The Function of Two Radical‐SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]‐Hydrogenase Cofactor

Abstract In the biosynthesis of the iron‐guanylylpyridinol (FeGP) cofactor, 6‐carboxymethyl‐5‐methyl‐4‐hydroxy‐2‐pyridinol (1) is 3‐methylated to form 2, then 4‐guanylylated to form 3, and converted into the full cofactor. HcgA‐G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S‐adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.


Experimental Procedures
Heterologous production and purification of HcgA Escherichia coli C41(DE3) cells were transformed with a plasmid (pRKISC) for overexpression of Fe-S cluster proteins, [1] and then further transformed with the expression plasmid pET28b(+) containing the synthesized M. maripaludis hcgA gene with a N-terminal His6 tag (GenScript), whose codon usage was optimized for expression in E. coli ( Figure S1) and cloned between the XhoI and NdeI restriction sites. The transformant was grown at 37 °C in Terrific Broth (1.2% Tryptone, 2.4% yeast extract, 0.5% glycerol and 89 mM potassium phosphate) supplemented with 50 µg/ml kanamycin and 10 µg/ml tetracycline. At optical density 600 (OD600) = 1.0, the expression of the hcgA gene was induced with 1 mM isopropyl ß-D-thiogalatopyranoside and supplemented with 0.12 g/L cysteine dihydrochloride, 0.1 g/L iron (II) sulfate, 0.1 g/L iron citrate and 0.1 g/L ferric ammonium citrate (final concentrations) and cultivated for 18 hours at 20 °C. After the incubation, the cells were harvested by centrifugation using Avanti JXN-26 centrifuge with JLA-10.500 rotor at 7,300 rpm for 30 min at 4 °C and stored at -20 °C. All purification steps were performed anaerobically in an anaerobic chamber (Coy Laboratories, Grass Lake, MI, USA). The frozen cells containing HcgA were resuspended in 20 mM sodium phosphate buffer pH 7.4 containing 0.5 M sodium chloride, 5% glycerol and 20 mM imidazole (Buffer A) and disrupted on ice by sonication for 10 min using SONOPULS GM200 (Bandelin) with KE76 tip with 50 cycles. The supernatant was collected by centrifugation in a Sorvall WX Ultra centrifuge with a T-647.5 rotor at 30,000 rpm for 30 min at 4 °C and loaded onto a Ni 2+ -charged HiTrap chelating column (cytiva, Freiburg im Breisgau, Germany) equilibrated with buffer A. The column was washed with buffer A and eluted with a linear gradient of imidazole from 20 to 500 mM in 20 mM sodium phosphate buffer pH 7.4 containing 0.5 M NaCl and 5% glycerol (w/v). The protein fractions were concentrated with a centrifugal filter unit (Merck Millipore, Darmstadt, Germany) and loaded onto a HiPrep 26/10 desalting column (GE Healthcare, Uppsala, Sweden) equilibrated with 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS)/NaOH buffer pH 7.4 containing 0.5 M NaCl and 20% glycerol (w/v). The protein was further purified by gel permeation chromatography using a HiPrep Sephacryl S-300 HR (GE Healthcare, Uppsala, Sweden) equilibrated with 50 mM MOPS/NaOH buffer pH 7.4 containing 0.5 M NaCl and 20% glycerol (w/v). The fractions were pooled and concentrated using a 30 kDa centrifugal filter unit. The protein was frozen in liquid N2 and stored at −20 °C.

Construction of a M. maripaludis strain for the expression of His6 tagged HcgG.
M. maripaludis hcgG gene was amplified with the primer pairs 0125_Fg (CCATCACATCGAAGGTCGTGGGCCCATGAAAGAACTCATAAAAAATTCATTAAATG) and 0125_Rg (TTTTATGACCTACAGATCTCCTAGGTTAAAGTAATGATACGGCATC), and cloned into pLW40neo digested with ApaI and AvrII as previously described. [2] The resulting plasmid (pWL40neoHishcgG) was transformed into M. maripaludis ∆upt (Mm901) by the polyethylene glycol (PEG) method as previously described with PEG 8000 (Millipore Sigma). [3] Homologous production and purification of HcgG M. maripaludis transformed with pWL40neoHishcgG was grown in 10 L glass fermenters with 9 L McCas medium [4] supplemented with 1 g/L neomycin, under constant gas flow of H2/CO2/H2S (80%/20%/0.1%) at 1.5 L/min. Cells were grown to an OD at 660 nm = 2. The fermenters were then cooled with ice water and the cells were harvested anaerobically by a continuous-flow centrifuge equipped with a Heraeus 3049 continuous flow rotor at 15,000 rpm at 4 °C under N2 atmosphere. The rotor was then transferred to an anaerobic chamber and the cells were resuspended in the residual medium in the rotor, then the cells were further centrifuged using an Avanti JXN-26 centrifuge with JLA-10.500 rotor at 15,000 rpm for 30 min at 4 °C. The cells were weighted and resuspended in 2-fold volume per weight of lysis buffer (50 mM Tris pH 7.4, 5 mM MgCl2, 0.5-2.5 U/ml DNAse I), aliquoted, flash frozen in liquid N2 and stored anaerobically at −75 °C. All purification steps were done anaerobically. Cell resuspension was thawed and disrupted using a Thermo IEC FRENCH® Press in a 40K cell at 20,000 psi for five cycles. Clear cell-free extracts were obtained by centrifugation in a Sorvall WX Ultra centrifuge with a T-647.5 rotor at 30,000 rpm for 30 min at 4 °C and subsequent filtration by 0.45 μm filters. The resulting supernatant was loaded onto two 5-ml Ni 2+ -charged HiTrap chelating column (cytiva, Freiburg im Breisgau, Germany) connected in series and equilibrated with 20 mM sodium phosphate buffer pH 7.4 containing 0.5 M sodium chloride, 5% glycerol (w/v) and 20 mM imidazole (Buffer A). The column was washed with buffer A and eluted with a linear gradient of imidazole from 20 to 500 mM in 20 mM sodium phosphate buffer pH 7.4 containing 0.5 M NaCl and 5% glycerol (w/v). The protein fractions were concentrated with a centrifugal filter unit (Merck Millipore, Darmstadt, Germany) and loaded onto a HiPrep Sephacryl S-300 HR (GE Healthcare, Uppsala, Sweden) equilibrated with 50 mM MOPS/NaOH buffer pH 7.4 containing 0.5 M NaCl, 20% glycerol (w/v), and 2 mM dithiothreitol. After SDS-PAGE, the fractions containing HcgG were pooled, concentrated using a 30 kDa centrifugal filter unit. The sample was frozen in liquid N2 and stored at −75 °C.

Preparation of the cell extract from Methanococcus maripaludis for in vitro biosynthesis
Methanococcus maripaludis hcgA and hcgG strains were cultivated in the 37 °C cultivation room using a medium with sodium formate as substrate under 80% N2 / 20% CO2, in which 100 mM Tris was added as buffer component. [5] Cultivation was performed in 5 L or 500 mL scale until an OD at 660 nm of 0.60.8 as described previously. [6] The actively growing cells were anaerobically harvested by a continuous-flow centrifuge (Heraeus 3049 continuous flow rotor at 15,000 rpm at 4°C), resuspended in medium again and sedimented by centrifugation (Beckmann JLA 10.500 rotor at 7,300 rpm and 4°C). The use of the culture medium for resuspension aimed to avoid lysis of the cells in low salt concentration buffer solutions. The cell pellets were finally anaerobically resuspended in a low salt concentration lysis buffer: 50 mM Tris/HCl pH 7.5, 5 mM MgCl2 and 2.5 U/mL DNaseI, to a final concentration of 0.5 g cells/mL buffer. One mL aliquots were frozen in liquid N2 and stored until use at −20°C. The frozen samples were anaerobically thawed on ice. Unbroken cells and membrane particles were removed by ultracentrifugation using a Sorvall TFT-80.4 rotor at 37,000 rpm and 4°C for 0.5 h. This supernatant is designated as cell extract and used for in vitro biosynthesis assay.

In vitro biosynthesis of the FeGP cofactor
In vitro biosynthesis of the FeGP cofactor was performed as previously described. [6] Briefly, 200 µL cell extract of M. maripaludis was supplemented with 1 mM Fe(SO4)2(NH4)2, 1 mM DTT, 2 mM sodium dithionite, 5 mM MgCl2, 2 mM SAM, 5 mM ATP, 10 µM precursors and 10 µM [Fe]-hydrogenase apoenzyme (final concentrations) under 50% H2 /50%CO, if no other conditions are mentioned. In the standard assays of in vitro biosynthesis using the cell extract from the hcgA or hcgG strains, 20 M HcgA or 20 M HcgG were added to the assay, respectively. In in vitro biosynthesis from precursors 1 and 2, 5 mM GTP were added to convert the precursors to 3 by endogenous HcgB. The solution was transferred to a vial containing 50% H2 / 50% CO or otherwise described atmosphere and sealed with a rubber stopper. The reaction mixtures were incubated at 40 °C for 1 hour. In the case of kinetic analysis of the HcgG reaction was performed at 20 °C (Figure 4c). The [Fe]-hydrogenase activity was determined in 1 cm light-path quartz cuvette with 0.7 ml sample containing 20 M methylene-H4MPT as the substrate in 120 mM potassium phosphate pH 6.0 amended with 1 mM EDTA at 40 °C under N2 as previously described. [7] The formation of methenyl-H4MPT + was determined by measuring the increase of the absorbance at 336 nm. The extinction coefficient of methenyl-H4MPT + is 21.6 mM -1 cm -1 . The reaction was started by the addition of the sample tested after dilution with the assay buffer. One unit of the activity is the amount of enzyme producing 1 mol of methenyl-H4MPT + per min.

In vitro reactions of HcgA or HcgG
Cell extract of M. maripaludis was filtrated with a 3 kDa cut-off filter to remove proteins. To determine the production of 5'deoxyadenosine by the HcgA and HcgG reactions or the production of precursor 1 in the HcgA reaction, 200 L reaction mixtures were prepared, which included 170 L cell extract, 3 kDa filtrated cell extract or lysis buffer (50 mM Tris/HCl pH 7.4, 5 mM MgCl2, 0.5-2.5 U/ml DNAse I), in addition 20 M HcgA or HcgG, 5 mM sodium dithionite, 2.5 mM SAM and 1 mM of the other components described specifically in the figure legends. The resulting reaction mixtures were sealed with 100 L mineral oil to avoid evaporation, then incubated at 37 °C overnight or for the time period described, either under an 95% N2/5% H2 atmosphere or 47.5% N2/2.5% H2/50% CO atmosphere. At the time indicated in the figures, 50 L aqueous samples were taken, and quenched by mixing with 200 L methanol, the resulting mixtures were incubated at 40 °C for 15 minutes for extraction of small components, and then subjected to 0.45 m filtration (Ultrafree-MC Centrifugal Filter, Millipore, Germany), evaporated to dryness by using a Heraeus Centrivac aerobically. The resulting residuals were dissolved in 50 L distilled water and stored at −20 °C until further LC-MS analysis. To check the effect of the other cellular enzymes, the labelled amino acids were mixed with the same volume of the cell extract and incubated for 2 hours in the anaerobic tent at room temperature. After centrifugation, the supernatant was filtrated with 3-kDa cut-off filter and used for the in vitro HcgA reaction.

Proteome analysis
Cell pellets were lysed and reduced by 5 mM tris(2-carboxyethyl)phosphine (TCEP) in the presence of 2% deoxycholate (DOC) at 90 °C for 10 minutes. After that, it was incubated at 25 °C for 30 minutes in 100 mM ammonium bicarbonate pH 8.2 and 10 mM iodacetamide (IAA) and then digested overnight at 30 °C with trypsin, MS approved (Serva). Before LC-MS analysis, samples were desalted using C18 microspin columns (Nest Group) according to the manufacturer's instructions. Dried and reconstituted peptides were then analyzed using liquid-chromatography-mass spectrometry carried out on a Orbitrap Exploris 480 instrument connected to an Ultimate 3000 RSLC nano and a nanospray ion source (Thermo Scientific). Peptide separation was performed on a reverse phase HPLC column (75 μm x 42 cm) packed with C18 resin (2.4 μm; Dr. Maisch) with a 90-minute gradient (formic acid / acetonitrile). MS data were searched against an in-house Methanococcus maripaludis S2 protein database using SEQUEST embedded into Proteome Discoverer 1.4 software (Thermo Scientific).

Determination of compounds 1, 2 and 5'-deoxyadenosine
Determination of compounds was performed using two different types of HResLC-MS. In both cases, the chromatographic separation was performed using a Kinetex EVO C18 column (150 × 1.7 mm, 3 μm particle size, 100 Å pore size, Phenomenex) connected to a guard column of similar specificity (20 × 2.1 mm, 5 μm particle size, Phenomoenex) a constant flow rate of 0.2 mL/min with mobile phase A being 0.1% formic acid in water and phase B being 0.1% formic acid methanol (Honeywell, Morristown, New Jersey, USA) at 40 °C. The injection volume was 5 µL. The mobile phase profile consisted of the following steps and linear gradients: 0 -0.5 min constant at 5% B; 0.5 -4 min from 5 to 90% B; 4 -5 min constant at 90% B; 5 -5.1 min from 90 to 5% B; 5.1 -10 min constant at 5% B. For determinations, a Thermo Scientific I-DX Orbitrap mass spectrometer was used. Ionization was performed using a high temperature electro spray ion source at a static spray voltage of 3300 V, Sheath gas at 50 (Arb), Auxiliary Gas at 25 (Arb), and Ion transfer tube and Vaporizer at 325 and 300 °C. Data dependent MS/MS measurements were conducted applying an orbitrap mass resolution of 120 000 using quadrupole isolation in a mass range of 100 -600 and combining it with a high energy collision dissociation (HCD). HCD was performed on the five most abundant ions per scan with a relative collision energy of 15%. Fragments were detected using the orbitrap mass analyzer at a predefined mass resolution of 30 000. Dynamic exclusion with and exclusion duration of 2.5 seconds after 1 scan with a mass tolerance of 10 ppm was used to increase coverage.        For sequence alignments, see Figure S10. The structure model is made by PyMOL. Figure S10. Multiple sequence alignments of HcgG from different microorganisms. The fully conserved amino-acid residues are shown in red. Potential cysteine residues responsible for the coordination of the possible [4Fe-4S]-binding site are highlighted by yellow stars under the sequences. The secondary structure elements were obtained from the AlphaFold model.