Ribonucleotide reductase modularity: Atypical duplication of the ATP-cone domain in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa, which causes serious nosocomial infections, is a gamma-proteobacterium that can live in many different environments. Interestingly P. aeruginosa encodes three ribonucleotide reductases (RNRs) that all differ from other well known RNRs. The RNR enzymes are central for de novo synthesis of deoxyribonucleotides and essential to all living cells. The RNR of this study (class Ia) is a complex of the NrdA protein harboring the active site and the allosteric sites and the NrdB protein harboring a tyrosyl radical necessary to initiate catalysis. P. aeruginosa NrdA contains an atypical duplication of the N-terminal ATP-cone, an allosteric domain that can bind either ATP or dATP and regulates the overall enzyme activity. Here we characterized the wild type NrdA and two truncated NrdA variants with precise N-terminal deletions. The N-terminal ATP-cone (ATP-c1) is allosterically functional, whereas the internal ATP-cone lacks allosteric activity. The P. aeruginosa NrdB is also atypical with an unusually short lived tyrosyl radical, which is efficiently regenerated in presence of oxygen as the iron ions remain tightly bound to the protein. The P. aeruginosa wild type NrdA and NrdB proteins form an extraordinarily tight complex with a suggested alpha4beta4 composition. An alpha2beta2 composition is suggested for the complex of truncated NrdA (lacking ATP-c1) and wild type NrdB. Duplication or triplication of the ATP-cone is found in some other bacterial class Ia RNRs. We suggest that protein modularity built on the common catalytic core of all RNRs plays an important role in class diversification within the RNR family.     

The periodic table of ribonucleotide reductases

In most organisms, transition metal ions are necessary cofactors of ribonucleotide reductase (RNR), the enzyme responsible for biosynthesis of the 2′-deoxynucleotide building blocks of DNA. The metal ion generates an oxidant for an active site cysteine (Cys), yielding a thiyl radical that is necessary for initiation of catalysis in all RNRs. Class I enzymes, widespread in eukaryotes and aerobic microbes, share a common requirement for dioxygen in assembly of the active Cys oxidant and a unique quaternary structure, in which the metallo- or radical-cofactor is found in a separate subunit, β, from the catalytic α subunit. The first class I RNRs, the class Ia enzymes, discovered and characterized more than 30 years ago, were found to use a diiron(III)-tyrosyl-radical Cys oxidant. Although class Ia RNRs have historically served as the model for understanding enzyme mechanism and function, more recently, remarkably diverse bioinorganic and radical cofactors have been discovered in class I RNRs from pathogenic microbes. These enzymes use alternative transition metal ions, such as manganese, or posttranslationally installed tyrosyl radicals for initiation of ribonucleotide reduction. Here we summarize the recent progress in discovery and characterization of novel class I RNR radical-initiating cofactors, their mechanisms of assembly, and how they might function in the context of the active class I holoenzyme complex.     

Inhibition of chlamydial class Ic ribonucleotide reductase by C‐terminal peptides from protein R2

Chlamydia trachomatis ribonucleotide reductase (RNR) is a class Ic RNR. It has two homodimeric subunits: proteins R1 and R2. Class Ic protein R2 in its most active form has a manganese–iron metal cofactor, which functions in catalysis like the tyrosyl radical in classical class Ia and Ib RNRs. Oligopeptides with the same sequence as the C‐terminus of C. trachomatis protein R2 inhibit the catalytic activity of C. trachomatis RNR, showing that the class Ic enzyme shares a similar highly specific inhibition mechanism with the previously studied radical‐containing class Ia and Ib RNRs. The results indicate that the catalytic mechanism of this class of RNRs with a manganese–iron cofactor is similar to that of the tyrosyl‐radical‐containing RNRs, involving reversible long‐range radical transfer between proteins R1 and R2. The competitive binding of the inhibitory R2‐derived oligopeptide blocks the transfer pathway. We have constructed three‐dimensional structure models of C. trachomatis protein R1, based on homologous R1 crystal structures, and used them to discuss possible binding modes of the peptide to protein R1. Typical half maximal inhibitory concentration values for C. trachomatis RNR are about 200 µ m for a 20‐mer peptide, indicating a less efficient inhibition compared with those for an equally long peptide in the Escherichia coli class Ia RNR. A possible explanation is that the C. trachomatis R1/R2 complex has other important interactions, in addition to the binding mediated by the R1 interaction with the C‐terminus of protein R2. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Starting a new chapter on class Ia ribonucleotide reductases

Ribonucleotide reductases (RNRs) use radical-based chemistry to convert ribonucleotides into deoxyribonucleotides, an essential step in DNA biosynthesis and repair. There are multiple RNR classes, the best studied of which is the class Ia RNR that is found in Escherichia coli, eukaryotes including humans, and many pathogenic and nonpathogenic prokaryotes. This review covers recent advances in our understanding of class Ia RNRs, including a recent reporting of a structure of the active state of the E. coli enzyme and the impacts that the structure has had on spurring research into the mechanism of long-range radical transfer. Additionally, the review considers other recent structural and biochemical research on class Ia RNRs and the potential of that work for the development of anticancer and antibiotic therapeutics.     

Control of metallation and active cofactor assembly in the class Ia and Ib ribonucleotide reductases: diiron or dimanganese?

Ribonucleotide reductases (RNRs) convert nucleotides to deoxynucleotides in all organisms. Activity of the class Ia and Ib RNRs requires a stable tyrosyl radical (Y?), which can be generated by the reaction of O2 with a diferrous cluster on the β subunit to form active diferric-Y? cofactor. Recent experiments have demonstrated, however, that in vivo the class Ib RNR contains an active dimanganese(III)-Y? cofactor. The similar metal binding sites of the class Ia and Ib RNRs, their ability to bind both MnII and FeII, and the activity of the class Ib RNR with both diferric-Y? and dimanganese(III)-Y cofactors raise the intriguing question of how the cell prevents mismetallation of these essential enzymes. The presence of the class Ib RNR in numerous pathogenic bacteria also highlights the importance of manganese for these organisms' growth and virulence.     

A Novel Clade of Unique Eukaryotic Ribonucleotide Reductase R2 Subunits is Exclusive to Apicomplexan Parasites

Apicomplexa are protist parasites of tremendous medical and economic importance, causing millions of deaths and billions of dollars in losses each year. Apicomplexan-related diseases may be controlled via inhibition of essential enzymes. Ribonucleotide reductase (RNR) provides the only de novo means of synthesizing deoxyribonucleotides, essential precursors for DNA replication and repair. RNR has long been the target of antibacterial and antiviral therapeutics. However, targeting this ubiquitous protein in eukaryotic pathogens may be problematic unless these proteins differ significantly from that of their respective host. The typical eukaryotic RNR enzymes belong to class Ia, and the holoenzyme consists minimally of two R1 and two R2 subunits (α₂β₂). We generated a comparative, annotated, structure-based, multiple-sequence alignment of R2 subunits, identified a clade of R2 subunits unique to Apicomplexa, and determined its phylogenetic position. Our analyses revealed that the apicomplexan-specific sequences share characteristics with both class I R2 and R2lox proteins. The putative radical-harboring residue, essential for the reduction reaction by class Ia R2-containing holoenzymes, was not conserved within this group. Phylogenetic analyses suggest that class Ia subunits are not monophyletic and consistently placed the apicomplexan-specific clade sister to the remaining class Ia eukaryote R2 subunits. Our research suggests that the novel apicomplexan R2 subunit may be a promising candidate for chemotherapeutic-induced inhibition as it differs greatly from known eukaryotic host RNRs and may be specifically targeted.     

Rv3389C from Mycobacterium tuberculosis, a member of the (R)-specific hydratase/dehydratase family

The (R)-specific 3-hydroxyacyl dehydratases/trans-enoyl hydratases are key proteins in the biosynthesis of fatty acids. In mycobacteria, such enzymes remain unknown, although they are involved in the biosynthesis of major and essential lipids like mycolic acids. First bioinformatic analyses allowed to identify a single candidate protein, namely Rv3389c, that belongs to the hydratases 2 family and is most likely made of a distinctive asymmetric double hot dog fold. The purified recombinant Rv3389c protein was shown to efficiently catalyze the hydration of (C(8)-C(16)) enoyl-CoA substrates. Furthermore, it catalyzed the dehydration of a 3-hydroxyacyl-CoA in coupled reactions with both reductases (MabA and InhA) of the acyl carrier protein (ACP)-dependent M. tuberculosis fatty acid synthase type II involved in mycolic acid biosynthesis. Yet, the facts that Rv3389c activity decreased in the presence of ACP, versus CoA, derivative and that Rv3389c knockout mutant had no visible variation of its fatty acid content suggested the occurrence of additional hydratase/dehydratase candidates. Accordingly, further and detailed bioinformatic analyses led to the identification of other members of the hydratases 2 family in M. tuberculosis.     

The structure and evolution of the murine inhibitor of carbonic anhydrase: A member of the transferrin superfamily

The original signature of the transferrin (TF) family of proteins was the ability to bind ferric iron with high affinity in the cleft of each of two homologous lobes. However, in recent years, new family members that do not bind iron have been discovered. One new member is the inhibitor of carbonic anhydrase (ICA), which as its name indicates, binds to and strongly inhibits certain isoforms of carbonic anhydrase. Recently, mouse ICA has been expressed as a recombinant protein in a mammalian cell system. Here, we describe the 2.4 Å structure of mouse ICA from a pseudomerohedral twinned crystal. As predicted, the structure is bilobal, comprised of two α‐β domains per lobe typical of the other family members. As with all but insect TFs, the structure includes the unusual reverse γ‐turn in each lobe. The structure is consistent with the fact that introduction of two mutations in the N‐lobe of murine ICA (mICA) (W124R and S188Y) allowed it to bind iron with high affinity. Unexpectedly, both lobes of the mICA were found in the closed conformation usually associated with presence of iron in the cleft, and making the structure most similar to diferric pig TF. Two new ICA family members (guinea pig and horse) were identified from genomic sequences and used in evolutionary comparisons. Additionally, a comparison of selection pressure (dN/dS) on functional residues reveals some interesting insights into the evolution of the TF family including that the N‐lobe of lactoferrin may be in the process of eliminating its iron binding function.     

A possible iron delivery function of the dinuclear iron center of HcgD in [Fe]-hydrogenase cofactor biosynthesis

HcgD, a homolog of the ubiquitous Nif3-like protein family, is found in a gene cluster involved in the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase. The presented crystal structure and biochemical analyses indicated that HcgD has a dinuclear iron-center, which provides a pronounced binding site for anionic ligands. HcgD contains a stronger and a weaker bound iron; the latter being removable by chelating reagents preferentially in the oxidized state. Therefore, we propose HcgD as an iron chaperone in FeGP cofactor biosynthesis, which might also stimulate investigations on the functionally unknown but physiologically important eukaryotic Nif3-like protein family members.     

Alteration of the binding specificity of cellular retinol-binding protein II by site-directed mutagenesis.

Rat cellular retinol-binding protein II (CRBP II) is an abundant 134-residue intestinal protein that binds all-trans-retinol and all-trans-retinal. It belongs to a family of homologous, 15-kDa cytoplasmic proteins that bind hydrophobic ligands in a noncovalent fashion. These binding proteins include a number of proteins that bind long chain fatty acids. X-ray analyses of the structure of two family members, rat intestinal fatty acid-binding protein and bovine myelin P2 protein, indicate that they have a high degree of conformational similarity and that the carboxylate group of their bound fatty acid interacts with a delta-guanidium group of at least 1 of 2 "buried" arginine residues. These 2 Arg residues are conserved in other family members that bind long chain fatty acids and in cellular retinoic acid-binding protein, but are replaced by Gln109 and Gln129 in CRBP II. We have genetically engineered two amino acid substitutions in CRBP II: 1) Gln109 to Arg and 2) Gln129 to Arg. The purified Escherichia coli-derived CRBP II mutant proteins were analyzed by fluorescence and nuclear magnetic resonance spectroscopy. Both mutants exhibit markedly decreased binding of all-trans-retinol and all-trans-retinaldehyde, but no increased binding of all-trans-retinoic acid. Arg substitution for Gln109 but not for Gln129 produces a dramatic increase in palmitate binding activity. Analysis of the endogenous fatty acids associated with the purified E. coli-derived proteins revealed that E. coli-derived intestinal fatty acid binding protein and the Arg109 CRBP II mutant are complexed with endogenous fatty acids in a qualitatively and quantitatively similar manner. These results provide evidence that this internal Arg may play an important role in the binding of long chain fatty acids by members of this protein family.     

Ribonucleotide reductases: divergent evolution of an ancient enzyme

Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron–sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their three-dimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family.     

Mitochondrial oxidative phosphorylation at Site I involving a fatty aldehyde/acid couple

A mechanism for respiration and oxidative phosphorylation at Site I is proposed which involves reduction by NADH of a thioester of a fatty acid to aldehyde. Oxidation of the aldehyde by non-heme iron forms fatty acid which initially binds to a membrane base. As the non-heme iron reduces, entropy is lost through the stretching of a lipid bilayer attached to the non-heme iron and the membrane and to which the fatty acid chain binds. Simultaneously energy is expended in separating carboxyl ion from the protonated base. The charge separation induces movements of protons and reactants which result in the formation of ATP. Subsequent oxidation of non-heme iron relaxes the stretched lipid and the entropy gain in the fatty acid contributes to reformation of its thioester. The mechanism accounts in detail for many observations.