Centres FeS et catalyse rédox (activation de la ribonucléotide réductase anaérobie en protéine radicalaire)

Récemment, il a été montré que les centres FeS sont capables de prende part à une catalyse rédox en participant à la réductolyse de la S-adénosyleméthionine (AdoMet) en un radical 5'-désoxyadénosyle (Ado). Le radical ainsi formé peut oxyder un résidu amino acide d'une chaine polypeptidique pour conduire à une protéine radicalaire. La ribonucléotide réductase (RNR) anaérobie d'E. coli est un des prototypes de cette nouvelle classe de protéines. Cette RNR acquiert un radical glycinyle au cours d'une étape d'activation nécessitant 4 partenaires : (i) un réducteur, (ii) un thiol, (iii) l'AdoMet et (iv) une enzyme activatrice qui contient un centre [4Fe-4S] par chaîne polypeptidique. Les mécanismes de transfert d'électron qui président à l'activation de l'enzyme ont été étudiés en fonction des différents partenaires et un mécanisme original pour la réductolyse de l'AdoMet a été proposé.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

La ribonucléotide réductase anaérobie d'Escherichia coli, une protéine à zinc (importance du site métallique pour la structure et l'activation de l'enzyme)

LES RIBONUCLEOTIDES REDUCTASES (RNRS) SONT DES ENZYMES UBIQUITAIRES ESSENTIELLES POUR LA SYNTHESE D'ADN. LA CROISSANCE EN ANAEROBIOSE D'ESCHERICHIA COLI DEPEND D'UNE RNR DE CLASSE III. L'ENZYME ACTIVEE CONTIENT UN RADICAL SENSIBLE A L'OXYGENE SITUE SUR LE RESIDU G681 DONT LA FORMATION IMPLIQUE L'INTERVENTION CONCERTEE D'UNE PROTEINE ACTIVATRICE FER-SOUFRE, DE S-ADENOSYLMETHIONINE, DE DITHIOTHREITOL (DTT), ET D'UN SYSTEME REDUCTEUR. LA STRUCTURE CRISTALLOGRAPHIQUE DE LA RNR DU BACTERIOPHAGE T4 A REVELE LA PRESENCE D'UN SITE METALLIQUE ZN(CYS)4 DANS LA PARTIE C-TERMINALE DE LA REDUCTASE. DANS CE TRAVAIL NOUS AVONS DEFINI DE NOUVELLES CONDITIONS DE PURIFICATION CONDUISANT A DES ENZYMES TRES ACTIVES, MONTRE QUE LE ZINC CONTROLE LA STRUCTURATION DE LA BOUCLE CTER CONTENANT LE SITE RADICALAIRE ET QUE, CONTRAIREMENT A CE QUI ETAIT ADMIS DEPUIS PLUS DE 15 ANS, LE DTT N'INTERVIENT PAS DANS LA FORMATION DU RADICAL GLY· MAIS PLUTOT DANS LES TRANSFERTS RADICALAIRES ENTRE GLY· ET LE SUBSTRAT.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Iron–Sulfur Clusters in “Radical SAM” Enzymes: Spectroscopy and Coordination

International audienceThere is increasing evidence for the versatility of the coordination of iron–sulfur clusters in biology. In addition to cysteine residues as the most favored ligand and providing sulfur coordination, oxygenbased (aspartate, tyrosinate …) and nitrogen-based (histidine, arginine …) residues have also been observed as ligands to the clusters. Furthermore, low-molecular-weight substrates (citrate in the case of aconitase) and cofactors (S-adenosylmethionine, SAM, in the case of “Radical SAM” enzymes) have been shown to bind to one of the iron atoms of the [4Fe–4S] clusters where they are then activated. In this chapter we discuss the potential as well as the limitations of ENDOR and HYSCORE spectroscopy for characterizing metalloprotein coordination and, more specifically, the cluster–SAM complexes that are essential intermediates in pyruvate formate lyase-activating enzyme, lysine 2,3 aminomutase, and ribonucleotide reductase activating enzyme. These three systems are prototypes for the “Radical SAM” enzyme superfamily, whose chemistry seems to be extensively utilized in the metabolism of all living organisms

Activation of the anaerobic ribonucleotide reductase by S-adenosylmethionine.

International audienc

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

International audienceThe 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. This review article summarizes recent important achievements obtained on these five enzymes with the main focus to delineate the role of this additional [4Fe-4S] cluster in catalysis

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

International audienceStrict 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. The 3D structure of the homologous enzyme from bacteriophage T4 has revealed the presence of a metal center bound to four conserved cysteine residues. In this report we identify the metal of the E. coli enzyme as Zn. We show that the presence of Zn in this site protects the protein from proteolysis and prevents the formation of disulfide bridges within it. Finally, we show with the fully Zn-loaded reductase that thioredoxin or small thiols are dispensable for the formation of the glycyl radical. However, they are necessary for obtaining high turnover numbers, suggesting that they intervene in radical transfer steps subsequent to the formation of the glycyl radical

Wybutosine biosynthesis: Structural and mechanistic overview

International audienceOver 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