Methylthiotransferase

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Methylthiotransferases are enzymes of the radical S-adenosyl methionine (radical SAM) superfamily. These enzymes catalyze the addition of a methylthio group to various biochemical compounds including tRNA and proteins. [1] Methylthiotransferases are classified into one of four classes based on their substrates and mechanisms. [2] All methylthiotransferases have been shown to contain two Fe-S clusters, one canonical cluster and one auxiliary cluster, that both function in the addition of the methylthio group to the substrate. [3]

Contents

Overview

Methylthiotransferases, also known as MTTases, are a subset of the radical SAM enzyme superfamily. These enzymes catalyze the addition of a methylthio group to either a protein or tRNA substrate. [1] Radical S-adenosylmethionine enzymes, otherwise known as radical SAM enzymes, are metalloproteins that cleave S-adenosyl-L-methionine into L-methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA). [3] 5'-dA is an intermediate in the reactions catalyzed by radical SAMs. 5'-dA removes a hydrogen from the substrate and allows for the addition of another group to that carbon on the substrate. [3] In order to complete their reactions, all radical SAMs require a reduced [4Fe-4S] cluster, which is found through a conserved cysteine motif, CX3CX2C. [3] Radical SAMs can have one or multiple Fe-S clusters. In this case, methylthiotransferases have multiple clusters. Radical SAMs are involved in many cellular processes in all three domains of life including metabolism and the biosynthesis of many cofactors used within the cell. [3]

There are four known classes of Methylthiotransferases; three classes are involved in the methylthiolation of tRNAs and one is involved in the methylthiolation of proteins. [2] All identified methylthiotransferases have two Fe-S active clusters and three characteristic domains within the protein. [1] [2] These three structural domains include an N-terminal uncharacterized protein family 0004 (UPF0004) domain that contains the auxiliary Fe-S cluster, a central radical SAM motif that contains the central active Fe-S motif, and a C-terminal "TRAM" domain that is thought to be involved in substrate recognition. [1] [2] Of the two Fe-S clusters, the central cluster binds the SAM that is used to generate the 5'-dA while the auxiliary cluster has a less studied functionality. Most research suggests that this auxiliary cluster functions as the direct donor of the sulfur during catalysis or it functions to coordinate an exogenous source of sulfur for use during catalysis. [4] In the comparatively well studied methylthiotransferase MiaB, the auxiliary cluster is thought to directly donate the sulfur of the methylthio group during catalysis. [4]

Proposed mechanism

Methylthiotransferases catalyze the addition of a methylthio group to various biochemical products. Transferring methylthio groups is a complicated reaction requiring multiple Fe-S clusters. Previous literature proposed that the enzymes would function sequentially, first adding a sulfur to the substrate and then adding a methyl group derived from the second SAM molecule. [5] This mechanism has not been supported by recent works. Studies now propose that a methyl group from the first SAM molecule is transferred to a sulfur within the auxiliary [4Fe-4S] cluster to form a methylthio group that is then transferred to the product via a radical mechanism facilitated by the 5'-dA radical intermediate produced from the cleavage of the second SAM molecule. [4] [6] The proposed mechanisms for MiaB and RimO slightly differ, with MiaB using a coordinated sulfur as the methylthio group [4] and RimO using an external sulfur attached to the unique iron atom within the cluster as the methylthio group. [6] Despite this difference, both use the same basic principles for the mechanism; create a methylthiolated intermediate using the auxiliary [4Fe-4S] cluster and then add the methylthio group to the substrate. [4] [6]

Known examples

Formation of the [3Fe-4S] auxiliary cluster of MiaB via the nucleophilic attack of a m3-bridging sulfide. Adapted from Zhang et al. 2020. MiaB Methylthiotransferase Mechanism.png
Formation of the [3Fe-4S] auxiliary cluster of MiaB via the nucleophilic attack of a μ3-bridging sulfide. Adapted from Zhang et al. 2020.

MiaB

MiaB is a methylthiotransferase that completes the methylthiolation of a modified adenosine base, N6-isopentenyl adenosine to C2-methylthio-N6-isopentenyl adenosine, in tRNA which involves the addition of a methylthiogroup to an inactivate C-H bond. [1] [3] [4] The modification of this base in tRNAs enhances codon-anticodon binding and maintenance of the ribosomal reading frame during translation of an mRNA into protein. [4] Unlike the other methylthiotransferases described here, MiaB donates the sulfur group for methylthiolation itself instead of using a secondary sulfur donor and also completes two SAM-dependent reactions within a single polypeptide. [3]

MtaB

MtaB is a methylthiotransferase that exists in bacteria, archaea, and eukarya that completes the methylthiolation of the modified adenosine base, N6-threonylcarbamoyladenosine, at position 37 of tRNAs that code for the ANN codons to 2-methylthio-N6-threonylcarbamoyladenosine. [1] [7] When compared to MiaB and RimO, MtaB is much less studied but is still potentially involved in various cellular processes. One potential application of studying this specific MTTase is that it is encoded by the gene CDKAL1 in humans, which is known to increase the reduction of insulin secretion when mutated or downregulated thus leading to a higher risk of the person developing type 2 diabetes. [1] [7]

RimO

Mechanism of RimO catalyzed addition of a methylthio group to the b-carbon of aspartate. Adapted from Landgraf et al. 2013. RimO thiomethyltransferase mechanism.png
Mechanism of RimO catalyzed addition of a methylthio group to the β-carbon of aspartate. Adapted from Landgraf et al. 2013.

RimO is a methylthiotransferase that completes the methylthiolation of the β-carbon of the Asp88 residue of the ribosomal S12 protein in bacteria, specifically E. coli. [1] [2] This MTTase is the first identified to create post-translational modifications as all other previously identified MTTases modify tRNAs. Though RimO acts on a different substrate than the other classes of MTTases, the primary structure of the protein and the mechanism behind its action are relatively similar. [1]

Related Research Articles

<i>S</i>-Adenosyl methionine Chemical compound found in all domains of life with largely unexplored effects

S-Adenosyl methionine (SAM), also known under the commercial names of SAMe, SAM-e, or AdoMet, is a common cosubstrate involved in methyl group transfers, transsulfuration, and aminopropylation. Although these anabolic reactions occur throughout the body, most SAM is produced and consumed in the liver. More than 40 methyl transfers from SAM are known, to various substrates such as nucleic acids, proteins, lipids and secondary metabolites. It is made from adenosine triphosphate (ATP) and methionine by methionine adenosyltransferase. SAM was first discovered by Giulio Cantoni in 1952.

<span class="mw-page-title-main">Nitrogenase</span> Class of enzymes

Nitrogenases are enzymes (EC 1.18.6.1EC 1.19.6.1) that are produced by certain bacteria, such as cyanobacteria (blue-green bacteria) and rhizobacteria. These enzymes are responsible for the reduction of nitrogen (N2) to ammonia (NH3). Nitrogenases are the only family of enzymes known to catalyze this reaction, which is a key step in the process of nitrogen fixation. Nitrogen fixation is required for all forms of life, with nitrogen being essential for the biosynthesis of molecules (nucleotides, amino acids) that create plants, animals and other organisms. They are encoded by the Nif genes or homologs. They are related to protochlorophyllide reductase.

Iron–sulfur proteins are proteins characterized by the presence of iron–sulfur clusters containing sulfide-linked di-, tri-, and tetrairon centers in variable oxidation states. Iron–sulfur clusters are found in a variety of metalloproteins, such as the ferredoxins, as well as NADH dehydrogenase, hydrogenases, coenzyme Q – cytochrome c reductase, succinate – coenzyme Q reductase and nitrogenase. Iron–sulfur clusters are best known for their role in the oxidation-reduction reactions of electron transport in mitochondria and chloroplasts. Both Complex I and Complex II of oxidative phosphorylation have multiple Fe–S clusters. They have many other functions including catalysis as illustrated by aconitase, generation of radicals as illustrated by SAM-dependent enzymes, and as sulfur donors in the biosynthesis of lipoic acid and biotin. Additionally, some Fe–S proteins regulate gene expression. Fe–S proteins are vulnerable to attack by biogenic nitric oxide, forming dinitrosyl iron complexes. In most Fe–S proteins, the terminal ligands on Fe are thiolate, but exceptions exist.

<span class="mw-page-title-main">Aconitase</span> Class of enzymes

Aconitase is an enzyme that catalyses the stereo-specific isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle, a non-redox-active process.

<span class="mw-page-title-main">Catalytic triad</span> Set of three coordinated amino acids

A catalytic triad is a set of three coordinated amino acids that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes. An acid-base-nucleophile triad is a common motif for generating a nucleophilic residue for covalent catalysis. The residues form a charge-relay network to polarise and activate the nucleophile, which attacks the substrate, forming a covalent intermediate which is then hydrolysed to release the product and regenerate free enzyme. The nucleophile is most commonly a serine or cysteine amino acid, but occasionally threonine or even selenocysteine. The 3D structure of the enzyme brings together the triad residues in a precise orientation, even though they may be far apart in the sequence.

<span class="mw-page-title-main">Methyltransferase</span> Group of methylating enzymes

Methyltransferases are a large group of enzymes that all methylate their substrates but can be split into several subclasses based on their structural features. The most common class of methyltransferases is class I, all of which contain a Rossmann fold for binding S-Adenosyl methionine (SAM). Class II methyltransferases contain a SET domain, which are exemplified by SET domain histone methyltransferases, and class III methyltransferases, which are membrane associated. Methyltransferases can also be grouped as different types utilizing different substrates in methyl transfer reactions. These types include protein methyltransferases, DNA/RNA methyltransferases, natural product methyltransferases, and non-SAM dependent methyltransferases. SAM is the classical methyl donor for methyltransferases, however, examples of other methyl donors are seen in nature. The general mechanism for methyl transfer is a SN2-like nucleophilic attack where the methionine sulfur serves as the leaving group and the methyl group attached to it acts as the electrophile that transfers the methyl group to the enzyme substrate. SAM is converted to S-Adenosyl homocysteine (SAH) during this process. The breaking of the SAM-methyl bond and the formation of the substrate-methyl bond happen nearly simultaneously. These enzymatic reactions are found in many pathways and are implicated in genetic diseases, cancer, and metabolic diseases. Another type of methyl transfer is the radical S-Adenosyl methionine (SAM) which is the methylation of unactivated carbon atoms in primary metabolites, proteins, lipids, and RNA.

<span class="mw-page-title-main">Enzyme catalysis</span> Catalysis of chemical reactions by specialized proteins known as enzymes

Enzyme catalysis is the increase in the rate of a process by a biological molecule, an "enzyme". Most enzymes are proteins, and most such processes are chemical reactions. Within the enzyme, generally catalysis occurs at a localized site, called the active site.

In enzymology, carbon monoxide dehydrogenase (CODH) (EC 1.2.7.4) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Adenylyl-sulfate reductase</span> Class of enzymes

Adenylyl-sulfate reductase is an enzyme that catalyzes the chemical reaction of the reduction of adenylyl-sulfate/adenosine-5'-phosphosulfate (APS) to sulfite through the use of an electron donor cofactor. The products of the reaction are AMP and sulfite, as well as an oxidized electron donor cofactor.

<span class="mw-page-title-main">Biotin synthase</span>

Biotin synthase (BioB) is an enzyme that catalyzes the conversion of dethiobiotin (DTB) to biotin; this is the final step in the biotin biosynthetic pathway. Biotin, also known as vitamin B7, is a cofactor used in carboxylation, decarboxylation, and transcarboxylation reactions in many organisms including humans. Biotin synthase is an S-Adenosylmethionine (SAM) dependent enzyme that employs a radical mechanism to thiolate dethiobiotin, thus converting it to biotin.

Lipoyl synthase is an enzyme that belongs to the radical SAM (S-adenosyl methionine) family. Within the radical SAM superfamily, lipoyl synthase is in a sub-family of enzymes that catalyze sulfur insertion reactions. Enzymes in this family contain two 4Fe-4S clusters, from which they obtain the sulfur groups that will be transferred onto the corresponding substrates. This particular enzyme participates in lipoic acid metabolism, so it transfers two sulfur atoms from its 4Fe-4S cluster onto the protein N6-(octanoyl)lysine through radical generation. This enzyme is usually localized to the mitochondria. Two organisms that have been extensively studied with regards to this enzyme are Escherichia coli and Mycobacterium tuberculosis. It is also found in other organisms, such as yeast and plants.

<span class="mw-page-title-main">Dioxygenase</span>

Dioxygenases are oxidoreductase enzymes. Aerobic life, from simple single-celled bacteria species to complex eukaryotic organisms, has evolved to depend on the oxidizing power of dioxygen in various metabolic pathways. From energetic adenosine triphosphate (ATP) generation to xenobiotic degradation, the use of dioxygen as a biological oxidant is widespread and varied in the exact mechanism of its use. Enzymes employ many different schemes to use dioxygen, and this largely depends on the substrate and reaction at hand.

Spore photoproduct lyase is a radical SAM enzyme that repairs DNA cross linking of thymine bases caused by UV-radiation. There are several types of thymine cross linking, but SPL specifically targets 5-thyminyl-5,6-dihydrothymine, which is also called spore photoproduct (SP). Spore photoproduct is the predominant type of thymine crosslinking in germinating endospores, which is why SPL is unique to organisms that produce endospores, such as Bacillus subtilis. Other types of thymine crosslinking, such as cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), are less commonly formed in endospores. These differences in DNA crosslinking are a function of differing DNA structure. Spore genomic DNA features many DNA binding proteins called small acid soluble proteins, which changes the DNA from the traditional B-form conformation to an A-from conformation. This difference in conformation is believed to be the reason why dormant spores predominantly accumulate SP in response to UV-radiation, rather than other forms of cross linking. Spores cannot repair cross-linking while dormant, instead the SPs are repaired during germination to allow the vegetative cell to function normally. When not repaired, spore photoproduct and other types of crosslinking can cause mutations by blocking transcription and replication past the point of the crosslinking. The repair mechanism utilizing spore photoproduct lyase is one of the reasons for the resilience of certain bacterial spores.

Radical SAM is a designation for a superfamily of enzymes that use a [4Fe-4S]+ cluster to reductively cleave S-adenosyl-L-methionine (SAM) to generate a radical, usually a 5′-deoxyadenosyl radical (5'-dAdo), as a critical intermediate. These enzymes utilize this radical intermediate to perform diverse transformations, often to functionalize unactivated C-H bonds. Radical SAM enzymes are involved in cofactor biosynthesis, enzyme activation, peptide modification, post-transcriptional and post-translational modifications, metalloprotein cluster formation, tRNA modification, lipid metabolism, biosynthesis of antibiotics and natural products etc. The vast majority of known radical SAM enzymes belong to the radical SAM superfamily, and have a cysteine-rich motif that matches or resembles CxxxCxxC. rSAMs comprise the largest superfamily of metal-containing enzymes.

23S rRNA (adenine2503-C2)-methyltransferase (EC 2.1.1.192, RlmN, YfgB, Cfr) is an enzyme with systematic name S-adenosyl-L-methionine:23S rRNA (adenine2503-C2)-methyltransferase. This enzyme catalyses the following chemical reaction

Glutamate 2,3-aminomutase is an enzyme that belongs to the radical s-adenosyl methionine (SAM) superfamily. Radical SAM enzymes facilitate the reductive cleavage of S-adenosylmethionine (SAM) through the use of radical chemistry and an iron-sulfur cluster. This enzyme family is implicated in the biosynthesis of DNA precursors, vitamin, cofactor, antibiotic and herbicides and in biodegradation pathways. In particular, glutamate 2,3 aminomutase is involved in the conversion of L-alpha-glutamate to L-beta-glutamate in Clostridium difficile. The generalized reaction is shown below:

Joan Blanchette Broderick is a professor of chemistry and biochemistry at Montana State University known for her work on bioinorganic chemistry, especially the chemistry of iron-sulfur interactions. She was elected a member of the National Academy of Sciences in 2022.

<span class="mw-page-title-main">Squire Booker</span> American biochemist

Squire Booker is an American biochemist at Penn State University. Booker directs an interdisciplinary chemistry research program related to fields of biochemistry, enzymology, protein chemistry, natural product biosynthesis, and mechanisms of radical dependent enzymes. He is an associate editor for the American Chemical Society Biochemistry Journal, is a Hughes Medical Institute Investigator, and an Eberly Distinguished Chair in Science at Penn State University.

<span class="mw-page-title-main">Deoxyadenosyl radical</span> Chemical compound

A deoxyadenosyl radical is a free radical that is structurally related to adenosine by removal of a 5′-hydroxy group from adenosine. This radical occurs in nature as a reactive intermediate. It is generated by radical SAM enzymes and by some varieties of vitamin B12. The deoxyadenosyl radical abstracts hydrogen atoms from substrates, causing rearrangements and other post transcriptional modifications required for biosynthesis.

5'-deoxyadenosine deaminase is an enzyme that catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine. To a lesser extent, the enzyme also catalyzes the deamination of 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine. The molecular mass of the DadD enzyme is approximately 230 kDa. DadD maintains 90% of its enzymatic activity after being heated at 60 degrees Celsius for ten minutes. The preferred pH for 5'deoxyadenosine deaminase is 9.0, with the enzyme denaturing at a pH of 11. The DadD enzyme has a preferred substrate of 5'deoxyadenosine, though it will also react with 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine at lower efficiencies.

References

  1. 1 2 3 4 5 6 7 8 9 Wang, Jiarui; Woldring, Rory P.; Román-Meléndez, Gabriel D.; McClain, Alan M.; Alzua, Brian R.; Marsh, E. Neil G. (2014-09-19). "Recent Advances in Radical SAM Enzymology: New Structures and Mechanisms". ACS Chemical Biology. 9 (9): 1929–1938. doi:10.1021/cb5004674. ISSN   1554-8929. PMC   4168785 . PMID   25009947.
  2. 1 2 3 4 5 Lee, Kyung-Hoon; Saleh, Lana; Anton, Brian P.; Madinger, Catherine L.; Benner, Jack S.; Iwig, David F.; Roberts, Richard J.; Krebs, Carsten; Booker, Squire J. (2009-10-27). "Characterization of RimO, a New Member of the Methylthiotransferase Subclass of the Radical SAM Superfamily". Biochemistry. 48 (42): 10162–10174. doi:10.1021/bi900939w. ISSN   0006-2960. PMC   2952840 . PMID   19736993.
  3. 1 2 3 4 5 6 7 Booker, Squire J; Cicchillo, Robert M; Grove, Tyler L (2007). "Self-sacrifice in radical S-adenosylmethionine proteins". Current Opinion in Chemical Biology. 11 (5): 543–552. doi:10.1016/j.cbpa.2007.08.028. PMC   2637762 . PMID   17936058.
  4. 1 2 3 4 5 6 7 Zhang, Bo; Arcinas, Arthur J.; Radle, Matthew I.; Silakov, Alexey; Booker, Squire J.; Krebs, Carsten (2020-01-29). "First Step in Catalysis of the Radical S-Adenosylmethionine Methylthiotransferase MiaB Yields an Intermediate with a [3Fe-4S]0-Like Auxiliary Cluster". Journal of the American Chemical Society. 142 (4): 1911–1924. doi:10.1021/jacs.9b11093. ISSN   0002-7863. PMC   7008301 . PMID   31899624.
  5. ↑ Fontecave, M.; Mulliez, E.; Atta, M. (2008-03-21). "New Light on Methylthiolation Reactions". Chemistry & Biology. 15 (3): 209–210. doi: 10.1016/j.chembiol.2008.02.011 . ISSN   1074-5521. PMID   18355719.
  6. 1 2 3 Landgraf, Bradley J.; Arcinas, Arthur J.; Lee, Kyung-Hoon; Booker, Squire J. (2013-10-16). "Identification of an Intermediate Methyl Carrier in the Radical S-Adenosylmethionine Methylthiotransferases RimO and MiaB". Journal of the American Chemical Society. 135 (41): 15404–15416. doi:10.1021/ja4048448. ISSN   0002-7863. PMC   4023531 . PMID   23991893.
  7. 1 2 Arragain, Simon; Handelman, Samuel K.; Forouhar, Farhad; Wei, Fan-Yan; Tomizawa, Kazuhito; Hunt, John F.; Douki, Thierry; Fontecave, Marc; Mulliez, Etienne; Atta, Mohamed (2010-09-10). "Identification of Eukaryotic and Prokaryotic Methylthiotransferase for Biosynthesis of 2-Methylthio- N 6 -threonylcarbamoyladenosine in tRNA". Journal of Biological Chemistry. 285 (37): 28425–28433. doi: 10.1074/jbc.M110.106831 . ISSN   0021-9258. PMC   2937867 . PMID   20584901.
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