Methane production by archaea depends on tetrahydromethanopterin (H 4 MPT), a pterin-containing cofactor that carries one-carbon units.Two redox reactions within the nine steps of H 4 MPT side chain biosynthesis have been hypothesized.Biochemical assays have demonstrated that the archaeal iron-sulfur flavoprotein dihydromethanopterin reductase X (DmrX or MM1854) catalyzes the final reaction of the pathway, the reduction of dihydromethanopterin to H 4 MPT, using dithiothreitol (DTT) as an artificial electron donor.The crystal structure of DmrB, a bacterial DmrX homolog that lacks iron-sulfur clusters, has led to a proposed ping-pong mechanism of electron transfer between FMNH 2 and the FMN prosthetic group of DmrB.However, an enzymatic assay to test the hypothetical DmrB mechanism is lacking because a suitable electron donor has not previously been identified.Furthermore, a second uncharacterized archaeal flavoprotein (MM1853) has been hypothesized to function in H 4 MPT side chain biosynthesis.In this work, to facilitate the development of assays to elucidate the functions of DmrB and MM1853, we tested a variety of electron donors, including dithiothreitol, ferredoxin, and a system consisting of NADH and an NADH-dependent flavin-reducing enzyme (Fre).Reduction of the DmrB prosthetic group (FMN) was measured as a decrease in absorbance at 460 nm.NADPH, NADH, and DTT were unable to reduce DmrB.However, NADH/Fre was able to reduce DmrB within 70 min (initial rate of 1.3 µM/min), providing the basis for a future DmrB activity assay.Carbon monoxide (CO)/CO dehydrogenase/ferredoxin reduced DmrB more rapidly within 6 min.Both electron transfer systems reduced a second flavin-containing archaeal protein MM1853, which is predicted to catalyze the third step of H 4 MPT biosynthe-How to cite this paper:

Methane-producing archaea are among a select group of microorganisms that utilize tetrahydromethanopterin (H4MPT) as a one-carbon carrier instead of tetrahydrofolate. In H4MPT biosynthesis, β-ribofuranosylaminobenzene 5'-phosphate (RFAP) synthase catalyzes the production of RFAP, CO2, and pyrophosphate from p-aminobenzoic acid (pABA) and phosphoribosyl-pyrophosphate (PRPP). In this work, to gain insight into amino acid residues required for substrate binding, RFAP synthase from Methanothermobacter thermautotrophicus was produced in Escherichia coli, and site-directed mutagenesis was used to alter arginine 26 (R26) and aspartic acid 19 (D19), located in a conserved sequence of amino acids resembling the pABA binding site of dihydropteroate synthase. Replacement of R26 with lysine increased the KM for pABA by an order of magnitude relative to wild-type enzyme without substantially altering the KM for PRPP. Although replacement of D19 with alanine produced inactive enzyme, asparagine substitution allowed retention of some activity, and the K M for pABA increased about threefold relative to wild-type enzyme. A molecular model developed by threading RFAP synthase onto the crystal structure of homoserine kinase places R26 in the proposed active site. In the static model, D19 is located close to the active site, yet appears too far away to influence ligand binding directly. This may be indicative of the protein conformational change predicted previously in the Bi-Ter kinetic mechanism and/or formation of the active site at the interface of two subunits. Due to the vital role of RFAP synthase in H4MPT biosynthesis, insights into the mode of substrate binding and mechanism could be beneficial for developing RFAP synthase inhibitors designed to reduce the production of methane as a greenhouse gas.

Methane-producing archaea and methylotrophic bacteria use tetrahydromethanopterin (H4MPT) and/or tetrahydrofolate (H4F) as coenzymes in one-carbon (C1) transfer pathways. The α-proteobacterium Methylobacterium extorquens AM1 contains a dihydromethanopterin reductase (DmrA) and two annotated dihydrofolate reductases (DfrA and DfrB). DmrA has been shown to catalyze the final step of H4MPT biosynthesis; however, the functions of DfrA and DfrB have not been examined biochemically. Moreover, sequence alignment (BLAST) searches have recognized scores of proteins that share up to 99% identity with DmrA but are annotated as diacylglycerol kinases (DAGK). In this work, we used bioinformatics and enzyme assays to provide insight into the phylogeny and substrate specificity of selected Dfr and DmrA homologs. In a phylogenetic tree, DmrA and homologs annotated as DAGKs grouped together in one clade. Purified histidine-tagged versions of the annotated DAGKs from Hyphomicrobium nitrativorans and M. nodulans (respectively, sharing 69 and 84% identity with DmrA) showed only low activity in phosphorylating 1,2-dihexanoyl-sn-glycerol when compared with a commercial DAGK from Escherichia coli. However, the annotated DAGKs successfully reduced a dihydromethanopterin analog (dihydrosarcinapterin, H2SPT) with kinetic values similar to those determined for M. extorquens AM1 DmrA. DfrA and DfrB showed little or no ability to reduce H2SPT under the conditions studied; however, both catalyzed the NADPH-dependent reduction of dihydrofolate. These results provide the first evidence that DfrA and DfrB function as authentic dihydrofolate reductases, while DAGKs with greater than 69% identity to DmrA may be misannotated and are likely to function in H4MPT biosynthesis.

The attempt to elucidate the biological function of enzymes involved in the biosynthesis of tetrahydromethanopterin for the use in one‐carbon metabolism by archaea and select bacteria has resulted in the crystal structure of the hypothetical protein A2617 from Methylibium petroleiphilum. The structure was initially solved at 2.0 Å resolution using a selenomethionine multi‐wavelength anomalous diffraction experiment, and further refined at 1.8 Å resolution using a native dataset. The Rfactor of the current model is 0.19 and the Rfree is 0.22. The crystal structure of A2617 is a homodimer with each monomer consisting of four α‐helices connected to a barrel fold of six anti‐parallel β‐sheets with an α‐helix by a pair of anti‐parallel β‐sheets. The monomers interface to form the homodimer at the region containing four α‐helices with the barrel folds flanking both ends of the structure. A deep cleft is present at the interface of the two monomers. The overall structure of A2617 resembles that of uncharacterized archaeal homologs; however, the structure is distinct due to the lack of bound FMN in the binding pocket that is found in the archaeal homolog crystal structures. Ligand docking studies using intermediates in the tetrahydromethanopterin biosynthetic pathway indicated that A2617 has the greatest binding affinity for 6‐hydroxymethyl‐7,8‐dihydropterin‐ribofuranosylaminobenzene‐5‐phosphate. This metabolite is the substrate for the third step of tetrahydromethanopterin side chain biosynthesis, which is an oxidoreductase reaction. The homology of A2617 with FMN‐containing archaeal proteins and the findings of the ligand binding studies provide support for the role of A2617 in catalyzing a reductive reaction in the biosynthesis of the tetrahydromethanopterin side chain.Support or Funding InformationThis research was supported by National Science Foundation grant number CHE‐1508801 and by a grant from the California State University Program for Education and Research in Biotechnology (CSUPERB).

The aminoglycoside 6'-N-acetyltransferase type Ib, AAC(6')-Ib, confers resistance to clinically relevant aminoglycosides and is the most widely distributed enzyme among AAC(6')-I-producing Gram-negative pathogens. An alternative to counter the action of this enzyme is the development of inhibitors. Glide is a computational strategy for rapidly docking ligands to protein sites and estimating their binding affinities. We docked a collection of 280,000 compounds from 7 sub-libraries of the Chembridge library as ligands to the aminoglycoside binding site of AAC(6')-Ib. We identified a compound, 1-[3-(2-aminoethyl)benzyl]-3-(piperidin-1-ylmethyl)pyrrolidin-3-ol (compound 1), that inhibited the acetylation of aminoglycosides in vitro with IC50 values of 39.7 and 34.9 µM when the aminoglycoside substrates assayed were kanamycin A or amikacin, respectively. The growth of an amikacin-resistant Acinetobacter baumannii clinical strain was inhibited in the presence of a combination of amikacin and compound 1.

Prior studies have indicated that MJ1099 from Methanocaldococcus jannaschii has roles in the biosynthesis of tetrahydromethanopterin and methanofuran, two key cofactors of one-carbon (C1) metabolism in diverse organisms including the methanogenic archaea. Here, the structure of MJ1099 has been solved to 1.7 Å resolution using anomalous scattering methods. The results indicate that MJ1099 is a member of the TIM-barrel superfamily and that it is a homohexamer. Bioinformatic analyses identified a potential active site that is highly conserved among MJ1099 homologs and the key amino acids involved were identified. The results presented here should guide further studies of MJ1099 including mechanistic studies and possibly the development of inhibitors that target the methanogenic archaea in the digestive tracts of humans and that are a source of the greenhouse gas methane.

Dihydromethanopterin reductase (Dmr) is a redox enzyme that plays a key role in generating tetrahydromethanopterin (H4MPT) for use in one-carbon metabolism by archaea and some bacteria. DmrB is a bacterial enzyme understood to reduce dihydromethanopterin (H2MPT) to H4MPT using flavins as the source of reducing equivalents, but the mechanistic details have not been elucidated previously. Here we report the crystal structure of DmrB from Burkholderia xenovorans at a resolution of 1.9 Å. Unexpectedly, the biological unit is a 24-mer composed of eight homotrimers located at the corners of a cubic cage-like structure. Within a homotrimer, each monomer-monomer interface exhibits an active site with two adjacently bound flavin mononucleotide (FMN) ligands, one deeply buried and tightly bound and one more peripheral, for a total of 48 ligands in the biological unit. Computational docking suggested that the peripheral site could bind either the observed FMN (the electron donor for the overall reaction) or the pterin, H2MPT (the electron acceptor for the overall reaction), in configurations ideal for electron transfer to and from the tightly bound FMN. On this basis, we propose that DmrB uses a ping-pong mechanism to transfer reducing equivalents from FMN to the pterin substrate. Sequence comparisons suggested that the catalytic mechanism is conserved among the bacterial homologs of DmrB and partially conserved in archaeal homologs, where an alternate electron donor is likely used. In addition to the mechanistic revelations, the structure of DmrB could help guide the development of anti-obesity drugs based on modification of the ecology of the human gut.

The microbial production of methane by methanogenic archaea is dependent on the synthesis of the pterin-containing cofactor tetrahydromethanopterin (H4MPT). The enzyme catalyzing the last step of H4MPT biosynthesis (dihydromethanopterin reductase) has not previously been identified in methane-producing microorganisms. Previous complementation studies with the methylotrophic bacterium Methylobacterium extorquens have indicated that an uncharacterized archaeal-flavoprotein-like flavoprotein (AfpA) from Methylobacillus flagellatus or Burkholderia xenovorans can replace the activity of a phylogenetically unrelated bacterial dihydromethanopterin reductase (DmrA). We propose that MM1854, a homolog of AfpA from Methanosarcina mazei, catalyzes the last step of H4MPT biosynthesis in methane-producing microorganisms. To test this hypothesis, a six-histidine (His6)-tagged version of MM1854 was produced. Bioinformatic analysis revealed the presence of one flavin mononucleotide (FMN)-binding site and two iron-sulfur cluster sites, consistent with an oxidoreductase enzyme. Purified His6-MM1854 occurred as a homodimer of 29-kDa subunits, and the UV-visible spectrum of the purified protein showed absorbance peaks at 380 and 460 nm, characteristic of oxidized FMN. NAD(P)H was incapable of directly reducing the flavin cofactor, but dithionite eliminated the FMN peaks, indicating successful electron transfer to MM1854. An electron transfer system of NADPH, spinach NADPH-ferredoxin oxidoreductase, and ferredoxin could also reduce the FMN peaks. A newly developed assay indicated that dithiothreitol-reduced MM1854 could transfer electrons to dihydromethanopterin. This assay was also effective with a heat-stable DmrX analog from Methanocaldococcus jannaschii (MJ0208). These results provide the first biochemical evidence that MM1854 and MJ0208 function as archaeal dihydromethanopterin reductases (DmrX) and that ferredoxin may serve as an electron donor.

Fragestellung: Die hypoglykämieassoziierte Mortalität bei Patienten mit Diabetes wird teilweise auf kardiale Rhythmusstörungen mit Sekundenherztod (sog. „Dead-in-bed Syndrome“) zurückgeführt. Rasch einsetzende Hypoglykämien können über Elektrolytverschiebungen und neurohumorale Mechanismen potentiell zur elektrischen Instabilität des Myokards beitragen, deren Kinetik nur unzureichend bekannt ist. In einer prospektiven, interventionellen Studie wurden die Effekte der akuten Hypoglykämie systematisch analysiert.

Fragestellung: Das kontinuierliche Glukosemonitoring (CGM) mittels Sensor wird zunehmend zur Therapieoptimierung bei Patienten mit Diabetes verwendet. Schnell einsetzende Hypoglykämien werden durch subkutane Glukosesensoren potentiell nur mit Verzögerung (time-lag) erkannt. Ziel der aktuellen Studie ist, die Kinetik der Glukosekonzentrationen bei kapillärer Point-of-Care Messung, Referenz-Plasmamessung und Sensormessung während einer insulininduzierten Hypoglykämie zu charakterisieren und mit hormonellen und klinischen Parametern zu korrelieren.

Dihydropterins are important intermediates in various metabolic pathways, including the biosynthesis of tetrahydrofolate and tetrahydromethanopterin, a key coenzyme in the one-carbon metabolism of methanogenic Archaea. Some procedures for the reduction of pterins to dihydropterins may produce undesirable tetrahydropterin contaminants. This work describes a procedure for the rapid reduction of pterins to dihydropterins while minimizing tetrahydropterin production that may be particularly useful in producing substrates for enzyme reactions when the dihydropterin substrate cannot be purchased commercially.

ABSTRACT Methylobacterium extorquens AM1 was used to explore the genetics of dephosphotetrahydromethanopterin (dH 4 MPT) biosynthesis. Strains with mutations in eight “archaeal-type” genes linked on the chromosome of M. extorquens AM1 were analyzed for the ability to synthesize dH 4 MPT, and six were found to be dH 4 MPT negative. Putative functions of these genes in dH 4 MPT biosynthesis are discussed.

Methylmalonyl-CoA epimerase (MCE) from the hyperthermophilic archaeon, Pyrococcus horikoshii, was expressed at high levels in Escherichia coli, purified, and partially characterized. The P. horikoshii MCE enzyme was a homodimer with an apparent molecular mass of 31,700 Da. The K m of the enzyme for methylmalonyl-CoA was 79 μM and the k cat was 240 s−1. The P. horikoshii enzyme was extremely heat-stable and withstood boiling for 60 min without detectable loss in activity.

Methylmalonyl–CoA epimerase (MCE) is broadly distributed in nature and has diverse cellular roles. Many MCE homologues are represented in public databases, but the biochemical function and physiological roles of the majority of these putative proteins have not been investigated. Here, a simplified assay for MCE is described. In this assay, MCE converted (2S)-methylmalonyl–CoA to (2R)-methylmalonyl–CoA which in turn was converted to succinyl–CoA by methylmalonyl–CoA mutase, an enzyme specific for the 2R isomer. MCE activity was quantified by measuring the disappearance of methylmalonyl–CoA by HPLC. To obtain the methylmalonyl–CoA mutase which was required as a reagent for the assay, an Escherichia coli strain was constructed that expressed high levels of this enzyme as a fusion protein with an 8× histidine tag. This allowed purification of the mutase in a single affinity chromatography step. Previously reported MCE assays required radioactive substrates and/or multiple reagent enzymes that were difficult to obtain. The assay reported here overcomes these difficulties and hence will facilitate studies of MCEs. Such enzymes play important roles in the metabolism of both prokaryotes and higher eukaryotes including humans.