Structure and function of the radical enzyme ribonucleotide reductase.

Ribonucleotide reductases (RNRs) catalyze all new production in nature of deoxyribonucleotides for DNA synthesis by reducing the corresponding ribonucleotides. The reaction involves the action of a radical that is produced differently for different classes of the enzyme. Class I enzymes, which are present in eukaryotes and microorganisms, use an iron center to produce a stable tyrosyl radical that is stored in one of the subunits of the enzyme. The other classes are only present in microorganisms. Class II enzymes use cobalamin for radical generation and class III enzymes, which are found only in anaerobic organisms, use a glycyl radical. The reductase activity is in all three classes contained in enzyme subunits that have similar structures containing active site cysteines. The initiation of the reaction by removal of the 3'-hydrogen of the ribose by a transient cysteinyl radical is a common feature of the different classes of RNR. This cysteine is in all RNRs located on the tip of a finger loop inserted into the center of a special barrel structure. A wealth of structural and functional information on the class I and class III enzymes can now give detailed views on how these enzymes perform their task. The class I enzymes demonstrate a sophisticated pattern as to how the free radical is used in the reaction, in that it is only delivered to the active site at exactly the right moment. RNRs are also allosterically regulated, for which the structural molecular background is now starting to be revealed.

Crystal structure of the epsilon subunit of the proton-translocating ATP synthase from Escherichia coli.

BACKGROUND: Proton-translocating ATP synthases convert the energy generated from photosynthesis or respiration into ATP. These enzymes, termed F0F1-ATPases, are structurally highly conserved. In Escherichia coli, F0F1-ATPase consists of a membrane portion, F0, made up of three different polypeptides (a, b and c) and an F1 portion comprising five different polypeptides in the stoichiometry alpha 3 beta 3 gamma delta epsilon. The minor subunits gamma, delta and epsilon are required for the coupling of proton translocation with ATP synthesis; the epsilon subunit is in close contact with the alpha, beta, gamma and c subunits. The structure of the epsilon subunit provides clues to its essential role in this complex enzyme. RESULTS: The structure of the E. coli F0F1-ATPase epsilon subunit has been solved at 2.3 A resolution by multiple isomorphous replacement. The structure, comprising residues 2-136 of the polypeptide chain and 14 water molecules, refined to an R value of 0.214 (Rfree = 0.288). The molecule has a novel fold with two domains. The N-terminal domain is a beta sandwich with two five-stranded sheets. The C-terminal domain is formed from two alpha helices arranged in an antiparallel coiled-coil. A series of alanine residues from each helix form the central contacting residues in the helical domain and can be described as an 'alanine zipper'. There is an extensive hydrophobic contact region between the two domains providing a stable interface. The individual domains of the crystal structure closely resemble the structures determined in solution by NMR spectroscopy. CONCLUSIONS: Sequence alignments of a number of epsilon subunits from diverse sources suggest that the C-terminal domain, which is absent in some species, is not essential for function. In the crystal the N-terminal domains of two epsilon subunits make a close hydrophobic interaction across a crystallographic twofold axis. This region has previously been proposed as the contact surface between the epsilon and gamma subunits in the complete F1-ATPase complex. In the crystal structure we observe what is apparently a stable interface between the two domains of the epsilon subunit, consistent with the fact that the crystal and solution structures are quite similar despite close crystal packing. This suggests that a gross conformational change in the epsilon subunit, to transmit the effect of proton translocation to the catalytic domain, is unlikely, but cannot be ruled out.

Ribonucleotide reductase--structural studies of a radical enzyme.

Ribonucleotide reductase contains a stable organic free radical essential for its activity located on a tyrosine residue in the small subunit of the enzyme called R2. The substrate binding site is, however, found in the catalytic subunit called R1. A long-range protein-mediated radical transfer pathway appears to be responsible for the delivery of the radical from the tyrosine in R2 to the substrate on R1. The active site is located deep inside the protein in a very stable beta/alpha-barrel structure and a hydrogen bonded system leads from the surface to Cys439 at the active site which is in excellent position to remove a hydrogen from the 3' of the ribose of a bound substrate nucleotide.

Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding.

BACKGROUND: Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis, catalyzing all de novo synthesis of deoxyribonucleotides. The enzyme comprises two dimers, termed R1 and R2, and contains the redox active cysteine residues, Cys462 and Cys225. The reduction of ribonucleotides to deoxyribonucleotides involves the transfer of free radicals. The pathway for the radical has previously been suggested from crystallographic results, and is supported by site-directed mutagenesis studies. Most RNRs are allosterically regulated through two different nucleotide-binding sites: one site controls general activity and the other controls substrate specificity. Our aim has been to crystallographically demonstrate substrate binding and to locate the two effector-binding sites. RESULTS: We report here the first crystal structure of RNR R1 in a reduced form. The structure shows that upon reduction of the redox active cysteines, the sulfur atom of Cys462 becomes deeply buried. The more accessible Cys225 moves to the former position of Cys462 making room for the substrate. In addition, the structures of R1 in complexes with effector, effector analog and effector plus substrate provide information about these binding sites. The substrate GDP binds in a cleft between two domains with its beta-phosphate bound to the N termini of two helices; the ribose forms hydrogen bonds to conserved residues. Binding of dTTP at the allosteric substrate specificity site stabilizes three loops close to the dimer interface and the active site, whereas the general allosteric binding site is positioned far from the active site. CONCLUSIONS: 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 ten-stranded beta/alpha barrel in ribonucleotide reductase protein R1.

The large subunit of ribonucleotide reductase (RNR) contains a ten-stranded beta/alpha barrel of a new type consisting of two antiparallel halves. The two halves of the barrel are pseudo 2-fold-related, have similar folds but different additional intervening secondary structure elements and loops. The inner diameter of the RNR barrel, 15 A to 20 A, is significantly larger than for the (beta alpha)3 barrels. The larger barrel forms a stable framework which holds an inserted hairpin loop rigidly and exposes active site residues at its tip. The barrel organization allows three cysteine residues to be positioned close to each other without forming unfavorable disulfide bridges between Cys439 on the tip of the inserted loop and the redox-active cysteine residues on the barrel strands. Redox-active cysteine residues separated by more than 200 residues are held in close proximity to each other on adjacent barrel strands.

Structure of ribonucleotide reductase protein R1.

Ribonucleotide reductase is the only enzyme that catalyses de novo formation of deoxyribonucleotides and is thus a key enzyme in DNA synthesis. The radical-based reaction involves five cysteins. Two redox-active cysteines are located at adjacent antiparallel strands in a new type of ten-stranded alpha/beta-barrel, and two others at the carboxyl end in a flexible arm. The fifth cysteine, in a loop in the centre of the barrel, is positioned to initiate the radical reaction.

Crystallization and crystallographic investigations of ribonucleotide reductase protein R1 from Escherichia coli.

Crystals of Escherichia coli ribonucleotide reductase protein R1 have been grown in complex with a synthetic peptide corresponding to the carboxyl end of protein R2. Good quality crystals could only be obtained after improvement of the purification protocol and are of the space group R32 with hexagonal cell axes a = b = 226 A and c = 341 A. They contain 3 subunits per asymmetric unit and diffract to 2.5 A resolution in synchrotron radiation. A multiple isomorphous replacement map at 5.5A, improved by solvent flattening, shows that the dimeric molecules are elongated, about 110 A long. The dimer is thin in the middle around the molecular two-fold axis. The subunit is shaped like a bowl, probably with the active site in its center.

The three-dimensional structure of notexin, a presynaptic neurotoxic phospholipase A2 at 2.0 A resolution.

The three-dimensional structure of notexin has been solved by molecular replacement methods. The structure has been refined at 2.0 A resolution to a crystallographic R-value of 16.5% with good stereo-chemistry. The core of the protein is very similar to other phospholipase A2s (PLA2 s) but several parts of the molecule are distinctly different. The most significant differences from PLA2 s from bovine pancreas and rattlesnake occur in the stretches 56-80 and 85-89. Residue 69, which has been shown to be important for phospholipase binding, has a different conformation and different interactions than in other known PLA2s. The C alpha positions for residues 86-88 differ by about 6 A from both the bovine and the rattlesnake enzyme. The crystals contain no Ca2+ ions. Instead, a water molecule occupies the calcium site.

New crystal forms of the small subunit of ribonucleotide reductase from Escherichia coli.

The small subunit of ribonucleotide reductase from Escherichia coli has been crystallized in two new crystal forms. The form most suitable for X-ray analysis belongs to the orthorhombic space group P2(1)2(1)2(1). It has the cell dimensions 74.3 A, 85.5 A, 115.7 A and diffracts to about 2.1 A resolution. The asymmetric unit most probably contains one dimer. Absorption spectra of single crystals confirm that the crystals contain a binuclear iron center. Crystals of the iron-depleted apoenzyme have also been obtained.

Crystallization and preliminary crystallographic data of ribonucleotide reductase protein B2 from Escherichia coli.

The B2 subunit of ribonucleotide reductase from Escherichia coli has been crystallized from ammonium sulfate solutions at pH 6.0. Crystals grew as orthorhombic plates with cell dimensions a = 58 A, b = 73 A, and c = 205 A. The asymmetric unit probably contains one B2 dimer of molecular weight 2 X 43,000. The packing of molecules in the crystals is compatible with an elongated shape of the dimer. The crystals diffract to 2.5 A and are suitable for structural work.