Purification, kinetic characterization, and site-directed mutagenesis of Methanothermobacter thermautotrophicus RFAP Synthase Produced in Escherichia coli.

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, ß-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.

Substrate Specificity Analysis of Dihydrofolate/Dihydromethanopterin Reductase Homologs in Methylotrophic α-Proteobacteria.

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.

Identification of an Inhibitor of the Aminoglycoside 6'-N-Acetyltransferase type Ib [AAC(6')-Ib] by Glide Molecular Docking.

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.

Structure of the methanofuran/methanopterin-biosynthetic enzyme MJ1099 from Methanocaldococcus jannaschii.

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 Šresolution 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.

Structure of dihydromethanopterin reductase, a cubic protein cage for redox transfer.

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.

Discovery and characterization of the first archaeal dihydromethanopterin reductase, an iron-sulfur flavoprotein from Methanosarcina mazei.

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.

Chemical reduction of pterins to dihydropterins as substrates for enzymatic reactions.

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.

Novel dephosphotetrahydromethanopterin biosynthesis genes discovered via mutagenesis in Methylobacterium extorquens AM1.

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.

Biochemical characterization of a dihydromethanopterin reductase involved in tetrahydromethanopterin biosynthesis in Methylobacterium extorquens AM1.

During growth on one-carbon (C1) compounds, the aerobic alpha-proteobacterium Methylobacterium extorquens AM1 synthesizes the tetrahydromethanopterin (H4MPT) derivative dephospho-H4MPT as a C1 carrier in addition to tetrahydrofolate. The enzymes involved in dephospho-H4MPT biosynthesis have not been identified in bacteria. In archaea, the final step in the proposed pathway of H4MPT biosynthesis is the reduction of dihydromethanopterin (H2MPT) to H4MPT, a reaction analogous to the reaction of the bacterial dihydrofolate reductase. A gene encoding a dihydrofolate reductase homolog has previously been reported for M. extorquens and assigned as the putative H2MPT reductase gene (dmrA). In the present work, we describe the biochemical characterization of H2MPT reductase (DmrA), which is encoded by dmrA. The gene was expressed with a six-histidine tag in Escherichia coli, and the recombinant protein was purified by nickel affinity chromatography and gel filtration. Purified DmrA catalyzed the NAD(P)H-dependent reduction of H2MPT with a specific activity of 2.8 micromol of NADPH oxidized per min per mg of protein at 30 degrees C and pH 5.3. Dihydrofolate was not a substrate for DmrA at the physiological pH of 6.8. While the existence of an H2MPT reductase has been proposed previously, this is the first biochemical evidence for such an enzyme in any organism, including archaea. Curiously, no DmrA homologs have been identified in the genomes of known methanogenic archaea, suggesting that bacteria and archaea produce two evolutionarily distinct forms of dihydromethanopterin reductase. This may be a consequence of different electron donors, NAD(P)H versus reduced F420, used, respectively, in bacteria and methanogenic archaea.

Characterization of two methanopterin biosynthesis mutants of Methylobacterium extorquens AM1 by use of a tetrahydromethanopterin bioassay.

An enzymatic assay was developed to measure tetrahydromethanopterin (H(4)MPT) levels in wild-type and mutant cells of Methylobacterium extorquens AM1. H(4)MPT was detectable in wild-type cells but not in strains with a mutation of either the orf4 or the dmrA gene, suggesting a role for these two genes in H(4)MPT biosynthesis. The protein encoded by orf4 catalyzed the reaction of ribofuranosylaminobenzene 5'-phosphate synthase, the first committed step of H(4)MPT biosynthesis. These results provide the first biochemical evidence for H(4)MPT biosynthesis genes in bacteria.

Outcomes of a research-driven laboratory and literature course designed to enhance undergraduate contributions to original research.

This work describes outcomes of a research-driven advanced microbiology laboratory and literature research course intended to enhance undergraduate preparation for and contributions to original research. The laboratory section was designed to teach fundamental biochemistry and molecular biology techniques in the context of an original research project. Site-directed mutants of a gene of interest were constructed, and the effects of mutations on the resulting enzymes were analyzed. Students were also introduced to the literature surrounding their project, electronic literature databases, and preparation of computer-generated slides for oral presentations. Student progress was evaluated through a laboratory report written as scientific manuscript, an oral presentation, a 10-page written review, and an essay examination. In the semester following the laboratory course, four of the 14 undergraduates joined the host laboratory to continue their projects as individual undergraduate researchers. Quantifiable outcomes of the course and subsequent undergraduate research included i) production of eight new site-directed mutants and preliminary characterization of the corresponding enzymes, ii) training of four individual undergraduate researchers prior to joining the laboratory, iii) publication of a manuscript with results from two undergraduate researchers, and iv) presentation of two posters with undergraduate co-authors at a national meeting. This research-driven approach may be applicable to enhance undergraduate contributions to other original research projects that have defined goals achievable within the timeframe of a single semester.

Sex determination using PCR.

PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can distinguish between male and female samples and determine the gender of an unknown DNA donor. The exercise is presented for upper division undergraduate majors in microbiology, biochemistry, and molecular biology, but can be adapted to different academic levels and disciplines. The use of student samples in the exercise can enhance learning of these techniques by making PCR and agarose gel electrophoresis directly relevant to the students.

Targeting methanopterin biosynthesis to inhibit methanogenesis.

This paper describes the design, synthesis, and successful employment of inhibitors of 4-(beta-D-ribofuranosyl)aminobenzene-5'-phosphate (RFA-P) synthase, which catalyzes the first committed step in the biosynthesis of methanopterin, to specifically halt the growth of methane-producing microbes. RFA-P synthase catalyzes the first step in the synthesis of tetrahydromethanopterin, a key cofactor required for methane formation and for one-carbon transformations in methanogens. A number of inhibitors, which are N-substituted derivatives of p-aminobenzoic acid (pABA), have been synthesized and their inhibition constants with RFA-P synthase have been determined. Based on comparisons of the inhibition constants among various inhibitors, we propose that the pABA binding site in RFA-P synthase has a relatively large hydrophobic pocket near the amino group. These enzyme-targeted inhibitors arrest the methanogenesis and growth of pure cultures of methanogens. Supplying pABA to the culture relieves the inhibition, indicating a competitive interaction between pABA and the inhibitor at the cellular target, which is most likely RFAP synthase. The inhibitors do not adversely affect the growth of pure cultures of the bacteria (acetogens) that play a beneficial role in the rumen. Inhibitors added to dense ruminal fluid cultures (artificial rumena) halt methanogenesis; however, they do not inhibit volatile fatty acid (VFA) production and, in some cases, VFA levels are slightly elevated in the methanogenesis-inhibited cultures. We suggest that inhibiting methanopterin biosynthesis could be considered in strategies to decrease anthropogenic methane emissions, which could have an environmental benefit since methane is a potent greenhouse gas.

Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

HPLC assay for methylmalonyl-CoA epimerase.

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 2 R 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 8x 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.

Purification, overproduction, and partial characterization of beta-RFAP synthase, a key enzyme in the methanopterin biosynthesis pathway.

Methanopterin is a folate analog involved in the C1 metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria. Although a pathway for methanopterin biosynthesis has been described in methanogens, little is known about the enzymes and genes involved in the biosynthetic pathway. The enzyme beta-ribofuranosylaminobenzene 5'-phosphate synthase (beta-RFAP synthase) catalyzes the first unique step to be identified in the pathway of methanopterin biosynthesis, namely, the condensation of p-aminobenzoic acid with phosphoribosylpyrophosphate to form beta-RFAP, CO2, and inorganic pyrophosphate. The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding beta-RFAP synthase has not yet been identified. In the present work, we report on the purification to homogeneity of beta-RFAP synthase. The enzyme was purified from the methane-producing archaeon Methanosarcina thermophila, and the N-terminal sequence of the protein was used to identify corresponding genes from several archaea, including the methanogen Methanococcus jannaschii and the sulfate-reducing archaeon Archaeoglobus fulgidus. The putative beta-RFAP synthase gene from A. fulgidus was expressed in Escherichia coli, and the enzymatic activity of the recombinant gene product was verified. A BLAST search using the deduced amino acid sequence of the beta-RFAP synthase gene identified homologs in additional archaea and in a gene cluster required for C1 metabolism by the bacterium Methylobacterium extorquens. The identification of a gene encoding a potential beta-RFAP synthase in M. extorquens is the first report of a putative methanopterin biosynthetic gene found in the Bacteria and provides evidence that the pathways of methanopterin biosynthesis in Bacteria and Archaea are similar.

Biodegradation of Halogenated Hydrocarbon Fumigants by Nitrifying Bacteria.

Three species of nitrifying bacteria were tested for the ability to degrade the halocarbon fumigants methyl bromide, 1,2-dichloropropane, and 1,2-dibromo-3-chloropropane. The soil nitrifiers Nitrosomonas europaea and Nitrosolobus multiformis degraded all three fumigants, while the marine nitrifier Nitrosococcus oceanus degraded only methyl bromide under the conditions tested. Inhibition of biodegradation by allylthiourea and acetylene, specific inhibitors of ammonia monooxygenase, suggests that ammonia monooxygenase is the enzyme which catalyzes fumigant degradation.