Crystallographic snapshots of a B12-dependent radical SAM methyltransferase.

By catalysing the microbial formation of methane, methyl-coenzyme M reductase has a central role in the global levels of this greenhouse gas1,2. The activity of methyl-coenzyme M reductase is profoundly affected by several unique post-translational modifications3-6, such as  a unique C-methylation reaction catalysed by methanogenesis marker protein 10 (Mmp10), a radical S-adenosyl-L-methionine (SAM) enzyme7,8. Here we report the spectroscopic investigation and atomic resolution structure of Mmp10 from Methanosarcina acetivorans, a unique B12 (cobalamin)-dependent radical SAM enzyme9. The structure of Mmp10 reveals a unique enzyme architecture with four metallic centres and critical structural features involved in the control of catalysis. In addition, the structure of the enzyme-substrate complex offers a glimpse into a B12-dependent radical SAM enzyme in a precatalytic state. By combining electron paramagnetic resonance spectroscopy, structural biology and biochemistry, our study illuminates the mechanism by which the emerging superfamily of B12-dependent radical SAM enzymes catalyse chemically challenging alkylation reactions and identifies distinctive active site rearrangements to provide a structural rationale for the dual use of the SAM cofactor for radical and nucleophilic chemistry.

Capturing a methanogenic carbon monoxide dehydrogenase/acetyl-CoA synthase complex via cryogenic electron microscopy.

Approximately two-thirds of the estimated one-billion metric tons of methane produced annually by methanogens is derived from the cleavage of acetate. Acetate is broken down by a Ni-Fe-S-containing A-cluster within the enzyme acetyl-CoA synthase (ACS) to carbon monoxide (CO) and a methyl group (CH3+). The methyl group ultimately forms the greenhouse gas methane, whereas CO is converted to the greenhouse gas carbon dioxide (CO2) by a Ni-Fe-S-containing C-cluster within the enzyme carbon monoxide dehydrogenase (CODH). Although structures have been solved of CODH/ACS from acetogens, which use these enzymes to make acetate from CO2, no structure of a CODH/ACS from a methanogen has been reported. In this work, we use cryo-electron microscopy to reveal the structure of a methanogenic CODH and CODH/ACS from Methanosarcina thermophila (MetCODH/ACS). We find that the N-terminal domain of acetogenic ACS, which is missing in all methanogens, is replaced by a domain of CODH. This CODH domain provides a channel for CO to travel between the two catalytic Ni-Fe-S clusters. It generates the binding surface for ACS and creates a remarkably similar CO alcove above the A-cluster using residues from CODH rather than ACS. Comparison of our MetCODH/ACS structure with our MetCODH structure reveals a molecular mechanism to restrict gas flow from the CO channel when ACS departs, preventing CO escape into the cell. Overall, these long-awaited structures of a methanogenic CODH/ACS reveal striking functional similarities to their acetogenic counterparts despite a substantial difference in domain organization.

Structural Dynamics of the Methyl-Coenzyme M Reductase Active Site Are Influenced by Coenzyme F430 Modifications.

Methyl-coenzyme M reductase (MCR) is a central player in methane biogeochemistry, governing methanogenesis and the anaerobic oxidation of methane (AOM) in methanogens and anaerobic methanotrophs (ANME), respectively. The prosthetic group of MCR is coenzyme F430, a nickel-containing tetrahydrocorphin. Several modified versions of F430 have been discovered, including the 172-methylthio-F430 (mtF430) used by ANME-1 MCR. Here, we employ molecular dynamics (MD) simulations to investigate the active site dynamics of MCR from Methanosarcina acetivorans and ANME-1 when bound to the canonical F430 compared to 172-thioether coenzyme F430 variants and substrates (methyl-coenzyme M and coenzyme B) for methane formation. Our simulations highlight the importance of the Gln to Val substitution in accommodating the 172 methylthio modification in ANME-1 MCR. Modifications at the 172 position disrupt the canonical substrate positioning in M. acetivorans MCR. However, in some replicates, active site reorganization to maintain substrate positioning suggests that the modified F430 variants could be accommodated in a methanogenic MCR. We additionally report the first quantitative estimate of MCR intrinsic electric fields that are pivotal in driving methane formation. Our results suggest that the electric field aligned along the CH3-S-CoM thioether bond facilitates homolytic bond cleavage, coinciding with the proposed catalytic mechanism. Structural perturbations, however, weaken and misalign these electric fields, emphasizing the importance of the active site structure in maintaining their integrity. In conclusion, our results deepen the understanding of MCR active site dynamics, the enzyme's organizational role in intrinsic electric fields for catalysis, and the interplay between active site structure and electrostatics.

Catalytic Activity of the Archetype from Group 4 of the FTR-like Ferredoxin:Thioredoxin Reductase Family Is Regulated by Unique S = 7/2 and S = 1/2 [4Fe-4S] Clusters.

Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe-4S]1+ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe-4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans, for which specialized target proteins were identified that are essential for growth and other diverse metabolisms.

Physiological and transcriptomic response to methyl-coenzyme M reductase limitation in Methanosarcina acetivorans.

Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis, the microbial metabolism responsible for nearly all biological methane emissions to the atmosphere. Decades of biochemical and structural research studies have generated detailed insights into MCR function in vitro, yet very little is known about the interplay between MCR and methanogen physiology. For instance, while it is routinely stated that MCR catalyzes the rate-limiting step of methanogenesis, this has not been categorically tested. In this study, to gain a more direct understanding of MCR's control on the growth of Methanosarcina acetivorans, we generate a strain with an inducible mcr operon on the chromosome, allowing for careful control of MCR expression. We show that MCR is not growth rate-limiting in substrate-replete batch cultures. However, through careful titration of MCR expression, growth-limiting state(s) can be obtained. Transcriptomic analysis of M. acetivorans experiencing MCR limitation reveals a global response with hundreds of differentially expressed genes across diverse functional categories. Notably, MCR limitation leads to strong induction of methylsulfide methyltransferases, likely due to insufficient recycling of metabolic intermediates. In addition, the mcr operon is not transcriptionally regulated, i.e., it is constitutively expressed, suggesting that the overabundance of MCR might be beneficial when cells experience nutrient limitation or stressful conditions. Altogether, we show that there is a wide range of cellular MCR concentrations that can sustain optimal growth, suggesting that other factors such as anabolic reactions might be rate-limiting for methanogenic growth. IMPORTANCE: Methane is a potent greenhouse gas that has contributed to ca. 25% of global warming in the post-industrial era. Atmospheric methane is primarily of biogenic origin, mostly produced by microorganisms called methanogens. Methyl-coenzyme M reductase (MCR) catalyzes methane formatio in methanogens. Even though MCR comprises ca. 10% of the cellular proteome, it is hypothesized to be growth-limiting during methanogenesis. In this study, we show that Methanosarcina acetivorans cells grown in substrate-replicate batch cultures produce more MCR than its cellular demand for optimal growth. The tools outlined in this study can be used to refine metabolic models of methanogenesis and assay lesions in MCR in a higher-throughput manner than isolation and biochemical characterization of pure protein.

Archaeal Connectase is a specific and efficient protein ligase related to proteasome ß subunits.

Sequence-specific protein ligations are widely used to produce customized proteins "on demand." Such chimeric, immobilized, fluorophore-conjugated or segmentally labeled proteins are generated using a range of chemical, (split) intein, split domain, or enzymatic methods. Where short ligation motifs and good chemoselectivity are required, ligase enzymes are often chosen, although they have a number of disadvantages, for example poor catalytic efficiency, low substrate specificity, and side reactions. Here, we describe a sequence-specific protein ligase with more favorable characteristics. This ligase, Connectase, is a monomeric homolog of 20S proteasome subunits in methanogenic archaea. In pulldown experiments with Methanosarcina mazei cell extract, we identify a physiological substrate in methyltransferase A (MtrA), a key enzyme of archaeal methanogenesis. Using microscale thermophoresis and X-ray crystallography, we show that only a short sequence of about 20 residues derived from MtrA and containing a highly conserved KDPGA motif is required for this high-affinity interaction. Finally, in quantitative activity assays, we demonstrate that this recognition tag can be repurposed to allow the ligation of two unrelated proteins. Connectase catalyzes such ligations at substantially higher rates, with higher yields, but without detectable side reactions when compared with a reference enzyme. It thus presents an attractive tool for the development of new methods, for example in the preparation of selectively labeled proteins for NMR, the covalent and geometrically defined attachment of proteins on surfaces for cryo-electron microscopy, or the generation of multispecific antibodies.

Analysis of GTP addition in the reverse (3'-5') direction by human tRNAHis guanylyltransferase.

Human tRNAHis guanylyltransferase (HsThg1) catalyzes the 3'-5' addition of guanosine triphosphate (GTP) to the 5'-end (-1 position) of tRNAHis, producing mature tRNAHis In human cells, cytoplasmic and mitochondrial tRNAHis have adenine (A) or cytidine (C), respectively, opposite to G-1 Little attention has been paid to the structural requirements of incoming GTP in 3'-5' nucleotidyl addition by HsThg1. In this study, we evaluated the incorporation efficiencies of various GTP analogs by HsThg1 and compared the reaction mechanism with that of Candida albicans Thg1 (CaThg1). HsThg1 incorporated GTP opposite A or C in the template most efficiently. In contrast to CaThg1, HsThg1 could incorporate UTP opposite A, and guanosine diphosphate (GDP) opposite C. These results suggest that HsThg1 could transfer not only GTP, but also other NTPs, by forming Watson-Crick (WC) hydrogen bonds between the incoming NTP and the template base. On the basis of the molecular mechanism, HsThg1 succeeded in labeling the 5'-end of tRNAHis with biotinylated GTP. Structural analysis of HsThg1 was also performed in the presence of the mitochondrial tRNAHis Structural comparison of HsThg1 with other Thg1 family enzymes suggested that the structural diversity of the carboxy-terminal domain of the Thg1 enzymes might be involved in the formation of WC base-pairing between the incoming GTP and template base. These findings provide new insights into an unidentified biological function of HsThg1 and also into the applicability of HsThg1 to the 5'-terminal modification of RNAs.

Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans.

The enzyme methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six posttranslationally modified amino acids near the active site. Recent studies have identified enzymes performing two of these modifications (thioglycine and 5-[S]-methylarginine), yet little is known about the formation and function of the remaining posttranslationally modified residues. Here, we provide in vivo evidence that a dedicated S-adenosylmethionine-dependent methyltransferase encoded by a gene we designated methylcysteine modification (mcmA) is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue might play a role in adaption to mesophilic conditions. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. High-resolution crystal structures of inactive MCR lacking the modified amino acids were indistinguishable from the fully modified enzyme, suggesting that interactions between the posttranslationally modified residues do not exert a major influence on the static structure of the enzyme but rather serve to fine-tune the activity and efficiency of MCR.

A versatile cis-prenyltransferase from Methanosarcina mazei catalyzes both C- and O-prenylations.

Polyprenyl groups, products of isoprenoid metabolism, are utilized in peptidoglycan biosynthesis, protein N-glycosylation, and other processes. These groups are formed by cis-prenyltransferases, which use allylic prenyl pyrophosphates as prenyl-donors to catalyze the C-prenylation of the general acceptor substrate, isopentenyl pyrophosphate. Repetition of this reaction forms (Z,E-mixed)-polyprenyl pyrophosphates, which are converted later into glycosyl carrier lipids, such as undecaprenyl phosphate and dolichyl phosphate. MM_0014 from the methanogenic archaeon Methanosarcina mazei is known as a versatile cis-prenyltransferase that accepts both isopentenyl pyrophosphate and dimethylallyl pyrophosphate as acceptor substrates. To learn more about this enzyme's catalytic activity, we determined the X-ray crystal structures of MM_0014 in the presence or absence of these substrates. Surprisingly, one structure revealed a complex with O-prenylglycerol, suggesting that the enzyme catalyzed the prenylation of glycerol contained in the crystallization buffer. Further analyses confirmed that the enzyme could catalyze the O-prenylation of small alcohols, such as 2-propanol, expanding our understanding of the catalytic ability of cis-prenyltransferases.

Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency.

Genetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

A Genetic Code Expansion-Derived Molecular Beacon for the Detection of Intracellular Amyloid-ß Peptide Generation.

Polypeptides generated from proteolytic processing of protein precursors, or proteolytic proteoforms, play an important role in diverse biological functions and diseases. However, their often-small size and intricate post-translational biogenesis preclude the use of simple genetic tagging in their cellular studies. Herein, we develop a labeling strategy for this class of proteoforms, based on residue-specific genetic code expansion labeling with a molecular beacon design. We demonstrate the utility of such a design by creating a molecular beacon reporter to detect amyloid-ß peptides, known to be involved in the pathogenesis of Alzheimer's disease, as they are produced from amyloid precursor protein (APP) along the endocytic pathway of living cells.

Probing the Active Site of Deubiquitinase USP30 with Noncanonical Tryptophan Analogues.

Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA have been evolved to generate genetically encoded noncanonical amino acids (ncAAs). Use of tryptophan (Trp) analogues with pyrrole ring modification for their spatial and polarity tuning in enzyme activity and substrate specificity is still limited. Herein, we report the application of an evolved PylRS, FOWRS2, for efficient incorporation of five Trp analogues into the deubiquitinase USP30 to decipher the role of W475 for diubiquitin selectivity. Structures of the five FOWRS-C/Trp analogue complexes at 1.7-2.5 Å resolution showed multiple ncAA binding modes. The W475 near the USP30 active site was replaced with Trp analogues, and the effect on the activity as well as the selectivity toward diubiquitin linkage types was examined. It was found that the Trp analogue with a formyl group attached to the nitrogen atom of the indole ring led to an improved activity of USP30 likely due to enhanced polar interactions and that another Trp analogue, 3-benzothienyl-l-alanine, induced a unique K6-specificity. Collectively, genetically encoded noncanonical Trp analogues by evolved PylRS·tRNACUAPyl pair unravel the spatial role of USP30-W475 in its diubiquitin selectivity.

Methanogenesis marker protein 10 (Mmp10) from Methanosarcina acetivorans is a radical S-adenosylmethionine methylase that unexpectedly requires cobalamin.

Methyl coenzyme M reductase (MCR) catalyzes the last step in the biological production of methane by methanogenic archaea, as well as the first step in the anaerobic oxidation of methane to methanol by methanotrophic archaea. MCR contains a number of unique post-translational modifications in its α subunit, including thioglycine, 1-N-methylhistidine, S-methylcysteine, 5-C-(S)-methylarginine, and 2-C-(S)-methylglutamine. Recently, genes responsible for the thioglycine and methylarginine modifications have been identified in bioinformatics studies and in vivo complementation of select mutants; however, none of these reactions has been verified in vitro Herein, we purified and biochemically characterized the radical S-adenosylmethionine (SAM) protein MaMmp10, the product of the methanogenesis marker protein 10 gene in the methane-producing archaea Methanosarcina acetivorans Using an array of approaches, including kinetic assays, LC-MS-based quantification, and MALDI TOF-TOF MS analyses, we found that MaMmp10 catalyzes the methylation of the equivalent of Arg285 in a peptide substrate surrogate, but only in the presence of cobalamin. We noted that the methyl group derives from SAM, with cobalamin acting as an intermediate carrier, and that MaMmp10 contains a C-terminal cobalamin-binding domain. Given that Mmp10 has not been annotated as a cobalamin-binding protein, these findings suggest that cobalamin-dependent radical SAM proteins are more prevalent than previously thought.

Methanosarcina acetivorans contains a functional ISC system for iron-sulfur cluster biogenesis.

BACKGROUND: The production of methane by methanogens is dependent on numerous iron-sulfur (Fe-S) cluster proteins; yet, the machinery involved in Fe-S cluster biogenesis in methanogens remains largely unknown. Methanogen genomes encode uncharacterized homologs of the core components of the ISC (IscS and IscU) and SUF (SufBC) Fe-S cluster biogenesis systems found in bacteria and eukaryotes. Methanosarcina acetivorans contains three iscSU and two sufCB gene clusters. Here, we report genetic and biochemical characterization of M. acetivorans iscSU2. RESULTS: Purified IscS2 exhibited pyridoxal 5'- phosphate-dependent release of sulfur from L-cysteine. Incubation of purified IscU2 with IscS2, cysteine, and iron (Fe2+) resulted in the formation of [4Fe-4S] clusters in IscU2. IscU2 transferred a [4Fe-4S] cluster to purified M. acetivorans apo-aconitase. IscU2 also restored the aconitase activity in air-exposed M. acetivorans cell lysate. These biochemical results demonstrate that IscS2 is a cysteine desulfurase and that IscU2 is a Fe-S cluster scaffold. M. acetivorans strain DJL60 deleted of iscSU2 was generated to ascertain the in vivo importance of IscSU2. Strain DJL60 had Fe-S cluster content and growth similar to the parent strain but lower cysteine desulfurase activity. Strain DJL60 also had lower intracellular persulfide content compared to the parent strain when cysteine was an exogenous sulfur source, linking IscSU2 to sulfur metabolism. CONCLUSIONS: This study establishes that M. acetivorans contains functional IscS and IscU, the core components of the ISC Fe-S cluster biogenesis system and provides the first evidence that ISC operates in methanogens.

Reactivity of [Fe4S4] Clusters toward C1 Substrates: Mechanism, Implications, and Potential Applications.

FeS proteins are metalloproteins prevalent in the metabolic pathways of most organisms, playing key roles in a wide range of essential cellular processes. A member of this protein family, the Fe protein of nitrogenase, is a homodimer that contains a redox-active [Fe4S4] cluster at the subunit interface and an ATP-binding site within each subunit. During catalysis, the Fe protein serves as the obligate electron donor for its catalytic partner, transferring electrons concomitant with ATP hydrolysis to the cofactor site of the catalytic component to enable substrate reduction. The effectiveness of Fe protein in electron transfer is reflected by the unique reactivity of nitrogenase toward small-molecule substrates. Most notably, nitrogenase is capable of catalyzing the ambient reduction of N2 and CO into NH4+ and hydrocarbons, respectively, in reactions that parallel the important industrial Haber-Bosch and Fischer-Tropsch processes. Other than participating in nitrogenase catalysis, the Fe protein also functions as an essential factor in nitrogenase assembly, which again highlights its capacity as an effective, ATP-dependent electron donor. Recently, the Fe protein of a soil bacterium, Azotobacter vinelandii, was shown to act as a reductase on its own and catalyze the ambient conversion of CO2 to CO at its [Fe4S4] cluster either under in vitro conditions when a strong reductant is supplied or under in vivo conditions through the action of an unknown electron donor(s) in the cell. Subsequently, the Fe protein of a mesophilic methanogenic organism, Methanosarcina acetivorans, was shown to catalyze the in vitro reduction of CO2 and CO into hydrocarbons under ambient conditions, illustrating an impact of protein scaffold on the redox properties of the [Fe4S4] cluster and the reactivity of the cluster toward C1 substrates. This reactivity was further traced to the [Fe4S4] cluster itself, as a synthetic [Fe4S4] compound was shown to catalyze the reduction of CO2 and CO to hydrocarbons in solutions in the presence of a strong reductant. Together, these observations pointed to an inherent ability of the [Fe4S4] clusters and, possibly, the FeS clusters in general to catalyze C1-substrate reduction. Theoretical calculations have led to the proposal of a plausible reaction pathway that involves the formation of hydrocarbons via aldehyde-like intermediates, providing an important framework for further mechanistic investigations of FeS-based activation and reduction of C1 substrates. In this Account, we summarize the recent work leading to the discovery of C1-substrate reduction by protein-bound and free [Fe4S4] clusters as well as the current mechanistic understanding of this FeS-based reactivity. In addition, we briefly discuss the evolutionary implications of this discovery and potential applications that could be developed to enable FeS-based strategies for the ambient recycling of unwanted C1 waste into useful chemical commodities.

An orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical amino acid mutagenesis in Escherichia coli.

We report the development of the orthogonal amber-suppressor pair Archaeoglobus fulgidus seryl-tRNA (Af-tRNASer)/Methanosarcina mazei seryl-tRNA synthetase (MmSerRS) in Escherichia coli. Furthermore, the crystal structure of MmSerRS was solved at 1.45 Å resolution, which should enable structure-guided engineering of its active site to genetically encode small, polar noncanonical amino acids (ncAAs).

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids.

In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as ß-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation.

Biochemical Characterization of the Methylmercaptopropionate:Cob(I)alamin Methyltransferase from Methanosarcina acetivorans.

Methanogenesis from methylated substrates is initiated by substrate-specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, the Methanosarcina acetivorans MtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate (MMPA):coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, with kcat of 0.315 s-1 and apparent Km for MPA of 12 µM. CoM did not serve as a methyl acceptor; thus, a second unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, was also demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine, and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were coexpressed. Biochemical characterization of this complex was not feasible, because the corrinoid cofactor of this complex was in the inactive Co(II) state and was not reactivated by incubation with strong reductants. The MtsF protein, composed of both corrinoid and methyltransferase domains, copurifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism.IMPORTANCE Methylmercaptopropionate (MMPA) is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady-state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in the methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate, methyl coenzyme M. Therefore, the additional enzymes required for methanogenic MMPA catabolism await discovery.

The l-Thr Kinase/l-Thr-Phosphate Decarboxylase (CobD) Enzyme from Methanosarcina mazei Gö1 Contains Metallocenters Needed for Optimal Activity.

The MM2060 (cobD) gene from Methanosarcina mazei strain Gö1 encodes a protein (MmCobD) with l-threonine kinase (PduX) and l-threonine-O-3-phosphate decarboxylase (CobD) activities. In addition to the unexpected l-Thr kinase activity, MmCobD has an extended carboxy-terminal (C-terminal) region annotated as a putative metal-binding zinc finger-like domain. Here, we demonstrate that the C-terminus of MmCobD is a ferroprotein containing ∼25 non-heme iron atoms per monomer of protein. The absence of the C-terminus substantially reduces, but does not abolish, enzymatic activities in vitro and in vivo. Single-residue substitutions of C-terminal putative Fe-binding cysteinyl and histidinyl residues resulted in the loss of Fe and changes in enzyme activity levels. Salmonella enterica ΔpduX and ΔcobD strains were used as heterologous hosts to assess coenzyme B12 biosynthesis as a function of 17 MmCobD variants tested. Some of the latter displayed 5-fold higher enzymatic activity in vitro and enhanced the growth rate of the S. enterica strains that synthesized them. Most of the MmCobD variants tested were up to 6-fold less active in vitro and supported slow growth rates of the S. enterica strains that synthesized them; some substitutions abolished enzyme activity. MmCobD exhibited an ultraviolet-visible absorption spectrum consistent with [4Fe-4S] clusters that appeared to be susceptible to oxidation by H2O2 and reduction by sodium dithionite. The presence of FeS clusters in MmCobD was corroborated by electron paramagnetic resonance and magnetic circular dichroism studies. Collectively, our results suggest that MmCobD contains one or more diamagnetic [4Fe-4S]2+ center(s) that may play a structural or regulatory role.

Toward a mechanistic and physiological understanding of a ferredoxin:disulfide reductase from the domains Archaea and Bacteria.

Disulfide reductases reduce other proteins and are critically important for cellular redox signaling and homeostasis. Methanosarcina acetivorans is a methane-producing microbe from the domain Archaea that produces a ferredoxin:disulfide reductase (FDR) for which the crystal structure has been reported, yet its biochemical mechanism and physiological substrates are unknown. FDR and the extensively characterized plant-type ferredoxin:thioredoxin reductase (FTR) belong to a distinct class of disulfide reductases that contain a unique active-site [4Fe-4S] cluster. The results reported here support a mechanism for FDR similar to that reported for FTR with notable exceptions. Unlike FTR, FDR contains a rubredoxin [1Fe-0S] center postulated to mediate electron transfer from ferredoxin to the active-site [4Fe-4S] cluster. UV-visible, EPR, and Mössbauer spectroscopic data indicated that two-electron reduction of the active-site disulfide in FDR involves a one-electron-reduced [4Fe-4S]1+ intermediate previously hypothesized for FTR. Our results support a role for an active-site tyrosine in FDR that occupies the equivalent position of an essential histidine in the active site of FTR. Of note, one of seven Trxs encoded in the genome (Trx5) and methanoredoxin, a glutaredoxin-like enzyme from M. acetivorans, were reduced by FDR, advancing the physiological understanding of FDR's role in the redox metabolism of methanoarchaea. Finally, bioinformatics analyses show that FDR homologs are widespread in diverse microbes from the domain Bacteria.