Putative S-Adenosyl Methionine Dependent Ironâ€Sulfur

Putative S-Adenosyl Methionine Dependent Iron–Sulfur Identification and Characterization of a Putative S-Adenosyl Methionine dependent Iron–Sulfur containing protein from  Methanococcus Jannaschii Qi Liu Research Proposal Dr. MishtuDey Dr. M. Lei Geng Dr. Leonard R. MacGillivray Dr. Amnon Kohen Dr. Daniel Quinn Introduction Biological methane formation is a microbial process that is catalyzed by microbes called methanogens, which belong to the third kingdom 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 different subunits in an ÃŽ ±2ÃŽ ²2ÃŽ ³2 arrangement and contains 2 mol of the nickel tetrapyrrole coenzyme F430, which are buried deeply within the protein complex[1]. There are five modified amino acids were found out on the ÃŽ ±-subunit and near the active site of MCR from methanothermobacter marburgensis based on the X-ray crystallographic studies. They are 1-N-methylhistitine (HisÃŽ ±400), 5-(S)-methylarginine (ArgÃŽ ±271), 2-(S)-methylglutamine (GluÃŽ ±400), S-methylcysteine (CysÃŽ ±452), where the side chains are methylated and one thioglycine (GlyÃŽ ±445) forming a thiopeptide bo nd (Figure 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 specific S-adenosylmethionine (SAM) dependent Figure1. Post Translational Modifications in MCR  enzymes. 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 kn own methanogenic genomes led to the identification of conserved open reading frame around 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 â€Å"CX3CX2C† (Figure 2), multiple sequence alignment of the putative gene from methanogens show the conserved CXGFCXXC, which is known to coordinate to [4Fe-4S] cluster. (Figure 3) Figure 2. Multiple Sequence Alignment of MJ0841 Homologues from Different Methanogens Figure 3. [4Fe–4S] cluster coordinated by three-cysteine motif CxxxCxxC. The fourth iron of the cluster interacts with a bound SAM. Specific Aims It 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 Discussion Expression and Purification of MJ0841 MJ0841, 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 (50ug/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 then induced at OD600 of 0.6 to 0.7 with addition of Isopropyl ÃŽ ²-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-mercaptomethanol 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 column 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 desired clean protein fractions, which were then combined an d set 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 lysis 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 purification fractions Reconstitution of the [4Fe-4S] Cluster of MJ0841 in Vitro The 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 molar 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 resulting 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] Cluster UV-vis spectroscopy was applied here for detecting the reduction of [4Fe-4S] cluster. A characteristic peak for [4Fe-4S] cluster was shown up near 412nm before 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-4S]2+ 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 collection EPR spectrums of as-isolated, oxidized 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-4S]2+ 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 protein 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 MJ0841 The characteristic reaction for detecting radical SAM enzymes is reductive cleavage of SAM into S-adenosylhomocysteine (SAH) and 5’-deoxyadenosyl radical (5’dAdo). Assays were conducted under strict anaerobic conditions. The reaction assay 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 identified by HPLC analysis. HPLC analysis of SAM cleavage assay products After quenching by TFA, the reaction mixture 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 5’dAdo. The UV-detector was set at wavelength 258nm, and the standard samples, SAM, SAH, and 5’dAdo, were run with the same condition as SAM cleavage reaction products. According to the retention time comparison between standard samples and products, formation of SAH and 5’dAdo 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 the relative intensities of 5’dAdo, SAH, and SAM standards. The green trace shows the assay with the use of reconstituted MJ0841, and the SAH and 5’dAdo were both observed. The purple trace shows the control assay without MJ0841, and there was not any of 5’dAdo formed (Figure 9). Figure 9. HPLC analysis of the SAM cleavage assays Conclusions Future Work Initial results seem to show the [4Fe-4S] cluster and the enzyme activity. Since SAM was cleaved enzymatically, the products will be detected by mass spectroscopy to confirm the formation SAH and 5’dAdo. 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.