Putative S-Adenosyl Methionine Dependent Ironâ€Sulfur

Putative S-Adenosyl Methionine Dependent smoothing ironSulfurIdentification and Characterization of a Putative S-Adenosyl Methionine dependent IronSulfur containing protein fromMethanococcus JannaschiiQi LiuResearch ProposalDr. MishtuDeyDr. M. Lei GengDr. Leonard R. MacGillivrayDr. Amnon KohenDr. Daniel QuinnIntroductionBiological methane take ination is a microbial process that is catalyzed by microbes called methanogens, which belong to the third solid ground of life, the Archaea. Methane is formed at the final catalytic step by methyl-coenzyme M reductase (MCR), in which coenzyme B (CoBSH, N-7-mercaptoheptanoylthreonine) donates two electrons to reduce methyl-coenzyme M. MCR is a 300kDa enzyme, which is composed of three dissimilar subunits in an 222 arrangement and contains 2 mol of the nickel tetrapyrrole coenzyme F430, which atomic number 18 buried deeply within the protein complex1. There are five modify amino acids were found out on the -subunit and near the active site of MCR from methanothermobacter marburgensis based on the X-ray crystallographic studies. They are 1-N-methylhistitine (His400), 5-(S)-methylarginine (Arg271), 2-(S)-methylglutamine (Glu400), S-methylcysteine (Cys452), where the side chains are methylated and one thioglycine (Gly445) forming a thiopeptide bond ( telephone number 1). Since the DNA sequence of the encoding MCR gene shows no unsusal condons or unusual codon usages at the positions in which the five modified amino acids were found, the modifications are introduced after translation 1. According to vivo labeling experiments with L-(methyl-D3)-methionine, people found that the methyl groups from the modified amino acids are introduced biosynthetically from the methyl group of methionine by specialised S-adenosylmethionine (surface-to-air missile) dependent Figure1. Post Translational Modifications in MCRenzymes. These methyl translational modifications are catalyzed by protein methylases that specifically recognize the amino acid sequences up and downstream of amino acid to be methylated. The genome of methanogens has many open reading frame predicted to be putative methyltransferases, which also agrees with the proposal that there are four different SAM-dependent protein methylases are involved in the post translation modification. A search of six known methanogenic genomes led to the identification of keep open reading frame about the MCR gene cluster. Some methangenic archaea contain two MCR isoenzymes, designated MCR1 and MCR2. This conserved hypothetical protein is found around MCR1 in Methanococcus jannaschii and Methanobacterium thermobacter. The open reading frame from Methanococcus jannaschii, MJ0841 is annotated as a conserved hypothetical protein, which is found to be related to the radical SAM enzyme superfamily. The signature motif of SAM radical enzymes is three cysteine motif CX3CX2C (Figure 2), multiple sequence connective of the putative gene from methanogens show the conse rved CXGFCXXC, which is known to coordinate to 4Fe-4S cluster. (Figure 3)Figure 2. Multiple Sequence Alignment of MJ0841 Homologues from Different MethanogensFigure 3. 4Fe4S cluster coordinated by three-cysteine motif CxxxCxxC.The fourth iron of the cluster interacts with a bound SAM.Specific AimsIt is interesting and important to determine the function of this hypothetical protein. We speculate that this hypothetical protein, MJ0841, could be a possible candidate responsible for the post-translation modification of the methylated amino acids, or, is involved in the formation of the thioglycine in MCR.Research Plan, Results and DiscussionExpression and katharsis of MJ0841MJ0841, a 1248bp gene, was cloned into pET28a vector. The resulting plasmid was transformed in E.coli BL21(DE3) cells for gene expression. In order to increase the iron content, MJ0841 was also co-expressed with PDB1282. Overnight cell culture grown at 37oC in Luria-Bertani (LB) medium containing both kanamycin (50 ug/ml) and ampicillin (100ug/ml) was inoculated, in a 100-fold dilution, into Terrific Broth (TB) media aerobically at 37oC. FeCl3 was also added to a final concentration of 100uM to be as the iron content for iron-sulfur cluster during the growth when OD600 was around 0.3. Protein expression was thusly induced at OD600 of 0.6 to 0.7 with addition of Isopropyl -D-1-thiogalactopyranoside (IPTG) to final concentration of 0.5mM. After overnight incubation at 37oC around 18hours, the cells were harvested by centrifugation at 5000rpm for 30mins at 4oC, and stored at -80oC. The following procedures were all carried out in oxygen free environment at 20oC.Purification was conducted anaerobically at oxygen level always below 2.0ppm in anaerobic chamber. 23g Cell were moved into anaerobic chamber and resuspended in 120mL lysis buffer (50mM tris-HCl, 300mM NaCl, 5% glycerol pH 7.5), and Phenylmethanesulfonyl fluoride (PMSF) 1mM final concentration, 3 tablets of protease inhibitor, 2-mercapto methanol 10mM) for 15mins. The cells were lysed by sonication for 15mins followed by centrifugation at 30,000 rpm for 40mins at 4oC to remove the cell debris. The supernatant was applied to a packed 15mL Ni-NTA resin tug equilibrated with lysis buffer. The column was then washed with 5 column volume of wash buffer (50mM tris-HCl, 300mM NaCl, 10mM IMD, 5% glycerol, pH 7.5). The brownish protein was eluted by gradient elution with 5 column volume of wash buffer and 5 column volume elution buffer (50mM tris-HCl, 300mM NaCl, 200mM IMD, 5% glycerol, pH 7.5). SDS-PAGE was applied to analyze the in demand(p) clean protein fractions, which were then combined and banding for overnight dialysis with dialysis buffer (50mM tris-HCl, pH 7.5, 5% glycerol) with slow stirring. The pooled fractions were concentrated using an Amicon centrifugal filter with a 30kDa molecular weight cut off (MWCO). The collected protein was further purified with 20mL packed Q-sepharose column equilibrated with ly sis buffer (50mM tris-HCl, 5% glycerol, pH 7.5). The column was then washed with 5 column volume of wash buffer (50mM tris-HCl, 200mM NaCl, 5% glycerol, pH 7.5). The brownish protein was eluted by gradient elution with 5 column volume of wash buffer and 5 column volume elution buffer (50mM tris-HCl, 700mM NaCl, 5% glycerol, pH 7.5). SDS-PAGE was applied to analyze the desired clean protein fractions, which were then combined for reconstitution (Figure 4).Figure 4. SDS-PAGE gel electrophoresis analysis of MJ0841 elaboration fractionsReconstitution of the 4Fe-4S Cluster of MJ0841 in VitroThe above apo-protein (16uM, 40ml) was incubated with final concentration of 5mM DTT for 1h with slow stirring at room temperature. Then, cystein was added into the above solution by dropwise to reach 10 submarine folds excess of protein. After 30mins incubation, 10 molar excess of Fe(NH4)2(SO4)2 was added slowly to provide enough iron content for iron sulfur cluster and incubated for 30mins. The r esulting solution was incubated with 10 molar excess of Na2S finally, and the brownish protein solution changed to dark brown after adding Na2S. The above final protein solution was kept in 4oC overnight around 14 hours for building up enough 4Fe-4S clusters. In order to remove the unbounded iron and sulfur, the overnight reconstituted protein was concentrated to 2.5 3ml of final volume and loaded onto a 5mL PT10 column equilibrated with lysis buffer (50mM tris-HCl, 5% glycerol, pH 7.5), and the final pure protein was combined.Reduction of 4Fe-4S ClusterUV-vis spectroscopy was applied here for detecting the reduction of 4Fe-4S cluster. A characteristic acme for 4Fe-4S cluster was shown up near 412nm ahead reducing. Sodium Dithionite was used as the reducing agent and the stock solution was prepared freshly right before adding into the protein. 100 equivalents of sodium dithionite were mixed with concentrated protein, and the peak at 412nm was reduced (Figure 5). According to the results from UV-vis, the 4Fe-4S cluster was built up by reconstitution, also the 4Fe-4S2+ was reduced to 4Fe-4S+ by the reduction of sodium dithionite.Figure 5. UV-vis spectra of purified reconsituted MJ0841 Figure 6. EPR spectrum of as-isolated MJ0841(blue trace) and purified reconstituted MJ0841 originating from a 3Fe-4S+ cluster.reduced with 100 equiv sodium dithionite (red trace)EPR samples preparation and spectral collectionEPR spectrums of as-isolated, oxidate and reduced form of MJ0841 are detected at 10K. All samples are prepared anaerobically. As-isolated protein was prepared with the protein without reconstitution, and exhibits a strong isotropic EPR signal, which is centered at g=2.01 same as the g=2.01 signal of the 3Fe-4S+ cluster form (Figure 6). The oxidized form protein containing 4Fe-4S2+ was prepared by injecting 200ul concentrated protein purified through PD10 column into EPR tube, and it normally shows silent EPR signal (Figure 7). Reduced form of as-isolated p rotein was performed by mixing with 100 equivalents of sodium dithionite with 200ul concentrated protein, which gives the reduction form of 4Fe-4S+ cluster and shows the characteristic EPR signal with g factors of g=2.03 and g=1.92 (Figure 8). Figure 7. EPR spectrum of oxidized form of MJ0841. Figure 8. EPR spectrum of reduced form of MJ0841.SAM cleavage activity of MJ0841The characteristic reaction for detecting radical SAM enzymes is reductive cleavage of SAM into S-adenosylhomocysteine (SAH) and 5-deoxyadenosyl radical (5dAdo). Assays were conducted under strict anaerobic conditions. The reaction stoppage contains the following 50mM Tris-HCl, 5mM DTT, 5Mm sodium dithionite, 0.5mM SAM. Reactions were initiated by addition of SAM and carried out at 20oC for 20 hours. The control reaction was run under the same conditions as the above assay, but without presence of protein. Trifluoroacetic acid (TFA) (final concentration 5% v/v) was added to quench the reactions, which then were i dentified by HPLC analysis.HPLC analysis of SAM cleavage assay products After quenching by TFA, the reaction variety show was centrifuged and the supernatant was applied into HPLC analysis. 10ul of assay mixture was injected into C18 column, which had been pre-equilibrated with equilibrium buffer (40mM ammonium acetate, pH=6.2). Then the column was washed with a linear gradient from 0-50% acetonitrile for 30mins at room temperature to detect SAH and 5dAdo. The UV-detector was set at wavelength 258nm, and the standard samples, SAM, SAH, and 5dAdo, were run with the same condition as SAM cleavage reaction products.According to the retention time comparison in the midst of standard samples and products, formation of SAH and 5dAdo were all detected via HPLC analysis when enzyme was present. In the absence of enzyme MJ0841, SAM was not consumed at all and there were no any products peak formed, which confirmed the SAM was cleaved by enzyme. The dark blue, red, light blue traces show th e relative intensities of 5dAdo, SAH, and SAM standards. The green trace shows the assay with the use of reconstituted MJ0841, and the SAH and 5dAdo were both observed. The purple trace shows the control assay without MJ0841, and there was not any of 5dAdo formed (Figure 9).Figure 9. HPLC analysis of the SAM cleavage assaysConclusions Future WorkInitial results seem to show the 4Fe-4S cluster and the enzyme activity. Since SAM was cleaved enzymatically, the products will be detected by chain reactor spectroscopy to confirm the formation SAH and 5dAdo. Furthermore, probable substrates of MJ0841 will be prepared, which should contain the amino acids that would be modified. The activity assays with substrates will be examined to detect the desired methylation reaction on substrates.