On the Role of Additional [4Fe-4S] Clusters with a Free Coordination Site in Radical-SAM Enzymes.

The canonical CysXXXCysXXCys motif is the hallmark of the Radical-SAM superfamily. This motif is responsible for the ligation of a [4Fe-4S] cluster containing a free coordination site available for SAM binding. The five enzymes MoaA, TYW1, MiaB, RimO and LipA contain in addition a second [4Fe-4S] cluster itself bound to three other cysteines and thus also displaying a potentially free coordination site.

Redox Behavior of the S-Adenosylmethionine (SAM)-Binding Fe-S Cluster in Methylthiotransferase RimO, toward Understanding Dual SAM Activity.

RimO, a radical-S-adenosylmethionine (SAM) enzyme, catalyzes the specific C methylthiolation of the D89 residue in the ribosomal S protein. Two intact iron-sulfur clusters and two SAM cofactors both are required for catalysis. By using electron paramagnetic resonance, Mössbauer spectroscopies, and site-directed mutagenesis, we show how two SAM molecules sequentially bind to the unique iron site of the radical-SAM cluster for two distinct chemical reactions in RimO.

Unanticipated coordination of tris buffer to the Radical SAM cluster of the RimO methylthiotransferase.

Radical SAM enzymes generally contain a [4Fe-4S](2+/1+) (RS cluster) cluster bound to the protein via the three cysteines of a canonical motif CxxxCxxC. The non-cysteinyl iron is used to coordinate SAM via its amino-carboxylate moiety. The coordination-induced proximity between the cluster acting as an electron donor and the adenosyl-sulfonium bond of SAM allows for the homolytic cleavage of the latter leading to the formation of the reactive 5'-deoxyadenosyl radical used for substrate activation.

The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date.

Wybutosine biosynthesis: structural and mechanistic overview.

Over the last 10 years, significant progress has been made in understanding the genetics, enzymology and structural components of the wybutosine (yW) biosynthetic pathway. These studies have played a key role in expanding our understanding of yW biosynthesis and have revealed unexpected evolutionary ties, which are presently being unraveled. The enzymes catalyzing the 5 steps of this pathway, from genetically encoded guanosine to wybutosine base, provide an ensemble of amazing reaction mechanisms that are to be discussed in this review article.

Two Fe-S clusters catalyze sulfur insertion by radical-SAM methylthiotransferases.

How living organisms create carbon-sulfur bonds during the biosynthesis of critical sulfur-containing compounds is still poorly understood. The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. Both belong to a subgroup of radical-S-adenosylmethionine (radical-SAM) enzymes that bear two [4Fe-4S] clusters.

4-Demethylwyosine synthase from Pyrococcus abyssi is a radical-S-adenosyl-L-methionine enzyme with an additional [4Fe-4S](+2) cluster that interacts with the pyruvate co-substrate.

Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called "Radical-SAM.

The methylthiolation reaction mediated by the Radical-SAM enzymes.

Over the past 10 years, considerable progress has been made in our understanding of the mechanistic enzymology of the Radical-SAM enzymes. It is now clear that these enzymes appear to be involved in a remarkably wide range of chemically challenging reactions. This review article highlights mechanistic and structural aspects of the methylthiotransferases (MTTases) sub-class of the Radical-SAM enzymes.

S-Adenosylmethionine-dependent radical-based modification of biological macromolecules.

Proteins and RNA molecules enjoy a variety of chemically complex post-translational and post-transcriptional modifications. The chemistry at work in these reactions, which was considered to be exclusively ionic in nature has recently been shown to depend on radical mechanisms in some cases. The overwhelming majority of these radical-based reactions are catalyzed by 'Radical-SAM' enzymes.

Identification of eukaryotic and prokaryotic methylthiotransferase for biosynthesis of 2-methylthio-N6-threonylcarbamoyladenosine in tRNA.

Bacterial and eukaryotic transfer RNAs have been shown to contain hypermodified adenosine, 2-methylthio-N(6)-threonylcarbamoyladenosine, at position 37 (A(37)) adjacent to the 3'-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. Using a combination of bioinformatic sequence analysis and in vivo assay coupled to HPLC/MS technique, we have identified, from distinct sequence signatures, two methylthiotransferase (MTTase) subfamilies, designated as MtaB in bacterial cells and e-MtaB in eukaryotic and archaeal cells. Both subfamilies are responsible for the transformation of N(6)-threonylcarbamoyladenosine into 2-methylthio-N(6)-threonylcarbamoyladenosine.

Post-translational modification of ribosomal proteins: structural and functional characterization of RimO from Thermotoga maritima, a radical S-adenosylmethionine methylthiotransferase.

Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P.

The Zn center of the anaerobic ribonucleotide reductase from E. coli.

Strict and facultative anaerobes depend on a class III ribonucleotide reductase for their growth. These enzymes are the sole cellular catalysts for de novo biosynthesis of the deoxyribonucleotides needed for DNA chain elongation and repair. In its active form, the class III ribonucleotide reductase from Escherichia coli contains a free radical located on the G681 residue which is essential for the activation of the ribonucleotide substrate toward its reduction.

S-adenosylmethionine: nothing goes to waste.

S-adenosylmethionine (SAM or AdoMet) is a biological sulfonium compound known as the major biological methyl donor in reactions catalyzed by methyltransferases. SAM is also used as a source of methylene groups (in the synthesis of cyclopropyl fatty acids), amino groups (in the synthesis of 7,8-diaminoperlagonic acid, a precursor of biotin), ribosyl groups (in the synthesis of epoxyqueuosine, a modified nucleoside in tRNAs) and aminopropyl groups (in the synthesis of ethylene and polyamines). Even though the mechanism of most of these reactions has not been extensively characterized, it is likely that the chemistry at work is mainly driven by the electrophilic character of the carbon centers that are adjacent to the positively charged sulfur atom of SAM.

Iron(II) carboxylate complexes based on a tetraimidazole ligand as models of the photosynthetic non-heme ferrous sites: synthesis, crystal structure, and Mössbauer and magnetic studies.

The preparations, X-ray structures, and detailed physical characterization are presented for new complexes involving an iron(II) center, a tetraimidazole ligand (TIM), and different carboxylates. [Fe(TIM)(C(6)H(5)CH(2)CO(2))](ClO(4)) (1) crystallizes in the Pbca space group with a = 10.8947(13), b = 20.

A metal-binding site in the catalytic subunit of anaerobic ribonucleotide reductase.

A Zn(Cys)(4) center has been found in the C-terminal region of the crystal structure of the anaerobic class III ribonucleotide reductase (RNR) from bacteriophage T4. The metal center is structurally related to the zinc ribbon motif and to rubredoxin and rubrerythrin. Mutant enzymes of the homologous RNR from Escherichia coli, in which the coordinating cysteines, conserved in almost all known class III RNR sequences, have been mutated into alanines, are shown to be inactive as the result of their inability to generate the catalytically essential glycyl radical.

Very high-field EPR study of glycyl radical enzymes.

This work shows that very high-field EPR spectroscopy allows a rather accurate determination of the g-tensor of protein radicals, including C-centered ones, and thus may be used as a probe for distinguishing a tyrosyl-, a glycyl-, or a tryptophanyl-radical. In this paper, we report the first complete analysis of the g-tensor of glycyl radical enzymes (anaerobic ribonucleotide reductase, pyruvate formate lyase, and benzylsuccinate synthase), thus providing new information on their EPR properties. Because the g-anisotropy is small, the complete resolution of the g-tensor could be only obtained at very high field (18.

The PLP-dependent biotin synthase from Escherichia coli: mechanistic studies.

Biotin synthase (BioB), an iron-sulfur enzyme, catalyzes the last step of the biotin biosynthesis pathway. The reaction consists in the introduction of a sulfur atom into two non-activated C-H bonds of dethiobiotin. Substrate radical activation is initiated by the reductive cleavage of S-adenosylmethionine (AdoMet) into a 5'-deoxyadenosyl radical.

Deoxyribonucleotide synthesis in anaerobic microorganisms: the class III ribonucleotide reductase.

For growth under oxygen-free atmosphere, some strict or facultative anaerobes depend on a class III ribonucleotide reductase for the synthesis of deoxyribonucleotides, the DNA precursors. Prototypes for this class of enzymes are ribonucleotide reductases from Escherichia coli and bacteriophage T4. This review article describes their structural and mechanistic properties as well as their complex allosteric regulation.

Biotin synthase is a pyridoxal phosphate-dependent cysteine desulfurase.

Biotin synthase (BioB) is an iron-sulfur dimeric enzyme which catalyzes the last step in biotin synthesis. The reaction consists of the introduction of a sulfur atom into dethiobiotin. It is shown here that BioB displays a significant cysteine desulfurase activity, providing it with the ability to mobilize sulfur from free cysteine.

Crystallographic, Electrochemical, and Pulsed EPR Study of Copper(II) Polyimidazole Complexes Relevant to the Metal Sites of Copper Proteins(,).

Copper(II) complexes of the following polyimidazole ligands have been synthesized: bis(imidazol-2-yl)methane (BIM), bis(imidazol-2-yl) ketone (BIK), 4-(imidazol-4-ylmethyl)-2-(imidazol-2-ylmethyl)imidazole (TRIM), bis[4-(imidazol-4-ylmethyl)imidazol-2-yl]methane (TIM), and bis[4-(imidazol-4-ylmethyl)imidazol-2-yl] ketone (TIK). Their crystal structures have been determined using X-ray diffraction. [Cu(ClO(4))(2)(BIM)(2)], 1, belongs to the triclinic space group P&onemacr; system, a = 7.