1 Inhibition of Methyl-CoM Reductase from Methanobrevibacter ruminantium by 2-
2 Bromoethanesulfonate
3 Tobias Gräwert,† Hans Peter Hohmann,§ Maik Kindermann,§ Stephane Duval,§ Adelbert
4 Bacher,† and Markus Fischer *,†
5 †Hamburg School of Food Science, Institute of Food Chemistry, Grindelallee 117, D 20146,
6 Hamburg, Germany
7 §DSM Nutritional Products, Wurmisweg 576, CH 4303 Kaiseraugst, Switzerland
8 * Corresponding author: Tel: +49 40 428384342; Fax: +49 40 428384342; E Mail:
9 [email protected] hamburg.de
10
11
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12 Supporting Information:
13 1. Materials and Methods
14 Anaerobic techniques: Experiments were conducted under anaerobic conditions using
15 flexible vinyl chambers type B from Coy Lab Products, Grass Lake, MI, United States
16 equipped with heavy duty gloves, manual airlocks, and an oil free vacuum pump type GAST
17 260 from Gast Manufacturing, Inc., Benton Harbor, MI, United States. The device was loaded
18 with a mixture of hydrogen/nitrogen 5:95 (v/v). The gas mixture inside the glovebox was
19 permanently recycled through bags containing palladium catalyst. The partial pressures of
20 hydrogen and oxygen were monitored at all times using Coy gas analyzers (model 10).
21 Operating conditions implicated oxygen partial pressures of less than 1 ppm. The temperature
22 inside the boxes was maintained at 15 °C for protein purification and at 23 °C for all other
23 procedures by heat exchanger systems. The import of equipment and reagents involved three
24 cycles of evacuation and gassing with the hydrogen/nitrogen gas mixture.
25 Microorganisms: Methanobrevibacter ruminantium (DSM 1093, ATCC 35063, JCM 13430)
26 was obtained from Leibniz Institut DSMZ Deutsche Sammlung von Mikroorganismen und
27 Zellkulturen GmbH, Inhoffenstraße 7B, 38124 Braunschweig, Germany. The organism was
28 maintained by serial culture in serum flasks. 1 Stock cultures were grown in the same way as
29 cultures for experiments (see below). Stock cultures were stored at 80 °C after centrifugation
30 at 5,000 rpm and subsequent resuspending in DSMZ medium 119 (Table S1) with 30%
31 glycerol. Subculturing was performed approximately once per month.
32 Medium: DSMZ Medium 119 (Table S1) was prepared according to the protocol given by
33 DSMZ (www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium119.pdf). The medium
34 was prepared at low oxygen partial pressure. Thus, 4 L of water were degassed by boiling for
35 20 min and were then transferred into a Coy glovebox. After dissolving all ingredients, pH
2
36 was adjusted to 7.0 with HCl. Upon addition of a fatty acid mixture, trace element solution
37 SL 10 and sewage sludge, the medium was filled into culture vessels and autoclaved. A fatty
38 acid mixture (Table S2) was prepared according to the protocol given by DSMZ
39 (www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium119.pdf) and stored inside the
40 glovebox. Trace element solution SL 10 (Table S3) was prepared according to DSMZ
41 Medium 320 and stored inside the glovebox:
42 www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium320.pdf. Sewage sludge was
43 obtained from a local wastewater treatment plant; sludge fluid was prepared inside a glovebox
44 by addition of 0.4 % yeast extract (8g) to 2 L of sludge and subsequent incubation of the
45 mixture for 48 h at 23 °C. After centrifugation at 13,000 rpm, the clear supernatant was
46 autoclaved and was then stored frozen.
47 Growing M. ruminantium in serum bottles: Culture medium was dispensed into serum
48 bottles (VWR International GmbH Hilpertstraße 20a, 64295 Darmstadt, product 612 0463)
49 inside a Coy glovebox. Serum bottles were sealed with butyl rubber seals (VWR, product
50 548 3100) and aluminum crimp caps (VWR, product 548 3096) using crimp tongues (VWR,
51 product 548 0073) (cf. Fig. 3). The vials underwent three cycles of evacuation to 100 mbar
52 and subsequent doping with 2 bar 80 ± 2 % H 2 / 20 ± 2 % CO 2 (Westfalen AG, Münster,
53 product A04010150, gas mix number G322637) using a manifold. The flasks were
54 subsequently autoclave sterilized and stored inside a glove box in the dark. Culture vessels
55 were inoculated at a ratio of 1:25 with liquid preculture using Luer slip syringes (B Braun
56 Omnifix F, 1 mL, Luer Slip 9204512N) and sterile needles (B Braun Sterican, 18G x 1”
57 16010915E). Cultures were grown for four days at 37 °C with shaking in the dark. For in vivo
58 IC 50 determination, serum vials were prepared, inoculated and incubated as mentioned above.
59 Prior to inoculation, inhibitor was added to final concentrations of 30 µM, 10 µM, 3 µM, 1
60 µM, 300 nM, 100 nM and 3nM, respectively by injection with a sterile syringe and needle.
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61 Culture vessels were then inoculated at a ratio of 1:25 with preculture using gas tight
62 syringes. Cultures were grown for four days at 37 °C with shaking in the dark. IC 50
63 determination was performed in duplicate.
64 Growing M. ruminantium in 1-Liter flasks: The screw caps of heavy duty 1 Liter Schott
65 flasks were modified to include a butyl rubber seal. Flasks containing 200 mL of medium
66 were autoclave sterilized and were subsequently doped with 80% H 2 / 20 % CO 2 using a gas
67 manifold (three evacuation/gassing cycles). Culture vessels were inoculated at a ratio of 1:25
68 with preculture using sterile syringes and needles. Cultures were grown for four days at 37 °C
69 with shaking in the dark. After two days, the gas phase was replaced by evacuation and
70 followed by doping with 2 bar 80 ± 2 % H 2 / 20 ± 2 % CO 2 (Westfalen AG, Münster, product
71 A04010150, Gemischnummer G322637) in order to remove the methane that had been
72 produced metabolically during the prior culture period.
73 Assessment of methane: Methane was monitored using a chromatograph HP 6890 GC FID
74 equipped with an FID detector and a Restek Rt Q Bond GC column (length, 30 m; inner
75 diameter, 0.32 mm). The column temperature was 150 °C; the temperature of the injector and
76 detector was 250 °C. The carrier gas was hydrogen; the flow rate was 1.0 mL/min. The
77 retention time of methane was 2.7 min. Alternatively, a chromatograph Chrompack equipped
78 with an FID detector and a Capillary Column Poraplot Q HT (length, 12.5 m; inner diameter,
79 0.32 mm) was used. The injector and column temperature was 40 °C, the detector temperature
80 was 250 °C. The hydrogen gas flow rate was 0.6 mL/min. The retention time of methane was
81 1.5 min. “Restek analyzed gas (99.9 ppm methane in helium, product 34477 PI)” was used for
82 calibration.
83 Preparation of crude cell extract: Each of 6 Schott flasks equipped a with butyl rubber seals
84 and containing 0.2 L of culture medium was inoculated with 5 ml of cell suspension (from
85 serum flasks) inside a glovebox by injection with a syringe through the butyl rubber seal. The 4
86 flasks were incubated at 37 °C with shaking for 4 days. The cells were harvested by
87 centrifugation. Specifically, the centrifuge beakers were filled inside the glovebox, and were
88 then centrifuged outside in a centrifuge that was continuously flushed with argon (Argon 4.6
89 from Westfalen AG). The pellet was resuspended in anaerobic loading buffer (100 mM
90 MOPS, pH 7.2, containing 400 mM NaCl) inside the glovebox. The suspension was sonicated
91 on ice (HD 2200, Bandelin Company, Danbury, USA; 65 % output, 6 pulses of 30 s each)
92 inside the glovebox. After centrifugation in a table top centrifuge inside the glovebox, the
93 supernatant was collected.
94 Protein purification: All procedures were performed under anaerobic conditions inside the
95 glove box. 2 Crude extract (30 ml) was applied to a column of Sepharose Q (GE Healthcare;
96 column volume, 25 ml) that had been equilibrated with 400 mM NaCl in 100 mM MOPS, pH
97 7.2. The column was washed with the above mentioned buffer and was then developed with a
98 gradient of 0.4 – 1.0 M NaCl in 100 mM MOPS, pH 7.2. Fractions of 3 ml were collected.
99 Fractions were analyzed for methane production as described in the following paragraph.
100 Enzyme assays: Assays were carried out according to Bonacker et al. 3 with the following
101 modifications: 250 mM MOPS buffer was used instead of 50 mM potassium phosphate and
102 10 mM methyl CoM was used instead of 11 mM. Dithiothreitol was omitted, since it lowered
103 the activity of methyl CoM reductase from M. ruminantium . Further, smaller assay vessels,
104 1,5 mL instead of 8mL were used and the assay temperature was lowered from 65 °C to 37 °C
105 to prevent precipitation of the M. ruminantium enzyme. A pH value of 7.2 instead of 7.0 was
106 used. Assay mixtures contained 250 mM MOPS, pH 7.2, 10 mM methyl CoM, 1 mM
107 coenzyme B, 0.3 mM aquocobalamin (0.4 mL in a GC vial with a total volume of 1.5 mL).
108 Protein and inhibitors were added as indicated. Methyl CoM served as the substrate for the
109 enzymatic generation of methane. The samples were incubated at 37 °C for 90 min. Aliquots
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110 of the overhead gas phase (100 µL, gas tight syringe von SGE Part No.: 005250) were
111 analyzed by gas chromatography (see above).
112 IC 50 measurements with crude cell extract resp. purified protein: For IC 50 determination,
113 assay mixtures were prepared that contained the inhibitor in final concentrations of 200 µM,
114 50 µM, 5 µM, 2µM, 500 nM, 200 nM, 20 nM and 2 nM, respectively. Assay mixtures
115 contained 250 mM MOPS, pH 7.2, 1 mM methyl CoM, 1 mM coenzyme B, 0.3 mM
116 aquocobalamin, inhibitor, and protein as indicated. Samples were incubated at 37 °C for 90
117 min. The concentration of methane in the headspace of the reaction vessels was plotted versus
118 the logarithm of the inhibitor concentration.
119 Statistics: Experiments were performed in triplicate. Subsequently, mean and standard
120 deviation were calculated. Best fits were calculated from mean of three independent
121 measurements using the program Dynafit (Biokin). 4
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123 2. Supporting Tables
124 Table S1: DSMZ Medium 119 Ingredient Amount
KH 2PO 4 0.5 g
MgSO 4 * 7 H 2O 0.4 g NaCl 0.4 g
NH 4Cl 0.4 g
CaCl 2 * 2 H 2O 50 mg
FeSO 4 * 7 H 2O 2.0 mg Trace element solution SL 10 1.0 ml Yeast extract 1.0 g Na acetate 1.0 g Na formate 2.0 g
NaHCO 3 4.0 g Fatty acid mixture 20 ml Resazurin 1.0 mg
Cysteine HCl * H 2O 0.5 g
Na 2S * 9 H 2O 0.5 g Distilled water ad 1.0 l 2 (methylthio)ethanesulfonate 1.0 mg Sludge fluid 50 ml 125
126 Table S2: Fatty acid mixture. pH was adjusted to 7.5 with NaOH Ingredient Amount Valeric acid 0.5 g Isovaleric acid 0.5 g 2 Methylbutyric acid 0.5 g Isobutyric acid 0.5 g Distilled water ad 20 ml 127
128 Table S3: Trace element solution SL 10. Ingredient Amount HCl (7.7 M) 10 ml
FeCl 2 * 4 H 2O 1.5 g
ZnCl 2 70 mg
MnCl 2 * 4 H 2O 0.1 g
H3BO 3 6.0 mg
CoCl 2 * 6 H 2O 190 mg
CuCl 2 * 2 H 2O 2.0mg
NiCl 2 * 6 H 2O 24 mg
Na 2MoO 4 * 2 H 2O 36 mg Distilled water ad 1.0 l 129 130
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131 3. References 132 133 1. Smith, P. H.; Hungate, R. E. Isolation and characterization of Methanobacterium
134 ruminantium n. sp. J. Bacteriol. 1958 , 75, 713–718.
135 2. Goubeaud, M.; Schreiner, G.;Thauer, R. K. Purified methyl coenzyme M reductase
136 is activated when the enzyme bound coenzyme F430 is reduced to the nickel(I)
137 oxidation state by titanium(III) citrate. Eur. J. Biochem. 1997 , 243, 110 114.
138 3. Bonacker, L.; Baudner, S.; Mörschel, E.; Böcher, R.; Thauer, R. K. Properties of the
139 two isoenzymes of methyl coenzyme M reductase in Methanobacterium
140 thermoautotrophicum. Eur. J. Biochem. 1993 , 217, 587 595.
141 4. Kuzmic, P. Program Dynafit for the analysis of enzyme kinetic data: application to
142 HIV proteinase. Anal. Biochem. 1996 , 237, 260 273.
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