Assembly of 2Fe-2S and 4Fe-4S Clusters in the Anaerobic Ribonucleotide Reductase from Escherichia c oli

The overwhelming majority of DNA photoproducts in UVirradiated spores is a unique thymine dimer called spore photoproduct (SP, 5-thymine-5,6-dihydrothymine). This lesion is repaired by the spore photoproduct lyase (SP lyase) enzyme that directly reverts SP to two unmodified thymines. The SP lyase is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily. In this study, by using a well characterized preparation of the SP lyase enzyme from Bacillus subtilis, we show that SP in the form of a dinucleoside monophosphate (spore photoproduct of thymidilyl-(3-5)-thymidine) is efficiently repaired, allowing a kinetic characterization of the enzyme. The preparation of this new substrate is described, and its identity is confirmed by mass spectrometry and comparison with authentic spore photoproduct. The fact that the spore photoproduct of thymidilyl-(3-5)-thymidine dimer is repaired by SP lyase may indicate that the SP lesion does not absolutely need to be contained within a singleor double-stranded DNA for recognition and repaired by the SP lyase enzyme.The DNA of all organisms is subject to modifications upon exposure to a wide variety of chemical and physical agents. Among them, solar ultraviolet radiation is known to induce dimerization reactions between adjacent pyrimidines (1). In the vast majority of living systems, the resulting photoproducts are cyclobutane pyrimidine dimers (CPDs) 5 and pyrimidine (6-4) pyrimidone photoproducts (Scheme 1A) that can be generated at any of the four bipyrimidine doublets (TT, CT, TC and CC), although the yields of the lesions depend on the bases involved (2). These lesions induce mutations and can be lethal because of blocking of the replication machinery. The photochemistry in bacterial spores is quite different. Indeed, in this dormant form produced by some bacteria such as Bacillus subtilis, the only photoproduct produced upon exposure to UV light corresponds to two thymines linked by the methyl group of one of the bases (3, 4). The formation of this specific lesion, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, SP) (Scheme 1A), is explained by specific features of the spores, including DNA conformation (A form), dehydration, the presence of dipicolinic acid in the core, and binding of small acid-soluble proteins to DNA (5-8). The formation of SP as the unique DNA lesion in irradiated spores is proposed to account for their extreme resistance to UV radiation. Indeed, spores express a specific repair enzyme, the spore photoproduct lyase (SP lyase) that directly reverts SP to two unmodified thymines upon germination (9, 10), much more efficiently than dimeric photoproducts are removed from other cell types by the classical nucleotide excision repair pathway. The specific photochemistry of DNA in spores combined with the action of SP lyase appears to be a major evolutionary advantage for spore-forming bacteria in resistance to UV radiation.In their N-terminal half, all SP lyase enzymes contain a strictly conserved a...

Section: Discussionmentioning

confidence: 95%

Dinucleotide Spore Photoproduct, a Minimal Substrate of the DNA Repair Spore Photoproduct Lyase Enzyme from Bacillus subtilis

Chandor

Berteau

Douki

et al. 2006

“…2A) 2ϩ clusters in other members of the AdoMetdependent radical enzymes, e.g. anaerobic ribonucleotide reductase-activating enzyme (40), pyruvate formate-lyase-activating enzyme (41), biotin synthase (42), and the tRNA-methylthiotransferase MiaB (39). EPR studies only showed a near isotropic S ϭ 1/2 resonance centered near g ϭ 2.01 ( (46), and this was further supported by VTVH MCD saturation magnetization data, which cannot be fit based solely on an S ϭ 1/2 ground state and indicate the presence of Kramers' S Ͼ 1/2 component (Fig.…”

Section: Effect Of Isc and Suf Proteins As Well As The Groes/el Chapementioning

confidence: 99%

Characterization of MOCS1A, an Oxygen-sensitive Iron-Sulfur Protein Involved in Human Molybdenum Cofactor Biosynthesis

Hänzelmann

Hernandez

Menzel

et al. 2004

138

124

The human proteins MOCS1A and MOCS1B catalyze the conversion of a guanosine derivative to precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine (AdoMet)-dependent radical enzymes, which catalyze the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus. MOCS1A was heterologously expressed in Escherichia coli and purified under aerobic and anaerobic conditions. Individual mutations of the conserved cysteines to serine revealed that all are essential for synthesis of precursor Z in vivo. The molybdenum cofactor in eukaryotic molybdoenzymes consists of a mononuclear molybdenum coordinated by the dithiolene moiety of a tricyclic pyranopterin, termed molybdopterin (MPT) 1 (1). In humans, defects in molybdenum cofactor biosynthesis lead to the pleiotropic loss of the molybdoenzymes sulfite oxidase, aldehyde oxidase, and xanthine dehydrogenase (2, 3). Affected patients usually die shortly after birth and show neurological abnormalities, such as attenuated growth of the brain, untreatable seizures, and dislocated ocular lenses (4). The first step during human molybdenum cofactor biosynthesis is catalyzed by MOCS1A and MOCS1B, leading to the synthesis of precursor Z, an oxygen-sensitive 6-alkyl pterin with a cyclic phosphate, from a guanosine derivative, most likely 5Ј-GTP (5-7).Analogous to other pteridine biosynthetic pathways, synthesis of precursor Z has been proposed to occur via a GTP cyclohydrolase-like reaction mechanism (5, 6). In contrast to these pathways, the C-8 atom of 5Ј-GTP is not released as formate but is retained and incorporated in a rearrangement reaction as the first carbon atom of the precursor Z side chain. In the second step of molybdenum cofactor biosynthesis catalyzed by MOCS3 (8) and MPT synthase (MOCS2) precursor Z is converted into MPT (9 -11). Finally molybdenum is incorporated into MPT by the multifunctional protein gephyrin (12, 13).MOCS1A contains two highly conserved cysteine motifs ( Fig. 1) proposed to be involved in iron-sulfur (FeS) cluster binding (14, 15), one is located near the N terminus (consensus sequence CX 3 CX 2 C where X denotes any amino acid), and one is near the C terminus (consensus sequence CX 2 CX 13 C). Several mutations identified in molybdenum cofactor deficiency patients are located in these conserved cysteine motifs indicating their functional importance for protein activity (2, 3). Based on sequence similarities to proteins such as biotin synthase, pyruvate formate-lyase-activating enzyme, and anaerobic ribonucleotide reductase-activating enzyme, MOCS1A has been classified as a member of the superfamily of S-adenosylmethionine (AdoMet)-dependent radical enzymes (16). In this class of enzymes, AdoMet serves as the free radical initiator and undergoes cleavage to methionine and a 5Ј-deoxyadenosyl radical that in turn propagates radical for...

“…Class III RNRs represent a third variant and harbors a stable glycyl radical within the so-called NrdD component that also harbors the active site region. An activating component, the 4Fe-4S NrdG protein, has the ability to generate the glycyl radical in NrdD via cleavage of the cofactor AdoMet (7)(8)(9). Class III RNR is an anaerobic enzyme, and the glycyl radical is extremely sensitive to oxygen.…”

mentioning

confidence: 99%

Cysteines Involved in Radical Generation and Catalysis of Class III Anaerobic Ribonucleotide Reductase

Andersson

Westman

Sahlin

et al. 2000

Class III ribonucleotide reductase (RNR) is an anaerobic glycyl radical enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides. We have investigated the importance in the reaction mechanism of nine conserved cysteine residues in class III RNR from bacteriophage T4. By using site-directed mutagenesis, we show that two of the cysteines, Cys-79 and Cys-290, are directly involved in the reaction mechanism. Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate. Our results provide strong experimental evidence for a similar radical-based reaction mechanism in all classes of RNR but also identify important differences between class III RNR and the other classes of RNR as regards the reduction per se. We also identify a cluster of four cysteines (Cys-543, Cys-546, Cys-561, and Cys-564) in the C-terminal part of the class III enzyme, which are essential for formation of the glycyl radical. These cysteines make up a CX 2 C-CX 2 C motif in the vicinity of the stable radical at Gly-580. We propose that the four cysteines are involved in radical transfer between Gly-580 and the cofactor S-adenosylmethionine of the activating NrdG enzyme needed for glycyl radical generation.