MiaB Protein from Thermotoga maritima

Pierrel, Fabien; Hernandez, Heather L.; Johnson, Michael K.; Fontecave, Marc; Atta, Mohamed

doi:10.1074/jbc.m301518200

Cited by 58 publications

(40 citation statements)

References 61 publications

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“…1), which is similar to that of, as isolated, MiaB, supports this notion. The spectrum of MiaB was ascribed to the [2Fe-2S] 2ϩ cluster (7). The [4Fe-4S] 2ϩ to [2Fe-2S] 2ϩ conversion is part of the oxygen-sensing mechanism of the transcription factor FNR (fumarate nitrate reduction), for instance (43,44).…”

Section: Discussionmentioning

confidence: 99%

Redox Reactions of the Iron-Sulfur Cluster in a Ribosomal RNA Methyltransferase, RumA

Agarwalla

Stroud

Gaffney

2004

Journal of Biological Chemistry

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RumA, an S-adenosyl-L-methionine-dependent methyltransferase specifically catalyzes the methylation of U1939 of 23 S ribosomal RNA to yield 5-methyluridine (m 5 U) 1 or ribothymidine (1). This enzyme was found to contain a [4Fe-4S] cluster, a prosthetic group usually not associated with methyltransferase enzymes. The x-ray structure of RumA revealed that the cluster is located in the RNA binding domain of the threedomain protein (2). It is held by four Cys-iron bonds provided by Cys 81 , Cys 87 , Cys 90 , and Cys 162 and is buried in a largely hydrophobic region, but one of its sulfur atoms is quite exposed to solvent. The closest distance between the cluster and the sulfur of the catalytic nucleophile, Cys 389 , is ϳ19 Å. RumA is a member of a growing list of nucleic acid modifying enzymes where the function of the iron-sulfur (FeS) cluster remains enigmatic.The reaction mechanism of m 5 U methyltransferases is similar to that of thymidylate synthase, and has been studied mainly employing the tRNA m 5 U54 methyltransferase, TrmA (3-5). It involves Michael addition of the catalytic Cys to carbon 6 (C-6) of pyrimidine, which activates C-5 for a nucleophilic attack on the methyl group of S-adenosyl-L-methionine. Following the methyl transfer, the enzyme is resolved by abstraction of a proton from C-5 and ␤-elimination. TrmA, as well as DNA and RNA 5-methylcytidine methyltransferases, which employ a similar catalytic mechanism (6), do not posses an FeS cluster or any other prosthetic group. Lack of a redox step in the catalytic mechanism, and absence of the cluster in enzymes that employ a similar mechanism preclude a direct role for [4Fe-4S] cluster in the RumA-catalyzed reaction.Two features distinguish the FeS cluster in RumA from the clusters in a different class of S-adenosyl-L-methionine-dependent enzymes that proceed by free radical mechanisms. The RumA cluster is ligated by 4 cysteines and the iron-binding sequence motif is CX 5 CX 2 CX n C. In contrast, the radical Sadenosyl-L-methionine enzymes, of which MiaB (7) and biotin synthase (8) are examples, have a [4Fe-4S] cluster built of a CX 3 CX 2 C sequence that leaves one iron of the cluster free to participate in catalysis. MiaB is a member of a family of enzymes catalyzing addition of sulfur to tRNA bases (9). The FeS sequence motifs in the DNA repair enzymes, endonuclease III (10) and MutY (11), are CX 6 CX 2 CX 5 C, somewhat similar to the RumA sequence. Although the FeS clusters in these enzymes are usually regarded as structural, recently it was shown that the redox potential of MutY is altered by DNA binding and that a change in charge of the [4Fe-4S] 3ϩ/2ϩ cluster might alter its affinity for DNA (12).The redox potentials of [4Fe-4S] clusters vary widely (13-16). Of particular interest in many contemporary studies of FeS proteins is understanding the factors that influence the potentials, and thus functions, of these clusters (17,18). Proteins with four Cys-iron bonds to the [4Fe-4S] cluster fall into two general redox groups, ones with an accessib...

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Section: Discussionmentioning

confidence: 99%

Redox Reactions of the Iron-Sulfur Cluster in a Ribosomal RNA Methyltransferase, RumA

Agarwalla

Stroud

Gaffney

2004

Journal of Biological Chemistry

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“…Expression and Purification Methods-The MiaBTm protein was overexpressed in E. coli BL21CodonPlus(DE3)-RIL TM and purified as previously described, apo and reconstituted forms were obtained as previously described (18). IscS and SufS have been purified as previously described and have been provided by Dr. S. Ollagnier-deChoudens (20, 21).…”

Section: Methodsmentioning

confidence: 99%

“…In the case of BioB enzyme, which also catalyzes C-H to C-S bond conversion reactions, this motif was shown to provide cysteine ligands for a catalytically essential [4Fe-4S] 2ϩ/1ϩ cluster (16). Such a cluster is present also in MiaB protein (17,18), and it is thus very likely that MiaB and BioB employ similar radical mechanisms for activation of sulfur and its insertion into their respective substrates.Recently, we cloned, expressed, and characterized by bio-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.…”

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confidence: 99%

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confidence: 99%

“…chemical and spectroscopic methods the MiaB protein from Thermotoga maritima called MiaBTm (18). The protein is able to assemble an O 2 -sensitive [4Fe-4S] cluster, which can enjoy both 2ϩ (oxidized) and 1ϩ (reduced) redox states.…”

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MiaB Protein Is a Bifunctional Radical-S-Adenosylmethionine Enzyme Involved in Thiolation and Methylation of tRNA

Pierrel

Douki

Fontecave

et al. 2004

Journal of Biological Chemistry

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157

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The last biosynthetic step for 2-methylthio-N 6 -isopentenyl-adenosine (ms 2 i 6 A), present at position 37 in some tRNAs, consists of the methylthiolation of the isopentenyl-adenosine (i 6 A) precursor. In this work we have reconstituted in vitro the conversion of i 6 A to ms 2 i 6 A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. We show that a synthetic i 6 A-containing RNA corresponding to the anticodon stem loop of tRNA Phe is also a substrate. This study demonstrates that MiaB protein is a bifunctional system, involved in both thiolation and methylation of i 6 A. In this process, one molecule of AdoMet is converted to 5 -deoxyadenosine, probably through reductive cleavage and intermediate formation of a 5 -deoxyadenosyl radical as observed in other "Radical-AdoMet" enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. The origin of the sulfur atom is discussed.In all organisms, the nucleosides of transfer RNA (tRNA) undergo different post-transcriptional modifications that are important for their biological activity (1). These modifications are part of the complex process of tRNA maturation and are introduced through the action of many different enzymes on the polynucleotide transcripts (2). Although modified bases are found at different positions in tRNA, the anticodon loop is the most heavily modified region of the molecule and contains the greatest variety of modified nucleosides. To date, a total of 86 structurally distinguishable modified nucleosides in tRNA from many diverse organisms of the three major phylogenetic domains of life have been identified (medstat.med.utah.edu/ RNAmods/). Among these modified nucleosides, the so-called thionucleosides containing a sulfur atom have been the subject of particular attention in the recent years.In Escherichia coli four different thiolated nucleosides have been characterized; these are 4-thiouridine (s 4 U), 1 2-thiocytidine (s 2 C), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), and 2-methylthio-N 6 -isopentenyl-adenosine (ms 2 i 6 A). If the synthesis of thiopyrimidines involves a non-redox substitution reaction of an oxygen atom by sulfur, the reaction leading to the synthesis of 2-methylthio-N 6 -isopentenyl-adenosine (ms 2 i 6 A) consists of a chemically highly challenging aromatic C-H to C-S bond conversion. This is an oxidation reaction whose mechanism has yet to be investigated. The modified nucleoside ms 2 i 6 A is found at position 37, next to the anticodon on the 3Ј-position in almost all eukaryotic and bacterial tRNAs that read codons beginning with U except tRNA I,V Ser (3).Initial studies on ms 2 i 6 A biosynthesis in E. coli and Salmonella typhimurium established a requirement for at least two enzymes (4). The first step consists in the addition of the isopentenyl group to the N 6 nitrogen of adenosine. This reaction is catalyzed by the well characterized tRNA-isopentenylpyrophosphate transferase enzyme, encoded by the miaA gene (5...

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Adenosyl Radical: Reagent and Catalyst in Enzyme Reactions

2010

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Lead in Adenosine is undoubtedly an ancient biological molecule that is a component of many enzyme cofactors; ATP, FADH, NAD(P)H, and coenzyme A, to name but a few, and, of course, of RNA. Here we present an overview of the role of adenosine in its most reactive form: as an organic radical formed either by homolytic cleavage of adenosylcobalamin (coenzyme B12, AdoCbl) or by single-electron reduction of S-adenosylmethionine (AdoMet) complexed to an iron-sulfur cluster. Although many of the enzymes we discuss are newly discovered, adenosine’s role as a radical cofactor most likely arose very early in evolution, before the advent of photosynthesis and the production of molecular oxygen which rapidly inactivates many radical enzymes. AdoCbl-dependent enzymes appear to be confined to a rather narrow repertoire of rearrangement reactions involving 1,2-hydrogen atom migrations. In contrast, there has been a recent explosion in the number radical AdoMet enzymes discovered that catalyze a remarkably wide range of chemically challenging reactions. Although all the radical AdoMet enzymes so far characterized come from anaerobically growing microbes and are very oxygen sensitive, there is tantalizing evidence that some of these enzymes may be active in aerobic organisms including humans.

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MiaB Protein from Thermotoga maritima

Cited by 58 publications

References 61 publications

Redox Reactions of the Iron-Sulfur Cluster in a Ribosomal RNA Methyltransferase, RumA

Redox Reactions of the Iron-Sulfur Cluster in a Ribosomal RNA Methyltransferase, RumA

MiaB Protein Is a Bifunctional Radical-S-Adenosylmethionine Enzyme Involved in Thiolation and Methylation of tRNA

Adenosyl Radical: Reagent and Catalyst in Enzyme Reactions

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