Etienne Mulliez

Activation of the Anaerobic Ribonucleotide Reductase fromEscherichia coli THE ESSENTIAL ROLE OF THE IRON-SULFUR CENTER FORS-ADENOSYLMETHIONINE REDUCTION

The anaerobic ribonucleotide reductase ofEscherichia coli catalyzes the synthesis of the deoxyribonucleotides required for anaerobic DNA synthesis. The enzyme is an α2β2 heterotetramer. In its active form, the large α2 subunit contains an oxygen-sensitive glycyl radical, whereas the β2 small protein harbors a [4Fe-4S] cluster that joins its two polypeptide chains. Formation of the glycyl radical in the inactive enzyme requiresS-adenosylmethionine (AdoMet), dithiothreitol, K+, and either an enzymatic (reduced flavodoxin) or chemical (dithionite or 5-deazaflavin plus light) reducing system. Here, we demonstrate that AdoMet is directly reduced by the Fe-S center of β2 during the activation of the enzyme, resulting in methionine and glycyl radical formation. Direct binding experiments showed that AdoMet binds to β2 with aK d of 10 μm and a 1:1 stoichiometry. Binding was confirmed by EPR spectroscopy that demonstrated the formation of a complex between AdoMet and the [4Fe-4S] center of β2. Dithiothreitol triggered the cleavage of AdoMet, leading to an EPR-silent form of β2 and, in the case of α2β2, to glycyl radical formation. In both instances, 3 methionines were formed per mol of protein. Our results indicate that the Fe-S center of β2 is directly involved in the reductive cleavage of AdoMet and suggest a new biological function for an iron-sulfur center, i.e redox catalysis, as recently proposed by others (Staples, R. C., Ameyibor, E., Fu, W., Gardet-Salvi, L., Stritt-Etter, A. L., Schürmann, P., Knaff, D. B., and Johnson, M. K. (1996) Biochemistry 35, 11425–11434).

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-N6-threonylcarbamoyladenosine, at position 37 (A37) 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 N6-threonylcarbamoyladenosine into 2-methylthio-N6-threonylcarbamoyladenosine. Recently, a variant within the human CDKAL1 gene belonging to the e-MtaB subfamily was shown to predispose for type 2 diabetes. CDKAL1 is thus the first eukaryotic MTTase identified so far. Using purified preparations of Bacillus subtilis MtaB (YqeV), a CDKAL1 bacterial homolog, we demonstrate that YqeV/CDKAL1 enzymes, as the previously studied MTTases MiaB and RimO, contain two [4Fe-4S] clusters. This work lays the foundation for elucidating the function of CDKAL1.

5-Lipoxygenase from potato tubers. Improved purification and physicochemical characteristics

Abstract Potato tubers are shown to contain at least three lipoxygenase isoenzymes. A very efficient extraction of lipoxygenase activity is obtained when a non-ionic detergent (0.1% Brij 99) is added to the homogenization buffer. The major isoenzyme, L 1 , has been purified in an almost homogeneous form with a good yield (18%) and a high specific activity (140–160 units/mg). It is efficiently stabilized by glycerol (20%, v/v). The purified L 1 isoenzyme is slightly contaminated by an 11-lipoxygenase, both having very close p I values (4.94 and 4.99, respectively). L 1 is a monomeric protein of M r 92 000 containing one iron atom per molecule. The native enzyme is in a pseudo-axial high-spin ferric state as indicated by EPR. Acting on linoleic acid, L 1 forms 9-hydroperoxyoctadecadienoic acid (9-HPOD) almost exclusively. With arachidonic acid, 5-hydroperoxyeicosatetraenoic acid (5-HPETE) is the major product (70–75%) beside small amounts of 8(ifS)- and 9-HPETE. Due to the contaminating activity, 11-HPETE (15%) is also present. Formation of both 8( S )-HPETE and leukotriene A 4 hydrolysis products accounts for the intrinsic 8-lipoxygenase activity of the L 1 isoenzyme.

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. One cluster binds S-adenosylmethionine and generates an Ado• radical via a well-established mechanism. However, the precise role of the second cluster is unclear. For some sulfur-inserting radical-SAM enzymes, this cluster has been proposed to act as a sacrificial source of sulfur for the reaction. In this paper, we report parallel enzymological, spectroscopic and crystallographic investigations of RimO and MiaB, which provide what is to our knowledge the first evidence that these enzymes are true catalysts and support a new sulfation mechanism involving activation of an exogenous sulfur cosubstrate at an exchangeable coordination site on the second cluster, which remains intact during the reaction.

The Anaerobic Ribonucleotide Reductase from Escherichia coli THE SMALL PROTEIN IS AN ACTIVATING ENZYME CONTAINING A [4Fe-4S]2+ CENTER

For deoxyribonucleotide synthesis during anaerobic growth, Escherichia coli cells depend on an oxygen-sensitive class III ribonucleotide reductase. The enzyme system consists of two proteins: protein α, on which ribonucleotides bind and are reduced, and protein β, of which the function is to introduce a catalytically essential glycyl radical on protein α. Protein β can assemble one [4Fe-4S] center per polypeptide enjoying both the [4Fe-4S]2+ and [4Fe-4S]1+ redox state, as shown by iron and sulfide analysis, Mössbauer spectroscopy (δ = 0.43 mm·s−1, ΔE Q = 1.0 mm·s−1, [4Fe-4S]2+), and EPR spectroscopy (g = 2.03 and 1.93, [4Fe-4S]1+). This iron center is sensitive to oxygen and can decompose into stable [2Fe-2S]2+ centers during exposure to air. This degraded form is nevertheless active, albeit to a lesser extent because of the conversion of the cluster into [4Fe-4S] forms during the strongly reductive conditions of the assay. Furthermore, protein β has the potential to activate several molecules of protein α, suggesting that protein β is an activating enzyme rather than a component of an α2β2 complex as previously claimed.For deoxyribonucleotide synthesis during anaerobic growth, Escherichia coli cells depend on an oxygen-sensitive class III ribonucleotide reductase. The enzyme system consists of two proteins: protein a, on which ribonucleotides bind and are reduced, and protein b, of which the function is to introduce a catalytically essential glycyl radical on protein a. Protein b can assemble one [4Fe-4S] center per polypeptide enjoying both the [4Fe-4S] and [4Fe-4S] redox state, as shown by iron and sulfide analysis, Mössbauer spectroscopy (d 5 0.43 mmzs, DEQ 5 1.0 mmzs , [4Fe-4S]), and EPR spectroscopy (g 5 2.03 and 1.93, [4Fe-4S]). This iron center is sensitive to oxygen and can decompose into stable [2Fe-2S] centers during exposure to air. This degraded form is nevertheless active, albeit to a lesser extent because of the conversion of the cluster into [4Fe-4S] forms during the strongly reductive conditions of the assay. Furthermore, protein b has the potential to activate several molecules of protein a, suggesting that protein b is an activating enzyme rather than a component of an a2b2 complex as previously claimed.

The Activating Component of the Anaerobic Ribonucleotide Reductase from Escherichia coli AN IRON-SULFUR CENTER WITH ONLY THREE CYSTEINES

Class III anaerobic ribonucleotide reductase small component, named protein β, contains a (4Fe-4S) center. Its function is to mediate electron transfer from reduced flavodoxin toS-adenosylmethionine, required for the introduction of a glycyl radical in the large component, named protein α, which then becomes active for the reduction of ribonucleotides. By site-directed mutagenesis we demonstrate that the three cysteines of the conserved CXXXCXXC sequence are involved in iron chelation. Such a sequence is also present in the activase of the pyruvate formate-lyase and in the biotin synthase, both carrying an iron-sulfur center involved in reductive activation ofS-adenosylmethionine. Even though they are able to bind iron in the (4Fe-4S) form, as shown by Mössbauer spectroscopy, the corresponding Cys to Ala mutants are catalytically inactive. Mutation of the two other cysteines of the protein did not result in inactivation. We thus conclude that the (4Fe-4S) cluster has, in the wild type protein, only three cysteine ligands and a fourth still unidentified ligand.

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., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826–1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 Å crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.

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. The recently described pyridoxal 5′‐phosphate‐bound enzyme was used to show that only one molecule of AdoMet, and not two, is required for the formation of one molecule of biotin. Furthermore 5′‐deoxyadenosine, a product of the reaction, strongly inhibited biotin formation, an observation that may explain why BioB is not able to make more than one turnover. However this enzyme inactivation is not irreversible.

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. Their evolutionnary relationship to class I and class II ribonucleotide reductases is also discussed.

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

Background: 4-Demethylwyosine synthase (TYW1) is a tRNA-modifying metalloenzyme involved in the biosynthesis of wyosine. Results: TYW1 enzyme belongs to the Radical-SAM superfamily with two Fe-S clusters involved in catalysis. Conclusion: The canonical Radical-SAM cluster binds and activates SAM co-factor, whereas the additional [4Fe-4S] cluster is shown to interact with the pyruvate co-substrate. Significance: This study helps to understand how radical-SAM enzymes with two Fe-S centers can synergistically achieve challenging radical insertion reactions. 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.” These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX3CX2C motif, and S-adenosyl-l-methionine (SAM) to generate a 5′-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed.