strate specificity. At the active site Bl contributes oxidation- reduction active sulthydryl groups. The two structural genes for E. coli ribonucleotide reductase,.
The EMBO Journal vol.5 no.8 pp.2037-2040, 1986
Identification of the stable free radical tyrosine residue in ribonucleotide reductase
Ake Larsson and Britt-Marie Sjoberg Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Box 590, S-751 24 Uppsala, Sweden Communicated by C.-I.Branddn
The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotidedirected mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyrl22 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the Bi and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain. Key words: ribonucleotide reductase/site-directed mutagenesis/ tyrosyl radical/protein engineering/electron paramagnetic resonance Introduction The enzyme ribonucleotide reductase is present in all dividing cells. It catalyzes the reduction of ribonucleotides to their corresponding deoxyribonucleotides. This constitutes the first unique step in the biosynthesis of DNA (Lammers and Follman, 1983). In Escherichia coli the active enzyme consists of a 1: 1 complex of the two non-identical subunits protein Bl and protein B2, each consisting of two identical polypeptide chains (Sjoberg et al., 1985a). One unique property of this type of ribonucleotide reductase is the presence of a stable free radical localized to a tyrosine residue in the B2 subunit. The tyrosyl radical is essential for enzymatic activity and is stabilized by an adjacent binuclear iron center (Petersson et al., 1980). The presence of tyrosyl radical has been verified in evolutionarily divergent species such as E. coli, bacteriophage T4, pseudo-rabies virus and mouse (Sjoberg et al., 1983; Sahlin et al., 1982; Lankinen et al., 1982; Graislund et al., 1982, 1985), and the conformations of the iron centers of protein B2 from all examined species have been found to be very similar (Graslund et al., 1985). Protein B2 from E. coli is a prototype for the small subunit of all iron-containing ribonucleotide reductases including the eukaryotic enzymes. Protein Bi binds ribonucleotide diphosphate substrates and nucleoside triphosphate effector molecules. It has two types of allosteric sites, regulating both the overall activity and the sub© IRL Press Limited, Oxford, England
strate specificity. At the active site Bl contributes oxidationreduction active sulthydryl groups. The two structural genes for E. coli ribonucleotide reductase, nrdA and nrdB for BI and B2 respectively, are encoded in an operon and are transcribed as a single polycistronic transcript (Hanke and Fuchs, 1983). The nrdA gene codes for an 87 kd polypeptide chain and the nrdB gene codes for a 43.5 kd polypeptide chain. The deduced amino acid sequence of protein B2 was aligned with amino acid sequences for B2 homologues of herpes simplex and Epstein Barr viruses and the clam Spisula solidissima (Sjoberg et al., 1985b). Since relatively few residues were conserved they were considered functionally important and it was suggested that the conserved residue Tyr 122 in E. coli protein B2 would harbour the tyrosyl radical. Here we describe the conversion of Tyr 122 in protein B2 to a phenylalanine, using oligonucleotide-directed mutagenesis. The mutant protein was characterized with electron paramagnetic resonance (EPR) and visible absorption spectroscopy as well as enzymatic methods. The results clearly show that Tyr 122 in E. coli protein B2 is the tyrosyl radical residue essential for catalytic activity.
Results and discussion Construction of a vector for manipulation and expression of ribonucleotide reductase subunits The E. coli nrd genes were earlier cloned in different types of phage and plasmid vectors (Platz and Sjoberg, 1980; Larsson, 1984; Sj6berg et al., 1986a; Salowe and Stubbe, 1986). Even though some of these constructs showed very efficient expression of protein B 1 or B2 we had several reasons for constructing a new vector. First, in cases of a deleterious mutation in one separately cloned gene, this subunit may titrate out the chromosomally encoded native subunit and consequently be lethal to the cells. Second, the amount of a manipulated gene product has to be great to facilitate its characterization without disturbance of the chromosomally encoded native enzyme. Third, earlier constructed recombinants were relatively large and very few unique restriction sites were available for manipulations of the genes. A low molecular weight recombinant plasmid expressing both subunits was therefore constructed. Since the nrd promoter is known to be efficiently transcribed (Larsson, 1984), a 5.7-kb fragment comprising the entire nrd operon was transferred to a 2.3 kb vector fragment originating from pBR322 (Figure 1). The recombinant pAL7 contains at least one unique restriction site centered in the nrdB gene and at least five unique restriction sites distributed in the nrdA gene. This enables transfer of shorter fragments of mutated genes to the wild-type genes and avoids extensive nucleotide sequencing of constructed mutants. The PvulI site of pBR322 is known to reside in the copy number control region and the use of this site raises the copy number two to three times (Covarrubias et al., 1981). The PvuII site was used in the construction of pAL7 in order to increase the expression of ribonucleotide reductase. Extracts from bacteria 2037
A.Larsson and B.-M.Sjoberg Hind III/(Kpnl)
C#
BstX
Fig. 1. Plasmid pAL7 constructed for expression of both subunits of E. coli ribonucleotide reductase. Escherichia coli DNA, 5.7 kb was cloned in 2.3 kb of pBR322, between the HindUI and PvuII sites in pBR322. A KpnI site in the E. coli DNA was fused with the HindIlI site in pBR322; using a 10 bp oligonucleotide, the KpnI site was destroyed. Marked in the figure are unique restriction sites and those restriction sites that were used in the plasmid construction. An asterisk denotes restriction sites deduced from the published sequence of the nrd operon. Table I. Expression of ribonucleotide reductase subunits
Plasmida
No plasmid pAL7 (Tyr 122) pAL83 (Tyr 122-Phe)
Protein content protein BI 1.5 53 83
/Ag/mg total B2
0.5 12 17
aPlasmids were maintained in GM33 bacteria.
carrying the pAL7 plasmid contained 12 jig of B2/mg total protein and 53 itg of B 1/mg total protein (Table I). The overproduction was 24 and 35 times respectively, as compared to the host strain, i.e. the contamination of chromosomally encoded enzyme is only 2-3 %. The differential expression of the two subunits has been observed before (Platz, 1981) and may be caused by the presence of 4 to 5 repetitive extragenic palindromic (REP, Stern et al., 1984) sequences in the intragenic region of the nrd operon (Hanke and Fuchs, 1983; Carlson et al., 1984). This is under present investigation. In summary the pAL7 plasmid overproduced both subunits of ribonucleotide reductase efficiently. Because of its variety of unique restriction sites in the nrd region, the pAL plasmid is especially suitable for protein engineering studies on ribonucleotide reductase. Substitution of Tyr 122 for phenylalanine in protein B2 The strategy chosen for site-directed mutagenesis of E. coli nrdB was the oligonucleotide-directed approach in M13 (Zoller and Smith, 1984). The complete nrdB gene (and part of the 3' end of nrdA) was cloned in M13mp 18. Two mutations were introduced with a 30-base mutagenic oligonucleotide. One converted the Tyr 122 TAT codon into a phenylalanine TTT codon, and the other introduced a unique XbaI site 10 nucleotides upstream of 2038
lmT
Fig. 2. Electron spin resonance spectra of E. coli cell suspensions. (A) GM33 bacteria (host strain). (B) bacteria containing the pAL7 plasmid (Tyr 122). (C) bacteria containing the pAL83 plasmid (Tyr 122-Phe). Bacteria were grown to late logarithmic phase in rich medium, washed in minimal medium without nitrogen, pelleted in EPR tubes, stored at -70°C and recorded at 77 K.
Tyr 122, with complete preservation of amino acid sequence at this point. The XbaI site was introduced to facilitate screening of the genetically silent Tyr 122- Phe mutation during later genetic manipulations. The mutation frequency was of the order of %, as expected for this type of approach. A stretch of 620 nucleotides in the mutant DNA was sequenced. It covered the nrdB sequence from the start of the B2 protein and downstream passed the Asp718 site (cf. Figure 1). The sequenced part of the nrdB gene was identical to the published sequence (Carlson et al., 1984) except for the four desired mismatches. The Tyr 122- Phe mutation in B2 was transferred on a 1.3 kb BamHIAsp718 fragment from the M13 derivative to the wild-type nrdB gene on the pAL7 plasmid. The new plasmid was denoted pAL83. The presence of the Tyr 122-Phe mutation in pAL83 was indirectly established by restriction analysis, in which case the concomitantly introduced unique XbaI site served as a marker. Mutant protein B2 lacks tyrosyl radical Ribonucleotide reductase subunits were expressed in bacteria carrying the plasmid pAL83 (Tyr 122- Phe) and the resulting extracts contained 17 ,.tg of B2/mg total protein and 83 jig of B1/mg total protein (Table I). The overproduction compared to the host strain was 38 times for the mutated B2 subunit and 56 times for the native Bi subunit. Both these values are higher than corresponding values in pAL7. It was earlier observed that a deleterious mutation in one of the subunits gave rise to an increased production of both subunits (Platz, 1981). It is conceivable that the cell tries to compensate for a defective enzyme by derepressing the operon. The mechanism for derepression of the nrd operon is so far unknown. A convenient assay for the tyrosyl radical of B2 is EPR spectroscopy on whole cells spun down in EPR tubes. Cells contain-
Stable free radical tyrosine residue Table H. Comparison of native and mutated B2 protein Protein
Polypeptide size
B2 (Tyr 122) B2 (Tyr 122-Phe)
(kd)
Iron content (mol/mol protein)
absorption bands
Iron-related
Interaction with protein BI
43.5 43.5
1.4 1.5
Yes Yes
Yes Yes
0.6
0.3
300
c co .0
o co 0
400
500
600
700
400
500
600
700
0.
0.:
300
Wavelength (nm) Fig. 3. Light absorption spectra of purified B2 protein from E. coli ribonucleotide reductase. (A) B2 (Tyr 122) (78 IAM). (B) B2 (Tyr 122- Phe) (76 WM). Both samples were in 50 mM Tris-HCI, pH 7.6, 20% glycerol. The buffer was used as a blank.
ing the wild-type pAL7 plasmid showed the typical EPR signal originating from the tyrosyl radical in protein B2 (Figure 2b). Cells containing the mutant pAL83 plasmid showed an EPR spectrum identical with the cellular background of the host strain (Figure 2a and c). These results clearly show that the mutation Tyr 122-Phe destroys the free radical. Mutant protein B2 has an intact iron center Absence of a tyrosyl radical in B2 protein carrying the Tyr 122- Phe mutation could be the result of an abnormal iron center or an incomplete protein. In order to exclude these possibilities we purified and characterized the mutated protein extensively. Preparations of B2(Tyr 122) and B2(Tyr 122- Phe) were made in parallel. SDS-gel electrophoresis showed that the preparations contained 90% B2 protein and that both the mutant and the native protein co-migrated with the protein B2 standard. The EPR spec-
Enzyme activity
(U/mg protein) 6380
