Discovery, structure and mechanism of a tetraether lipid synthase.

Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.

Efficient Incorporation of Clickable Unnatural Amino Acids Enables Rapid and Biocompatible Labeling of Proteins in Vitro and in Bacteria.

Site-specific incorporation of unnatural amino acids (uAAs) bearing a bioorthogonal group has enabled the attachment - typically at a single site or at a few sites per protein - of chemical groups at precise locations for protein and biomaterial labeling, conjugation, and functionalization. Herein, we report the evolution of chromosomal Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (aaRS) for the alkyne-bearing uAA, 4-propargyloxy-l-phenylalanine (pPR), with ∼30-fold increased production of green fluorescent protein containing three instances of pPR compared with a previously described M. jannaschii-derived aaRS for pPR, when expressed from a single chromosomal copy. We show that when expressed from multicopy plasmids, the evolved aaRSs enable the production - using a genomically recoded Escherichia coli and the non-recoded BL21 E. coli strain - of elastin-like polypeptides (ELPs) containing multiple pPR residues in high yields. We further show that the multisite incorporation of pPR in ELPs facilitates the rapid, robust, and nontoxic fluorescent labeling of these proteins in bacteria. The evolved variants described in this work can be used to produce a variety of protein and biomaterial conjugates and to create efficient minimal tags for protein labeling.

A substrate binding model for the KEOPS tRNA modifying complex.

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.

The Elongator subunit Elp3 is a non-canonical tRNA acetyltransferase.

The Elongator complex catalyzes posttranscriptional tRNA modifications by attaching carboxy-methyl (cm5) moieties to uridine bases located in the wobble position. The catalytic subunit Elp3 is highly conserved and harbors two individual subdomains, a radical S-adenosyl methionine (rSAM) and a lysine acetyltransferase (KAT) domain. The details of its modification reaction cycle and particularly the substrate specificity of its KAT domain remain elusive. Here, we present the co-crystal structure of bacterial Elp3 (DmcElp3) bound to an acetyl-CoA analog and compare it to the structure of a monomeric archaeal Elp3 from Methanocaldococcus infernus (MinElp3). Furthermore, we identify crucial active site residues, confirm the importance of the extended N-terminus for substrate recognition and uncover the specific induction of acetyl-CoA hydrolysis by different tRNA species. In summary, our results establish the clinically relevant Elongator subunit as a non-canonical acetyltransferase and genuine tRNA modification enzyme.

TYW1: A Radical SAM Enzyme Involved in the Biosynthesis of Wybutosine Bases.

Transfer RNA is extensively modified by the actions of a variety of enzymes. The radical S-adenosyl-l-methionine enzyme TYW1 modifies tRNAPhe forming the characteristic tricyclic ring via the condensation of carbons 2 and 3 of pyruvate. This chapter details methods that are required for studies of TYW1.

Identification of the Radical SAM Enzymes Involved in the Biosynthesis of Methanopterin and Coenzyme F420 in Methanogens.

Methanogenic archaea represent a source of unique and fascinating anaerobic biochemistry that includes the involvement of many radical S-adenosyl-l-methionine (SAM) enzymes, some of which have well-established functions, while the majority have currently unknown or only partially understood functions. Here, we describe our strategy for the identification of the radical SAM enzyme that catalyzes the two methylation reactions in methanopterin biosynthesis in Methanocaldococcus jannaschii. Additionally, we describe the similar strategy carried out for the identification of the two radical SAM enzymes required for the biosynthesis of the 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) moiety of coenzyme F420 in M. jannaschii. This approach can be employed for future functional identification of radical SAM enzymes with currently unknown functions.

Promiscuity of methionine salvage pathway enzymes in Methanocaldococcus jannaschii.

The methionine salvage pathway (MSP) is critical for regeneration of S-adenosyl-l-methionine (SAM), a widely used cofactor involved in many essential metabolic reactions. The MSP has been completely elucidated in aerobic organisms, and found to rely on molecular oxygen. Since anaerobic organisms do not use O2, an alternative pathway(s) must be operating. We sought to evaluate whether the functions of two annotated MSP enzymes from Methanocaldococcus jannaschii, a methylthioinosine phosphorylase (MTIP) and a methylthioribose 1-phosphate isomerase (MTRI), are consistent with functioning in a modified anaerobic MSP (AnMSP). We show here that recombinant MTIP is active with six different purine nucleosides, consistent with its function as a general purine nucleoside phosphorylase for both AnMSP and purine salvage. Recombinant MTRI is active with both 5-methylthioribose 1-phosphate and 5-deoxyribose 1-phosphate as substrates, which are generated from phosphororolysis of 5'-methylthioinosine and 5'-deoxyinosine by MTIP, respectively. Together, these data suggest that MTIP and MTRI may function in a novel pathway for recycling the 5'-deoxyadenosine moiety of SAM in M. jannaschii. These enzymes may also enable biosynthesis of 6-deoxy-5-ketofructose 1-phosphate (DKFP), an essential intermediate in aromatic amino acid biosynthesis. Finally, we utilized a homocysteine auxotrophic strain of Methanosarcina acetivorans Δma1821-22Δoahs (HcyAux) to identify potential AnMSP intermediates in vivo. Growth recovery experiments of the M. acetivorans HcyAux were performed with known and proposed intermediates for the AnMSP. Only one metabolite, 2-keto-(4-methylthio)butyric acid, rescued growth of M. acetivorans HcyAux in the absence of homocysteine. This observation may indicate that AnMSP pathways substantially differ among methanogens from phylogenetically divergent genera.

Driving Forces of Translocation Through Bacterial Translocon SecYEG.

This review focusses on the energetics of protein translocation via the Sec translocation machinery. First we complement structural data about SecYEG's conformational rearrangements by insight obtained from functional assays. These include measurements of SecYEG permeability that allow assessment of channel gating by ligand binding and membrane voltage. Second we will discuss the power stroke and Brownian ratcheting models of substrate translocation and the role that the two models assign to the putative driving forces: (i) ATP (SecA) and GTP (ribosome) hydrolysis, (ii) interaction with accessory proteins, (iii) membrane partitioning and folding, (iv) proton motive force (PMF), and (v) entropic contributions. Our analysis underlines how important energized membranes are for unravelling the translocation mechanism in future experiments.

Structural basis for tRNA-dependent cysteine biosynthesis.

Cysteine can be synthesized by tRNA-dependent mechanism using a two-step indirect pathway, where O-phosphoseryl-tRNA synthetase (SepRS) catalyzes the ligation of a mismatching O-phosphoserine (Sep) to tRNACys followed by the conversion of tRNA-bounded Sep into cysteine by Sep-tRNA:Cys-tRNA synthase (SepCysS). In ancestral methanogens, a third protein SepCysE forms a bridge between the two enzymes to create a ternary complex named the transsulfursome. By combination of X-ray crystallography, SAXS and EM, together with biochemical evidences, here we show that the three domains of SepCysE each bind SepRS, SepCysS, and tRNACys, respectively, which mediates the dynamic architecture of the transsulfursome and thus enables a global long-range channeling of tRNACys between SepRS and SepCysS distant active sites. This channeling mechanism could facilitate the consecutive reactions of the two-step indirect pathway of Cys-tRNACys synthesis (tRNA-dependent cysteine biosynthesis) to prevent challenge of translational fidelity, and may reflect the mechanism that cysteine was originally added into genetic code.

Dynamic distinctions in the Na+/Ca2+ exchanger adopting the inward- and outward-facing conformational states.

Na+/Ca2+ exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na+ or Ca2+ specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.

A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion.

The conserved SecYEG protein-conducting channel and the accessory proteins SecDF-YajC and YidC constitute the bacterial holo-translocon (HTL), capable of protein-secretion and membrane-protein insertion. By employing an integrative approach combining small-angle neutron scattering (SANS), low-resolution electron microscopy and biophysical analyses we determined the arrangement of the proteins and lipids within the super-complex. The results guided the placement of X-ray structures of individual HTL components and allowed the proposal of a model of the functional translocon. Their arrangement around a central lipid-containing pool conveys an unexpected, but compelling mechanism for membrane-protein insertion. The periplasmic domains of YidC and SecD are poised at the protein-channel exit-site of SecY, presumably to aid the emergence of translocating polypeptides. The SecY lateral gate for membrane-insertion is adjacent to the membrane 'insertase' YidC. Absolute-scale SANS employing a novel contrast-match-point analysis revealed a dynamic complex adopting open and compact configurations around an adaptable central lipid-filled chamber, wherein polytopic membrane-proteins could fold, sheltered from aggregation and proteolysis.

Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation.

The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.

Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase.

Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a kcat /KM of 3.2 × 10(7) and 3.0 × 10(7) s(- 1) M(- 1) , respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site-directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10-fold decrease in kcat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828-840. © 2016 Wiley Periodicals, Inc.

From Suicide Enzyme to Catalyst: The Iron-Dependent Sulfide Transfer in Methanococcus jannaschii Thiamin Thiazole Biosynthesis.

Bacteria and yeast utilize different strategies for sulfur incorporation in the biosynthesis of the thiamin thiazole. Bacteria use thiocarboxylated proteins. In contrast, Saccharomyces cerevisiae thiazole synthase (THI4p) uses an active site cysteine as the sulfide source and is inactivated after a single turnover. Here, we demonstrate that the Thi4 ortholog from Methanococcus jannaschii uses exogenous sulfide and is catalytic. Structural and biochemical studies on this enzyme elucidate the mechanistic details of the sulfide transfer reactions.

Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii.

GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/ß fold consisting of seven ß-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time.

S-Inosyl-L-Homocysteine Hydrolase, a Novel Enzyme Involved in S-Adenosyl-L-Methionine Recycling.

UNLABELLED: S-Adenosyl-L-homocysteine, the product of S-adenosyl-L-methionine (SAM) methyltransferases, is known to be a strong feedback inhibitor of these enzymes. A hydrolase specific for S-adenosyl-L-homocysteine produces L-homocysteine, which is remethylated to methionine and can be used to regenerate SAM. Here, we show that the annotated S-adenosyl-L-homocysteine hydrolase in Methanocaldococcus jannaschii is specific for the hydrolysis and synthesis of S-inosyl-L-homocysteine, not S-adenosyl-L-homocysteine. This is the first report of an enzyme specific for S-inosyl-L-homocysteine. As with S-adenosyl-L-homocysteine hydrolase, which shares greater than 45% sequence identity with the M. jannaschii homologue, the M. jannaschii enzyme was found to copurify with bound NAD(+) and has Km values of 0.64 ± 0.4 mM, 0.0054 ± 0.006 mM, and 0.22 ± 0.11 mM for inosine, L-homocysteine, and S-inosyl-L-homocysteine, respectively. No enzymatic activity was detected with S-adenosyl-L-homocysteine as the substrate in either the synthesis or hydrolysis direction. These results prompted us to redesignate the M. jannaschii enzyme an S-inosyl-L-homocysteine hydrolase (SIHH). Identification of SIHH demonstrates a modified pathway in this methanogen for the regeneration of SAM from S-adenosyl-L-homocysteine that uses the deamination of S-adenosyl-L-homocysteine to form S-inosyl-L-homocysteine. IMPORTANCE: In strictly anaerobic methanogenic archaea, such as Methanocaldococcus jannaschii, canonical metabolic pathways are often not present, and instead, unique pathways that are deeply rooted on the phylogenetic tree are utilized by the organisms. Here, we discuss the recycling pathway for S-adenosyl-L-homocysteine, produced from S-adenosyl-L-methionine (SAM)-dependent methylation reactions, which uses a hydrolase specific for S-inosyl-L-homocysteine, an uncommon metabolite. Identification of the pathways and the enzymes involved in the unique pathways in the methanogens will provide insight into the biochemical reactions that were occurring when life originated.

Identification of a unique radical S-adenosylmethionine methylase likely involved in methanopterin biosynthesis in Methanocaldococcus jannaschii.

Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-L-methionine (SAM) enzyme superfamily member encoded by the MJ0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX3CX2C motifs present in MJ0619 did not inhibit C-7 methylation, while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in the loss of C-7 methylation, suggesting that the first motif could be involved in C-9 methylation, while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and that methylation does not require cobalamin. When E. coli cells expressing MJ0619 were grown with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor.

A possible iron delivery function of the dinuclear iron center of HcgD in [Fe]-hydrogenase cofactor biosynthesis.

HcgD, a homolog of the ubiquitous Nif3-like protein family, is found in a gene cluster involved in the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase. The presented crystal structure and biochemical analyses indicated that HcgD has a dinuclear iron-center, which provides a pronounced binding site for anionic ligands. HcgD contains a stronger and a weaker bound iron; the latter being removable by chelating reagents preferentially in the oxidized state. Therefore, we propose HcgD as an iron chaperone in FeGP cofactor biosynthesis, which might also stimulate investigations on the functionally unknown but physiologically important eukaryotic Nif3-like protein family members.

Identification of a 5'-deoxyadenosine deaminase in Methanocaldococcus jannaschii and its possible role in recycling the radical S-adenosylmethionine enzyme reaction product 5'-deoxyadenosine.

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5'-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5'-deoxyadenosine to 5'-deoxyinosine as its major product but will also deaminate 5'-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5'-deoxyadenosine deaminase (DadD). The Km for 5'-deoxyadenosine was found to be 14.0 ± 1.2 µM with a kcat/Km of 9.1 × 10(9) M(-1) s(-1). Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5'-deoxyadenosine. Since 5'-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5'-deoxyadenosine, whereupon the 5'-deoxyribose moiety of 5'-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.

Hydrolysis at one of the two nucleotide-binding sites drives the dissociation of ATP-binding cassette nucleotide-binding domain dimers.

The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides "sandwiched" at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.