Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effectors and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.

The enzyme ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, which are each inactive alone. R1 contains the active site and R2 contains a stable tyrosyl radical essential for catalysis. The reduction of ribonucleotides is radical-based, and a long range electron transfer chain between the active site in R1 and the radical in R2 has been suggested. To find evidence for such an electron transfer chain in Escherichia coli ribonucleotide reductase, we converted two conserved tyrosines in R1 into phenylalanines by sitedirected mutagenesis. The mutant proteins were shown to be enzymatically inactive. In addition, the mechanism-based inhibitor 2 -azido-2 -deoxy-CDP was incapable of scavenging the R2 radical, and no azido-CDPderived radical intermediate was formed. We also show that the loss of enzymatic activity was not due to impaired R1-R2 complex formation or substrate binding. Based on these results, we predict that the two tyrosines, Tyr-730 and Tyr-731, are part of a hydrogenbonded network that constitutes an electron transfer pathway in ribonucleotide reductase. It is demonstrated that there is no electron delocalization over these tyrosines in the resting wild-type complex.The enzyme ribonucleotide reductase is essential for all living organisms. By catalyzing the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the enzyme furnishes cells with precursors for DNA synthesis. To maintain a stable and balanced supply of nucleotides during cell proliferation, the enzyme is cell cycle regulated (1) and also under strict allosteric regulation (for recent review see Ref.2). The Escherichia coli ribonucleotide reductase holoenzyme complex consists of two nonidentical dimeric proteins, R1 and R2, which are each inactive alone. The larger protein R1 contains substrate and allosteric effector binding sites. The substrate binding site of R1 includes a cysteine triad that is involved in catalysis (3-5). The smaller protein R2 contains an essential tyrosyl radical that is generated and stabilized by an oxo-bridged diiron center (6 -9). The oxidized tyrosyl radical of R2 probably participates in the reaction mechanism as a transient electron sink.The reaction catalyzed by ribonucleotide reductase is the reduction of the 2Ј-hydroxyl group of a ribonucleoside diphosphate. The mechanism involves the initial generation of a transient protein radical in R1 close to the bound substrate. The protein radical abstracts a hydrogen atom from the 3Ј position of the substrate thereby generating an oxidized substrate radical that enables leaving the protonated 2Ј-hydroxyl group. The resulting substrate radical cation intermediate is reduced by a redox-active cysteine pair, which in turn is oxidized to a disulfide (10). The 3Ј-hydrogen atom is reintroduced by the same amino acid that initially abstracted it and that again forms the transient protein radical (11).The crystal structure of the R2 protein shows that the R2 radical is buried inside the protein structure about 10 Å from the closest s...

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Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His 6 -tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore 'expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization.

A hydrogen-bonded catalytic radical transfer pathway in Escherichia coli ribonucleotide reductase (RNR) is evident from the three-dimensional structures of the R1 and R2 proteins, phylogenetic studies, and site-directed mutagenesis experiments. Current knowledge of electron transfer processes is difficult to apply to the very long radical transfer pathway in RNR. To explore the importance of the hydrogen bonds between the participating residues, we converted the protein R2 residue Asp237, one of the conserved residues along the radical transfer route, to an asparagine and a glutamate residue in two separate mutant proteins. In this study, we show that the D237E mutant is catalytically active and has hydrogen bond connections similar to that of the wild type protein. This is the first reported mutant protein that affects the radical transfer pathway while catalytic activity is preserved. The D237N mutant is catalytically inactive, and its tyrosyl radical is unstable, although the mutant can form a diferric-oxo iron center and a R1-R2 complex. The data strongly support our hypothesis that an absolute requirement for radical transfer during catalysis in ribonucleotide reductase is an intact hydrogen-bonded pathway between the radical site in protein R2 and the substrate binding site in R1. Our data thus strongly favor the idea that the electron transfer mechanism in RNR is coupled with proton transfer, i.e. a radical transfer mechanism.