STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF 2 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 dinuc lear 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 le ucine in nrdB-1 and Leu304 to phenylalanine in nrdB-2. Both mutations are the results of single amino acid replacements of non-conserved res idues. The three-dimensional structures of [L348]R2 and [F304]R2 have been determined to 0.26-nm and 0.28-nm resolution, respectively. Compa red with wild-type R2, [L348]R2 binds with higher affinity to R1, prob ably due to increased flexibility of its C-terminus. Since the three-d imensional structure, iron-center properties and radical properties of [L348]R2 are comparable to those of wild-type R2, the low catalytic a ctivity of the holoenzyme is probably caused by a perturbed interactio n between R2 and R1. The [F304]R2 enzyme has increased radical sensiti vity and low catalytic activity compared with wild-type R2. In [F304]R 2 the only significant change in structure is that the evolutionary co nserved Ser211 forms a different hydrogen bond to a distorted helix. T he 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.