.
With all NrdAB enzymes that were investigated in detail both
thioredoxin and glutaredoxin are potential hydrogen
donors(19) . With the NrdEF enzyme from S. typhimurium thioredoxin was essentially inactive, whereas glutaredoxin was
active(6) . However, compared to NrdAB enzymes, the K for glutaredoxin was 1 order of magnitude higher
for the NrdEF enzyme. High concentrations of DTT can function as an
artificial hydrogen donor for all class I enzymes investigated so
far(19) .
DE2 from L. lactis had by itself very low CDP-reductase activity that depended on the presence of DTT (Fig. 3B). The ``background'' activity, without addition of DTT, seen in Fig. 3B is explained by the presence of a small amount of DTT in the DE2 preparation, required for stabilization of the protein. When DE1 was added together with DE2, DTT gave a strong stimulation of the reaction. This effect, together with the small molecular mass of DE1 suggested that this protein functioned as an intermediary between DTT and the actual ribonucleotide reductase present in DE2, similar to thioredoxin and glutaredoxin in other class I and class II systems.
For DE1, reverse phase chromatography had given a product that was homogeneous on electrophoresis on a denaturing SDS gel, with an apparent molecular mass of 10 kDa. The 40-step long N-terminal sequence shown in Table 2is in accordance with the sequence deduced from the base sequence of the gene described below with the exception of Cys-19 that from the DNA sequence is expected to be Trp. Cys-10 and Cys-13 correspond to the cysteines in the Cys-X-X-Cys sequence characteristic for all glutaredoxins and thioredoxins(19) . Overall, the N-terminal sequence presents good alignments with that of glutaredoxins (see ``Discussion'') and definitely identifies DE1 as a glutaredoxin-related redoxin that functions as an intermediate between DTT and DE2 to provide the electrons required for the reduction of ribose.
The DE2-peptides in Table 2were obtained by trypsin digestion of the proteins from two bands of an SDS gel as described under ``Experimental Procedures.'' These bands had mobilities close to those for class I R1E and R2F proteins, and the peptides are labeled accordingly in Table 2. Computer comparison of the peptides obtained from the larger protein gave the best alignments with known amino acid sequences present in R1E from S. typhimurium(5) , E. coli(4) , and M. tuberculosis(7) . A similar comparison of peptides from the smaller protein localized them to R2F. The numbering system in Table 2refers to the position of the corresponding peptides in the S. typhimurium sequence, with underlined residues common to S. typhimurium and L. lactis. These results identify the ribonucleotide reductase from L. lactis as an NrdEF enzyme.
Figure 4: Schematic organization of the genes encoding the NrdEF-ribonucleotide reductase system of L. lactis, as demonstrated by PCR amplification. Deduced intergenic distances are 0.6 and 0.3 kb between the gene for DE1 (nrdH) and nrdE, and nrdE and nrdF, respectively. Peptide sequences shown in Table 2are positioned, as well as derived primers used to amplify bands of 2.8 and 1.3 kb. Shadowed portions of arrows denote one-strand sequenced areas of the extremes of PCR bands.
Figure 5: A, schematic representation of the single specific primer-PCR method used for cloning nrdH. EcoRI-HindIII digested chromosomal DNA was ligated with EcoRI digested pBSK plasmid and amplified with primers PrRev and PrE giving rise to a fragment that contained the nrdH gene. B, nucleotide sequence of the L. lactis nrdH gene and its upstream region. The deduced amino acid sequences of nrdH and ORF2 are shown. Translation initiation and stop codons are boxed. Putative ribosome binding sites (RBS) are overlined, as well as the binding sites for primers PrA and PrE.
The nucleotide sequence of the 680-base pair fragment present between the chromosomal EcoRI site and the PrE primer binding site is shown in Fig. 5B and has been deposited in the EMBL data base under accession number X92690. The DE1 gene is formed by 219 nucleotides, encoding a putative protein of 72 residues with a predicted molecular mass of 8.3 kDa. A ribosome binding site is located 7 base pairs upstream of the ATG-triplet coding for the first methionine. Also several putative TATA boxes are found. Downstream of this gene, the 5`-extreme of an additional ORF is found, also preceded by a less conserved ribosome binding site. The amino acid sequence predicted by this ORF shows a considerable similarity (56%) to that of the ORF2 present in the nrdEF operons of E. coli and S. typhimurium(4) , with, respectively, 32.4 and 29.4% identities. The G + C content of the entire sequenced fragment is 31.3%, that for the DE1 coding region is 34.3%, close to the 37% established for L. lactis(28) . Also the codon usage of the DE1 gene is in agreement with that established for L. lactis (data not shown). Both results indicate that the incorporation of the gene for DE1 into the L. lactis genome is not a recent event.
As described under ``Discussion,'' the DE1 sequence shows considerable homology to various glutaredoxin sequences. Glutaredoxins transfer electrons from NADPH via glutathione to the R1 protein of class I enzymes of the NrdAB-type(19) . High concentrations of glutaredoxin 1 can fullfil this function for the R1E protein of S. typhimurium(6) . The amino acid sequence of DE1 strongly suggests that it is a similar redoxin protein. We propose the designation nrdH for the gene coding for DE1.
The fact that an nrdD mutant of L. lactis was able to grow anaerobically may suggest that the
gene is dispensible for anaerobic growth of the bacteria. A similar
result was recently obtained with E. coli containing an
interrupted nrdD gene. (
)It then came as a surprise
to find that the anaerobic growth of the L. lactis mutant was
supported by a class I ribonucleotide reductase. The general wisdom has
it that these enzymes require oxygen for the generation of their
tyrosyl radical(2) . We could not demonstrate any other
reductase activity in mutant extracts and it is therefore a fair
conclusion that the class I enzyme provided the deoxyribonucleotides
for DNA replication under our anaerobic growth conditions.
The most
probable explanations for this apparent paradox is that small amounts
of oxygen remained during the ``anaerobic'' incubation and
that the NrdEF enzyme has a very high affinity for oxygen, sufficient
to make possible the generation of the tyrosyl radical under those
conditions. When in recent experiments sodium sulfide was added to the
medium to scavenge traces of oxygen the growth of the nrdD mutant was inhibited severely, whereas
the wild type strain grew normally. This suggests that a functional nrdD gene is indeed required under stricter anaerobic
conditions.
We then found also that extracts from wild type L.
lactis contained the same kind of activity as the the nrdD mutant leading to the general
conclusion that the active ribonucleotide reductase of L. lactis belongs to the NrdEF group of reductases. These enzymes differ in
several respects from the NrdAB enzymes originally discovered in E.
coli and also found in all eukaryotes. The differences are large
enough to justify a definition of two subgroups of class I enzymes,
with NrdAB enzymes forming subclass Ia and the NrdEF enzymes subclass
Ib. Members of each subclass are primarily recognized from their amino
acid sequence. As to known functional differences, they concern the
allosteric effect of dATP and the nature of the small protein that
shuttles electrons from NADPH to the reductase. dATP is a general
inhibitor for class Ia enzymes but is a positive effector for CDP
reduction by class Ib enzymes(2, 6) . In this respect
they behave as class II enzymes(29) . Furthermore, the redoxin
identified in this paper as an electron transporter for the L.
lactis reductase is different from the thioredoxin and
glutaredoxin used by class Ia enzymes(19) .
This redoxin was separated from the L. lactis reductase proper by chromatography on DEAE-cellulose early during purification. The reduction of CDP by the reductase then showed an almost absolute requirement for the small protein (Fig. 3B) that could not be satisfied by DTT. This is an unusual behavior for class I (and II) reductases, since in all cases known so far high concentrations of DTT can at least partially short-circuit the specific redoxin and directly reduce redox-active thiols of the reductase. With its 72 amino acids, the new redoxin is smaller than any other similar electron transport protein. The two cysteines in positions 10 and 13 harbor the redox-active thiols that carry out the transthiolation required for the maintenance of the active thiols of the reductase. The gene for the redoxin (nrdH) forms part of the nrdEF operon.
On searching for amino acid sequence homology it became apparent that the protein coded by nrdH presents a considerable degree of similarity with various forms of glutaredoxins and glutaredoxin-like proteins (Fig. 6). All alignments were made such that the two redox-active cysteines of the various proteins occupy identical positions. It then appears that the sequence of the new redoxin shows 48.6% similarity and 27.8% identity with that of glutaredoxin 3 (30) and 40.3% similarity and 16.7% identity with that of glutaredoxin 1 (31) . However, the greatest similarity is found with the ORF1 products of the nrdEF operons of E. coli (63.9% similarity, 36.1% identity) and S. typhimurium (62.5% similarity, 33.3% identity). The genes for the three proteins also occupy identical positions within the operon. We propose that ORF1 is a nrdH gene and that all three proteins have a similar redoxin function for class Ib reductases.
Figure 6: Amino acid sequence alignments of the NrdH product from L. lactis with putative proteins coded by ORF1 of the E. coli (ORF1 Ec) and S. typhimurium (ORF1 St) nrdEF operons as well as with glutaredoxin 1 (GRX1) and glutaredoxin 3 (GRX3) of E. coli. Consensus shows by capital letters complete conservation between all five proteins and by lower case letters conservation between the L. lactis protein and the two ORF1 proteins. The alignments were started from the two redox-active cysteines in the active site (boxed with broken lines). Boxed amino acid residues of GRX1 show the glutathione binding site of this protein (32) .
Whereas the amino
acid sequence classifies the NrdH proteins as a glutaredoxin-like
protein, they can hardly be classified as glutaredoxins. As the name
implies, glutaredoxins use glutathione for the reduction of the
disulfide bond between the two cysteines of the active center during
the shuttling of electrons. However, extracts of L. lactis contain no glutathione.()(
)Furthermore, the
NrdH proteins do not contain the amino acid sequences of glutaredoxin 1 (boxed in Fig. 6) responsible for glutathione
binding(32) . Also the sequence in the active center
(Cys-Met/Val-Gln-Cys) differs from the typical Cys-Pro-Tyr-Cys of
glutaredoxins. The sequence also differs from the Cys-Gly-Pro-Cys of
thioredoxins. Naming the new redoxin must await the outcome of
experiments now in progress that aim at the definition of the enzyme
system that reduces the disulfide bond in the active site.
Several other glutaredoxin-like proteins have been described in the literature, often with unknown functions. Among them the protein from Methanobacterium thermoautotrophicum(33) may be involved in ribonucleotide reduction. Also in this case the protein does not contain the amino acids thought to be required for glutathione binding and the microorganism lacks glutathione. Another potential glutaredoxin-like protein corresponds to the ORF2 gene of the rubredoxin operon of Clostridium pasteurianium(20) . In this case, the neighbor gene of the operon (ORF1) codes for a thioredoxin reductase-like protein.
The first class Ib enzymes were discovered in S. typhimurium and E. coli as ``silent'' enzymes whose physiological function is still not understood. The genes are poorly transcribed and chromosomal gene expression is not sufficient to complement mutants in the genes coding for the active class Ia enzymes. Recently, ribonucleotide reductases of other microorganisms were characterized as class Ib enzymes and our work now adds L. lactis to this group. Among microorganisms, class Ia enzymes have so far been found only in Enterobacterioaceae and the closely related H. influenzae. It seems possible that members of class Ib are the prevalent class I enzymes of microorganisms.