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Maturation of iron hydrogenases

Published on 24 January 2019


Dr. Mohamed Atta
CEA researcher
Laboratoire Chimie et Biologie des métaux
17 avenue des Martyrs
38 054 Grenoble cedex 09
: (33) 4 38 78 91 15

Enzymolgy of radical-based modification of biological macromolecules

Our group is interested in the understanding the enzymology of some of these systems through the study of two methylthiotransferases (MTTases) performing radical sulphur insertion on large macromolecules (nucleic acid or protein) namely MiaB and RimO (Figure 1) and also in its possible extension to radical insertion of a two carbon unit into a modified GTP of specific tRNAs performed by the enzyme TYW1 during the biosynthesis of wyosine. In addition, we are interested in identifying the sulphur donor of the tRNA-modifying enzyme TtcA whose activity depends on the presence of an unusual Fe-S center. Finally, we are also engaged in exploring two systems the anaerobic ribonucleotide reductase and spore photoproduct lyase involved in DNA biosynthesis and DNA repair respectively.

Figure 1.

A part TtcA enzyme all the proteins described above belongs to the Radical-SAM superfamily discovered in 2001. These enzymes produce a 5'-deoxyadenosyl radical by reductive cleavage of S-Adenosyl methionine (SAM) bound to a subsite iron of a [4Fe-4S] cluster attached to the protein through the three cysteines of the canonical motif CysxxxCysxxCys. These enzymes basically behave as radical initiators to activate their substrate by H-atom abstraction. The very high reactivity of 5'-deoxyadenosyl radical allows practically any site of the target substrate to be activated for modification (
Figure 2). However, the counterpart is that the selectivity of the reaction is much more difficult to control. In fact, the mechanistic and structural investigation of these fascinating radical-based enzymes may help to understand the following: first, how primary free radicals can be generated in one protein and directed to specific sites into a macromolecular target; second, how intermediate free radicals are controlled to generate the desired product; third, how all these radicals avoid deleterious redox quenching reactions.

Figure 2.

MTTases project (MiaB and RimO)

In this case, the substrate (tRNA or protein), once activated, is supposed to react with a source of sulphur to yield the thiolated product. We have recently shown that, as for other radical sulphur insertion systems, MTTases bind a second [4Fe-4S] cluster linked to the protein by three other conserved cysteines. Using advanced EPR techniques, we have been able to show that this second cluster is able to repeatedly bind sulfide thus allowing these enzymes to act catalytically in vitro. Moreover, we have shown that the reactions proceed via methylthio transfer. Indeed, these enzymes use SAM for two different reactions. On each cycle, SAM acts both as an electrophile to methylate the bound sulfide, and as precursor of an adenosyl radical to activate the substrate. The product results from radical coupling of the methylthio moiety to the activated substrate. Indeed, our recent published data show for the first time that biological radical sulphur insertion proceeds in a catalytic manner and does not involve sacrificial destruction of the additional cluster as has been generally admitted until now. However, although MTTases are true enzymes they are rapidly inactivated probably because of the detrimental high sulfide concentration needed to observe catalysis in vitro. We are presently trying to identify the physiological sulfide donor of the system and we would like also to understand how these enzymes cope with the use of SAM for two different reactivities. It is important to highlight that in addition to the MiaB and RimO enzymes we have identified three new MTTases families as follows:

(i) MtaB found in eubacteria with the B. subtilis yqeV gene as its prototype;
(ii) e-MtaB found in higher eukaryotes and archaebacteria with the murine CDKAL1 gene as its prototype; and
(iii) MTL1 found so far exclusively in ε proteobacteria. CDKAL1 is of special interest because genetic polymorphisms in this gene increase the reduction of insulin secretion and increase the risk of developing type 2 diabetes.

TYW1 project

The biosynthetic pathway of Wyosine and its derivatives (yW) was revealed and shown to require multiple steps of unprecedented enzymatic transformations. In this process TYW1 catalyzes the second step which is an intriguing reaction that consists of building a new aromatic ring on the guanine base (Figure 1-C) by transforming m1G37-tRNA substrate into 4-demethylwyosine (imG37-14-tRNA). TYW1 has been identified as an S-adenosylmethionine (SAM)-dependent (Radical-SAM) enzyme and features two catalytically important, oxygen-sensitive [4Fe-4S]2+/+ clusters, each ligated by only three cysteine residues. The N-terminal [4Fe-4S] cluster, bound to the polypeptide by CysX12CysX12Cys motif. The non-cysteinyl-coordinated Fe site of this cluster was shown to bind the pyruvate co-substrate for its activation. Downstream of the cluster II, the CysX3CysX2Cys motif is present and is the hallmark of the Radical-SAM enzymes responsible for the coordination of a second [4Fe-4S] cluster. Our recent data strongly suggests that the non-cysteinyl-coordinated Fe site of one of the N-terminal [4Fe-4S] clusters is able to interact with the pyruvate co-substrate for the reaction. A possible role for this additional [4Fe-4S] cluster in the activity of the enzyme is suggested and is under investigation. The work on TYW1 provides a new example of a Radical-SAM enzyme using its two FeS centers to synergistically achieve difficult radical insertion reactions.

TtcA project

From genetic studies TtcA gene has been suggested to encode a protein with an Fe-S cluster, we have indeed shown that the enzyme is an Fe-S. The cluster is absolutely required for activity however its role in the mechanism is unknown. We are presently investigating two possibilities either the cluster has a structural role or it is involved in the catalysis.
The objective of this project is to use X-ray crystallography for studying radical transfer reactions in the anaerobic ribonucleotide reductase (anRNR) from Thermatoga maritima. RNRs are essential enzymes as they are the sole de novo providers of deoxyribonucleotides to the cell. Tm anRNR is a complex made of two proteins, the reductase part (protein α) contains a metal binding site that controls the access and the stability of an essential glycyl radical nearby and a small radical SAM protein (protein ß) which catalyses the formation of this radical.
Both, the nature of the metal-binding site in the reductase proper and the cysteine motif of the activating protein are atypical when compared to other members of the anRNRs (class III) allowing us to suggest that this protein defines a sub-group in that class. Thus this project is two-fold: to characterize an unprecedented anRNR and to study the radical transfer pathways leading to the formation of the glycyl radical in one hand and to the reduction of the substrates in the other. Since these enzymes are controlled by allostery, these radical transfer reactions must be finely tuned according to the nature of the allosteric effectors.