Structural and functional characterization of two mutated R2 proteins of Escherichia coli ribonucleotide reductase.
The R2 protein of ribonucleotide reductase from Escherichia coli is a homodimeric tyrosyl-radical-containing enzyme with two identical dinuclear iron centers. Two randomly generated genomic mutants, nrdB-1 and nrdB-2, that produce R2 enzymes with low enzymatic activity, have been cloned and characterized to identify functionally important residues and areas of the enzyme. The mutations were identified as Pro348 to leucine in nrdB-1 and Leu304 to phenylalanine in nrdB-2. Both mutations are the results of single amino acid replacements of non-conserved residues. The three-dimensional structures of [L348]R2 and [F304]R2 have been determined to 0.26-nm and 0.28-nm resolution, respectively. Compared with wild-type R2, [L348]R2 binds with higher affinity to R1, probably due to increased flexibility of its C-terminus. Since the three-dimensional structure, iron-center properties and radical properties of [L348]R2 are comparable to those of wild-type R2, the low catalytic activity of the holoenzyme is probably caused by a perturbed interaction between R2 and R1. The [F304]R2 enzyme has increased radical sensitivity and low catalytic activity compared with wild-type R2. In [F304]R2 the only significant change in structure is that the evolutionary conserved Ser211 forms a different hydrogen bond to a distorted helix. The results obtained with [F304]R2 indicate that structural changes in E. coli R2 in the vicinity of this helix distortion can influence the catalytic activity of the holoenzyme.
Larsson
A
A
Department of Molecular Biology, University of Stockholm, Sweden.
Climent
I
I
Nordlund
P
P
Sahlin
M
M
Sjöberg
B M
BM
D001426:Bacterial Proteins / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals; D009154:Mutation; D011487:Protein Conformation; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D005609:Free Radicals; D012264:Ribonucleotide Reductases