Purification and properties of 5,10-methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase, two coenzyme F420-dependent enzymes, from Methanosarcina barkeri

Biochimica et Biophysica Acta. 1079 (1991) 293-302 © 1991 El~vier Science Publishers B.V. All rights reserved 0167-4838/91/$03.50 ADONIS 016748389 !002864

293

BBAPRO 34003

Purification and properties of 5,10-methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase, two coenzyme F420-dependent enzymes, from Methanosarcina barkeri Ben W.J. te Br/Smmelstroet, Wim J. Geerts, Jan T. Keltjens, Chris van der Drift and Godfried D. Vogels Department of Microbiololo', Faculty of Science. Unicersit)' of Nijmegen. Nijmegen, The Netherlands (Received 25 February 19911

Key words: Methylenetetrahydromethanopterin dehydrogenase: MethylenetetraFydromethanopterin reductase: 5,6.7,8-Tetrahydromethanopterin; Coenzyme F~,o; (M. barkeri)

5,10-Methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase have been purified to homogeneity by a factor of 86 and 68, respectively, from methanol.grown Methanosarcina barkeri cells. The dehydrogenase was isolated as a hexamer of a single 35 kDa subunit, whereas the reductase was composed of four identical 38 kDa subunits. The purified oxygen-stable enzymes catalyzed the oxidation of $,10-methylenetetrahydromethanopterin and methyltetrahydromethanopterin with Vm~ values 9f 3000 and 200/z mot m i n - t mg -t, respectively. The methanogenic electron carrier coenzyme F4~0 was a specific electron acceptor for both enzymes. Steady state kinetics for the two enzymes were in agreement with ternary complex (sequential) p,~chanisms. Methylene reductase and methylene dehydrogenase are proposed to function in the methanol oxidation step to CO z.

Introduction

The pathway of CO 2 conversion into rr othane (Eqn. 1) by methanogenic bacteria is presently well understood [1-3]. in the initial step CO, is reduced and concomitantly bound to the one-carbon carrier methanofuran, and forrnylmethanofuran is formed, in a series of 5,6,7,8-tetrahydromethanopterin (H4MPT)dependent reactions the fo.rmyt group is first transferred to H4MPT resulting in formyI-H4MPT. The latter compound is subsequently converted by cyclohy-

drolysis into 5,10-methenyI-H4MPT (mcthcnyiH 4MPT), which is reduced via 5,10-methylene-H4MPT (methylene-H,~MPT) into methyl-H4MPT, in the hydrogenotrophic organism Methanobacterium thermoautotrophicum methenyi-H4MPT reduction may be catalyzed by two distinct enzymes that either use reduced coenzyme F4,_. [4-6] or hydrogen gas [7] as the reductant. For methylenc-H4MPT reduction in the organism a F420-dependent reductase has been described [8,9]. Following the transfer of the methyl group of methylH 4 M P T to 2-mercaptoethanesulfonic acid (Coenzyme M), methylcoenzyme M is produced, which is the substrate of the terminal step of methanogenesis

Abbreviations: H4MPT, 5,6,7,8-tetrahydromethanopterin: F4~, 7.8didemethyl-5-deazariboflavin-5'-phosphory,llactylglutamate: F*, 8-hydro~-5-deazariboflavin-5'-phosphate; F,, 8-hydroxy-5-deazariboflavim F4~H 2, 1,5-dihydro.coenzyme F4:o: F~,~, 8-OH adenylylated F42a: CHAPS. 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesuifonate; HPLC, high-pertormance liquid chromatography: FPLC. fast-protein liquid chromatography; SDS, sodium dodec2,'! sulfate: PEI. polyethyleneimide.

4H, . C O , --. CHa 4- 2H,O

Correspondence: J.T. Keltjens, Departmenl of Microbit~log3-. Faculty

A number of methanogenic bacteria - including Methanosarci~,,, barkeri, the organism studied here are capable of growth on methanol (Eqn. 2). Three-

of Science, University of Nijmegen. Toernooiveld. NL-6525 ED Nijmegen, The Netherlands.

( J G ' ' ~- - 131 kJ/mol CHa)

(1) 4('H~OH ~ 3 C H ~ * C O , +21-t ,O

(-1¢

TM

= - 107 kJ/mol CH,s) ~2~

294 quarters of the substrate is reduced, via methylcoenzyme M, to methane. The rest of the substrate is oxidized to CO, in order to generate the reducing equivalents for the methyl group reduction. The way methanol oxidation proceeds is not completely established. In M. barked grown on methanol formylmethanofuran dehydrogenase [10], formylmelhanofuran: H4MPT formyltransferase [11] and methenylH4MPT cyclohydrolase [12] are present in high activities and have been isolated and characterized. This implies that the oxidation pathway at least partly proceeds by the CO., reduction route outlined above, though in the opposite direction, in addition, methylene-H4MPT reductase (methylene reductase), which catalyzes reaction (3), has been purified from the methanol-grown organism [13]. Methanol conversion into CO 2, thus, may start from the level of methylH4MPT. Oxidation of the lafter gives methyleneH4MPT, which subsequently has to be oxidized into methenyl-tt~ MPT (reaction 4) catalyzed by methyleneH,,MPT dehydrogenase (methylene dehydrogenase). The latter reaction has not as yet been studied in detail in M. barkeri. m e t h y I - H 4 M P T + c o e n z y m e F42o 5 , 1 0 - m e t h y l e m . - H 4 M P T + c o e n z y m e F4zI~H

(3)

5,10-met h y l e n e - H ,t M P T + c o e n z y m e F,t2~j •-~ 5,10-me,hen~,l-H 4 M P T + c o e n z y m e F.~2oH 2

(4)

In the paper by Ma and Thauer [13] reaction (3) was only measured in the direction of methylene-H4MPT reduction. Here, we focused on the reaction as it may occur under physiological conditions during growth on methanol, viz. methyl-H4MPT oxidation. In addition the presence, purification and some of the properties of methylene dehydrogenase of M. barked are described. Materials and Methods

Organisms and extracts. M. thermoautotrophicurn strain AH (DSM 1053) and M. barken strain MS (DSM 800) were grown in a 300 I fermentor on synthetic medium under 80% hydrogen/20% CO 2 [14], and under an 80% nitrogen/20% C n 2 atmosphere with 250 mM methanol [15], respectively. Cells were harvested at the end of exponential growth and stored at -70°C under a nitrogen atmosphere until use. Cellfree extracts of M. barkeri were prepared by suspending whole cells (1 : 1, w/v) in 100 mM Tris-HCl buffer (pH 7) and passing the suspension twice through a French Pressure Cell operating at 138 MPa. The suspension was centrifuged for 20 min at 18000 x g and 40C for removal of cell debris. Boiled cell-free extracts

for cofactor purification were prepared under anoxic condi!ions from cells of M. thermoautotrophicum by suspending whole cells (1"1, w/v) in 30 mM sodium acetate buffer (pH 4) that contained I mM dithiothreitol. The suspension was placed in a boiling water bath for 1 h and, after co31ing, the slurry was centrifuged anaerobically for 40 rain at 18000 × g and 4°C. The supernatant was carefully decanted in an anaerobic glove box and was stored at - 7 0 ° C under a nitrogen atmosphere until further use. Purification of the substrates. H4MPT and F42o were purified from boiled cell-free extracts of M. thermoautotrophicum as described before [5,8]. Methyl-H4MPT was prepared and purified under strict anoxic conditions from methylene-H4MPT, which is non-enzymically formed from H4MPT and excess formaldehyde, according to two previously reported procedures [5,8]. The first method consisted of reduction of methyleneH4MPT under hydrogen (200 kPa) at 60°C in the presence of catalytic amounts of F420 (12 ~tM) and cell-free extract of M. thermoautotrophicum that had been freed of cofactors by repeatedly washing with 20 mM potassium phosphate buffer (pH 7) on an Amicon PM 30 filter [8]. In the second procedure methyleneH aMPT was incubated under nitrogen with catalytic concentrations of F4z0 and methylene dehydrogenase [5] and methylene reductase [8] purified trom M. thermoautotrophicum. Under these conditions the substrate was converted into methyl-H4MPT and methenyl-H 4MPT [12]. Reduced F420 (F420H2) was prepared anaerobically by reduction of F42o with sodium borohydride and subsequent C ~ column chromatography as described elsewhere [8]. 8-OH adenylylated F420 was enzymieally prepared from F42o and ATP and subsequently purified as specified in [16]. Enzyme assays. Reactions were followed spectrophotometrically by measuring the increase of the absorption at 335 nm, which is the absorbance maximum of methenyI-H4MPT (~335 = 21.6 mM -~ cm - l ) [17], the decrease of the absorption at 401 nm as a result of F420 reduction (~401 = 27.6 m M - t cm-i)[18], or simultaneously at both wavelengths. When reaction rates were calculated on the basis of the absorption at 335 nm, values were corrected for the contribution of reduced F420 (E335 = 8.0 mM-~ c m - t ) at this wavelength. The assays were performed in stoppered cuvettes under nitrogen. Anoxic reaction mixtures were prepared inside a glove box. After prewarming the cuvettes to 37°C reactions were started by the addition of enzyme fraction (10-50 #1)by use of a gas-tight syringe flushed with nitrogen. Standard reaction mixtures (2.0 ml) for the methylene dehydrogenase assay contained 116 nmol F4z,, 140 nmol H4MPT and 10 ~mol formaldehyde in 100 mM Bistris propane buffer (pH 6.5).

295 Methylene reductase activity was measured in reaction mixtures (2,0 ml) containing 130 nmol methylH4MPT and 116 nmol F420 in 100 mM Tris-HCI buffer (pH 7.0) and excess methylene dehydrogenase (7,5 #g, 5.5 units) purified from M ,hermealttotrophicum [5] Purification of the enzymes. Methylene dehydrogenase and redactase were purified aerobically at 4°C by procedures summarized in Tables I and II, respectively. If not stated otherwise all of the following buffers used routinely contained 0.5 mM CHAPS and, after the TSK-butyl step, 10% ethylene glycol; the compounds were added to stabilize enzyme activity. To 10 ml cell-free extract of M. barkeri, 10 ml saturated ammonium sulfate in water was added dropwise under gentle stirring over a period of approx. 4 h. After stirring for an additional 12 h the solution was centrifuged at 49000 × g and 4°C for 20 min. The supernatant containing both enzymes was applied to a Fractogel TSK-butyl 650 column (6.5 × 1.8 cm) equilibrated with 3 M ammonium sulfate in 20 mM potassium phosphate buffer (pH 7). The coiumn was washed with 20 mi 3 M ammonium sulfate and developed with a linear gradient (500 ml) of 2 to 0 M ammor, ium sulfate in 20 mM potassium phosphate buffer (pH 7). The dehydrogenase and reductase coeluted at a concentration of 1 to 0.4 M ammonium sulfate. Fractions containing both enzymes were pooled, concentrated and desalted by ultrafiltration (Amicon PM 10) after addition of ethylene glycol to a final concentration of 10%. The desalted and concentrated enzyme preparation was applied to a column (I 1 x 2.8 cm) packed with Q-Sepharose Fast Flow. In order to separate the dehydrogenase and the reductase the column was developed successiwly with a ~inear gradient of 0 to 150 mM NaCl (60 ml), an isocratic step of 150 mM NaCI (180 ml), a linear gradient of 150 to 200 mM NaCi (10 ml) and finally an isocratic elution of 200 mM NaCI (720 ml) all in 20 mM potassium phosphate buffer (pH 7) and 10% ethylene glycol. The dehydrogenase was collected from the first (150 mM NaCI) and the reductase from the second isocratic (200 mM~ step. Fractions that were eluted with the 150 to 200 mM linear gradient contained both enzyme activities and were discarded. Hereafter, the purification was continued separately for both enzymes. The two enzyme pools were concentrated by ultrafiltration (Amicon PM t0) and loaded on to a Sephacryl S-300 column (90 × 2.8 cm). The column was eluted (flow rate 0.3 m l / m i n ) with 200 mM KCt in 25 mM Tris-HCl (pH 7) and 10% ethylene glycol. Active fractions from both separations were pooled and desalted by carefully washing with 25 mM Tris-HC! (pH 7) and 10% ethylene glycol on an Amicon PM 10 filter. Methylene dehydrogenase and reductase were brought to homogeneity by FPLC on a PEI column (10 x 1.0 cm). The column was developed at a flow rate of 1,0 m i / m i n with a linear gradient of 0 to 1 M KCI in

50 mM Tris-HCl (pH 7) and 10% ethylene glycol. Both the dehydrogcnase and the reductase were eluted at 9(X) mM KCI. HPLC analyses, ttPLC analysis of the coenzymes was carried out either (A) at ambient temperature on a Waters liquid chromatograph equipped with Model 6000 and M45 pumps, a M660 Gradient Programmer, a U6K injector, and a model 451) variable-wavelength detector or (B) at 40°C on a HP 1084B liquid chrc~matggraph (Hewlett Packard)connected to a Diode Array Detector (HP 1040A). in both ,,,ystcms separation took place on a precolumn (50 x 2.1 ram) of 37 to 50 p m Corasil C1~ (Waters) coupled to a 10 /zm LiChrosorb RP-18 (Merck) column (250 × 5 ram). Elution was performed with a linear gradient (2.0 m l / m i n ) in 10 rain of oxygen-free 0 to 25% methanol in 40 mM sodium formate (pH 3). Peaks were recorded at 260 am.

Protein determination. Protein was determined by the Bio-Rad Protein assay (Bio-Rad Laboratories, Richmond, CA) based on the method of Bradford [19]. Bovine serum albumin was used as a standard. Molecular weight determinations. Apparent molecular weights of the subunits were determined by gel electrophoresis on a 12.5% polyacrylamide Phast gel containing 0.1% SDS. Prior to application to the gel native proteins were denaturated by boiling for 5 rain in 10 mM sodium phosphate buffer (oH 7) that contained 1% SDS, 5%/3-mercaptoethanol, 1 mM EDTA and 10% glycerol. Electrophoresis and staining of the get were performed on a Phast System separation and control ~mit from Pharmacia (Uppsala, Sweden). The following proteins served as the ~tandards: aiactalbumin (14 400), soybeai~ trypsin inhibitor (20 100), carbonic anhydrase (30 000), ovalbumin (43 000), bovine serum albumin (67000) and phosphorylase b (94000). The molecular weights of the native proteins were estimated by get filtration on Sephacr3"l S-200 calibrated with cytochrome c (12400), carbonic anhydrase (30000), bovine serum albumin (67000), alcohol dehydroge nase ( 150 000), ,a-amylase (200 000) and Blue dextran as the marker for the void volume. Materials. If not mentioned otherwise chemicals were of the highest grade available. FMN, NADH, NADPH and alcohol dehydrogenase were from Boehringer ( M mheim, F.R.G.). FAD, NADP+, NAD +, Bistris pro, ane, cy_tochrome c and SDS were purchased from Sigma (St. Louis, MO). TSK-butyl 650 and /t-amylase were from Merck AG (Darmstadt, F.R.G.). O-Sepharose, Sephacryl S-300, Sephacryl S-200, DEAE-Sephadex, Blue dextran, polyacrylamide Phast gels, the protein molecular weight calibration kit for SDS gel electrophoresis and Phast gel blue R for staining of the gels were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden), WP PEI FPLC column material, 40 # m Bonded-phase octadecyl Cts and methanol

296 (HPLC-grade) were from J.T. Baker (Deventer, The Netherlands). CHAPS, carbonic anhydrase, bovine serum albumin and dithiothreitol were purchased from Serva Feinbiochemica (Heidelberg. F.R.G.) and benzyl viologen was from BDH Chemical (Poole, U.K.). F, and F ÷ were kindly provided by S.W.M. Kengen from our laboratory. Gasses were obtained from Hock Loos (Schiedam, The Netherlands). Nitrogen and hydrogencontaining gasses were made oxygen-free by passage at I50°C over a prereduced R3-11 and at ambient temperature over a RO-20 catalyst, respectively: catalysts were a gift from BASF (Ludwigshafen, F.R.G.). Results

Purification and properties of meihylene-H4MPT dehydrogenase and methylene-H4MPT reductase jrom M. barkeri Methylene dehydrogenase and methylene reductase were purified from M. barked as oxygen-stable enzymes as summarized in Tables I and II, respectively. When kept at the appropriate conditions (0.5 mM CHAPS plus 10% ethylene glycol), no losses in activities occurred on storage at 4°C for more than 3 months. Both enzymes behaved as soluble proteins since activi-

ties were exclusively recovered in the supernatant after cell breakage and subsequent centrifugation. Methylene dehydrogenasc was purified by a factor 86 in 40% yield and up to a specific activity of 670 /stool methylene-H4MPT oxidized per min per mg protein. After this stage only one band could be detected after denaturating SDS-polyacrylamide gel electrophoresis with an apparent M, = 35 000 (Fig. 1). For the native enzyme a molecular mass of 200 kDa was estimated on the basis of Sephacryl S-200 gel chromatography, which indicates that the enzyme is a hexamer of identical 35 kDa subunits. Methylene reductasc and dehydrogenase could be purified by similar methods; both enzymes behaved quite the same during the various steps. Resolution, though not complete, could be achieved by careful elution during Q-Sepharo,~e Fasl Flow ior~-chromatography. Fractions also containing dehydrogenase activity were discarded, which substantially contributed to the loss in yield during this step and the modest overall recovery (29%) of the reductase. The 68-fold purified enzyme had a specific activity of 31.5 #mol min -t mg -L, when measured in the direction of methylH4MPT oxidation. The preparation showed only one band after SDS-gel eleetrophoresis with an apparent

TABLE I

Purification of methylene-H ~MPT d('hydrogenase from M. barkeri Purification used !0 ml crude celt-free extract of M. h.rkeri. Activities were measured in the direction of methylene-H4MPT oxidation and F.~zo reduction. Units are expressed as tamol methenyI-H.~MPT formed per min Step

Total protein (mg)

Total activity (units)

Crude extract Ammonium sulfate (50% supern atant) TSK-butyl 65[) Q-Sepharose Sephac:5'l S-300 FPLC WP-PE!

170

1332

142 76 8.2 1.9 0.8

I 185 946 892 577 536

Specific activity (units/mg)

Factor (-fold)

Yield (c-/)

7.8

1.0

I(W)

8.3 12.4 109 304 670

1, I 1.6 13.0 39

89 71 67 43 40

N6

TABLE !i

Purification of methylene-H ~MPT reductase from M, barkeri Purification used i0 ml crude cell-free extract of 34, barkeri. Activities were measured in the direction of methyl-tt4MPT oxidation in the presence of purified methylene dehydrogenase from M. thermoautolrophicum (5.5 ~nils). Units are expressed as ~mol methenyI-H ~ MPT formed per min Step

Total protein (rag)

Total activity (units}

Crude extract Ammonium sulfate (50% supernatant) TSK-butyl 650 O-Sepharose SephacD'! S-3[~) FPLC WP-PE1

i 70

79

142 76 6.8 2.6 0.73

70 64 30 25 23

Specific activity (unit~/mg)

Factor (-fold)

Yield (G-)

0.46

! .0

i 00

(I.49 0.84 4.41 9.6 31.5

I. ! 1.8 0.6 21 68

89 81 38 32 29

297 1

2

Molecular weight ~0

3

x I0-~. i

.

o

•

""~i~:~i~i: 4T°P .:?!:;,;

B O

6

,, ~?-~ ,

,,lip

L.

.

2

~.~

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:

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Front

02 03 O~ 05

05

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Fig. I. Sodium dodecyl sulfate gel ¢lectrophoresis of me'nyIenc-tt 4MPI ~ deh~;drogcna.,,e and mcth~ iene-H 4 MPT rcducta~e from .~t. harkcri. Gel electrophoresis was performed as described under Mat, rials and Mclhods and subsequently ,,mined with ('ooma',sic brilliant blue R-250= (A) The lanes represent the following en~'mes: (I) methylene dehydrogenase (27 ng). (2) e n ~ m i c ~,tandards (kDa) comi~sed of phosphors.lase h ('44). bovine serum albumin (67), ovalbumin (43). carbohic anhydrase (3(I). tDpsin inhibitor (211.1)and a-lactalbumin (14.4) and (3) methylent: reductase (32 ng). (B) Estimation of the s,bunit molecular size of methylene dehydrogenase ~,: I and meth.vlcne reductase ( ~ ) : the c h e m i c standard~ (e) are as given in A.

M~ = 38000 (Fig. 1). The native enzyme was eluted from a Sephacryl S-200 column at about 16(1 kDa. which implies that the reductase consists of t'our 38 kDa subun;ts. From the purification factors it follows that both enzymes each comprise about I% of the total cell protein, which is in accordance with a central metabolic role. Purified and concent.ated enzymes were both colorless and ultraviolet spectra only showed an absorption band around 280 r.~; characteristic bands that would account for the presence of prosthetic groups like flavins or iron-sulfur clusters were absent.

Substrates and products of the methylene dehydrogenase and methylene redtwtase reactions Methylene dehydrogenase was routinely measured in the directien of methylene-H,MPT oxidation and F4z0 reduction. The spectrophotometric assay resulted in an increase of the absorbance at 335 nm due to methenyl-H4MPT formation and a simultaneous decrease in the absorbance at 41)1 nm as a result of F4_,. reduction in accordance with a stoichiometry according to Eqn. (4). No reaction was observed when F.~z. was omitted from the assay. Anaerobic addition of F.~.2t~H2 after completion of the reaction resulted in a decrease of absorption v_t 335 nm and an increased absorption at 401 rim, indicating that the opposite reaction also could

take place. Hence. methylene dehydrogenase from M.

barkeri, like the enzyme from M. thermoautotrophicum [5,6], is capable of catalyzing the oxidation of methylene-H.~ MPT and reduction of melhenyl-H 4MPT. Previously [13] methylene reductase from M. barkeri was studied in the direction of methylene-H,MPT reduction coupled to F~2,H ., oxidation. The enzyme, howexer, may also catalyze the reverse reaction, viz. methyI-H4MPT oxidation. In fact. the enzyme was routinely assayed here in this direction. The spcctrophotometric assay performed in the presence of excess methylene dchydrogenase resulted into the formarion of methcnyl-H4MPT and F4z,~H=. As reported befi~re [8]. the stoichiomet~ deduced from thc spectral changes was in accordance with Eqn. (5) (data not sho'xn ): meIhsI-H~MPT+ 2 F.:, .~ 5.11)-melhen.'~l-it4MPT + 2 F4zl~ll 2

(5)

It is of interest to note that no methyl-H ~MPT oxidation was observed when methylene dehydrogenase was omitted, either here or with the enz2,.me from M. thermoautotrophicum [8]. Incubation of methylene-H~MPT, which was nonenzymically formed from H4MPT and excess formaldehyde, with methylene dehydrogenase and -reductase purified from ~t. barkeri and catalytic amounts of F4~~

298 rr, Al 1%1 . . . .

Absorbance at 26~r~ ....................

100

7~

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......

_

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,

25

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265

290

315

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Wavel, ength

19,82

i

,

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1G25 11117

15.25

t ~

t ~

t,,

8

12

Fig 3. Uhraviolel ab~)rption spectra of methyl-lt4MPT and its formaldehyde adduct. Spectra of lhe peak eluting at about 16 rain in Fig 2D were recorded with a diode array spectrophotometer connected to the liP liquid chromatograph (system B). The spectrum (-- - - - - ) was measured at the upslope of the peak and corresponds with mcthyI-H~MPT [3]: ( ) was recorded at the downslope,

.

®~

0

{rim)

16

...~

20

2t,

Time (mini Fil~ 2. IIPLC analysis of the pr~lucts obtained after incubalmn of melhylene-H4MPT and F~,-uwith methylene-HzMPT dehydrogenase and methylene-H~MPT reductase. The reaction mixture (2 ml)composed as specified for the dehydrogenase assay under Materials and Methods and containing 50 ng dehydrogenase and 1 pg reductase was incubated for 15 rain at . 7 C . (A) HaMPT (28 nmol): (B) methylene-H4MPT (28 nmoD prepared from H~MPT with 35-iotd exee~ formaldehyde: (C) methyl-H4MPT (5.2 nmol: R t = i5.88 mini and F4z(~ (11.6 nmol. R , - 19.82 mini; (D) 75 /al of the reactton mixture; the compound ~ith R~ = 18.17 is 5.10-methenyl-tt~MPT: (E) melhyl-H~MPT (5.2 nmol) treated with I ,umoi fo~matdeh)de. Analyses were performed as de~ribed under Materials and Methods using system CA),

followed by analysis of the reaction mixture by HPLC, showed the disproportionation of methylene-H4MPT into methenyl-H4MPT (retention time R, = t8.17 mini and methyl-H4MPT (R,--- 16 mini (Fig. 2D). During incubation actually two compounds were formed with an R t of about 16 rain. One compound with an R, = 15.88 min, which was present as a shoulder of a major peak, had the retention time and the characteristic absorbance maximum at 295 nm [31 of authentic methyi-H.~MPT (Fig. 3). The ultraviolet absorption

spectrum of the major component (Rt = 16.25 mini was quite different (Fig. 3). That component was also non-enzymically produced from methyi-H4MPT and formaldehyde (Fig. 2E) and represents an adduct of formaldehyde to methyI-H~MPT, presumably to its pterin moiety. Binding of formaldehyde is a reversible one. MethyI-H4MPT prepared from the methylene derivative and subsequently purified by column chromatography lacked the Rt = 16.25 min compound. The disproportionation of m e t h y l e n e - H 4 M P T into methenyI-H.tMPT and both methyl derivatives went to completion. At the end of the reaction methylenett4MPT was no longer detected; methenyI-H4MPT and the methyl forms were present in a 1:1 ratio as estimated from the peak areas (Fig. 2D). F4,v} was strictly, though catalytically, required in the disproportionation and no reaction occurred in its absence (not shown). F4:0 is a quite specific co-substrate of the methylene dehydrogenase and -reductase. The natural electron carriers FAD. FMN. NAD ~ and NADP ~ as well as the artificial electron carrier benz'yi viologen could not substitute for F~2~ (not shown). F* and F4, two F4~ derivati'~es which lack the lactyiglutamyl and the 5'phosphoryllactylglutamyl residues in the sidechain, respectively, ,,,,'ere partially active (Table !i!). In contrast, F3~~ did not serve as an electron acceptor.

Kinetic properties of the methylene dehydrogenase and methylene reductase reactions Methylene dehydrogenase and reducta.~ showed pH optima at 6 and 6.7, respectively. For both enzymes the reaction rates increased at increasing temperatures up to 60-65°C (Fig. 4). Hereabove the velocities rapidly decreased, presumably as a result of thermal denaturation. From the slopes of Arrhenius plots activation energies amounting to 22.5 and 35 k J / m o l may be

299 '..iv Pnm/nrn0~'~

TABLE !!!

1/app V~.~ !~m,'n'm..:',l -

®

Relatire reactmn rales qf the 5./O-methylene-HzMPT dehydr¢,eenaw and 5.lO-rnethylene-H4MPT reducta.~e with F~2o dertrati~ e~ Reaction rates for the methylene dehydrogena,,e reaction g.crc mc.=sured in the direction of methylene~H4MP'l- oxidation in the presence of 50 ng enzyme. 76.5 /zM methylene-H~MPT and electron carrier as indicated. Me!hy!ene reductase wa~, measured in the direction of methyI-H4MPT oxidation in the p r e ~ n c e of i p g methylene dehydrogenase (5.5 U) purified from M thermoautotrophicum, 65 p M meIhyI-H4MPT and electron carrier as sT,ccified Reaction rates in the presence of F~2~,~vere set as 100':~- and equalled 32 n m o l / m i n and 31 nmol/min for the methylene dehydrogenase and -reductase reactions, respectively

3

O2

-

"* 78

li

0 O2

06

e"

03

:;"

2

i ,

I~

~0

0

~1

02

0'3

O't,

0'5

Relative reaction rate ( % )

Electron carrier (/zM)

F.,z. F~ F0 F3~

20

(63) (58) (63) (63)

dehydrogenase

reductase

t00 51 39 0

100 13 0 0

1It [~nlnrnoit . . . . . . . . . . . . . . . . . . . .

® n~

calculated for the dehydrogenase and reductase, respectively. Lineweaver-Burk plots of the methylene dehydrogenase reaction with varying concentrations of methyiene-H4MPT and F420 yielded a series of non-parallel lines intersecting the abscissa at one point left of the ordinate (Fig. 5A and C). Reciprocal repiots of the Actnwly (Ulmgl /

1200 /' / ' ¢

//

/O'

/

/

./

~.,.

/

./

600

i

._

02

0

01

02

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O~

05

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IJ.

Fig. 5. Line~eavcr-Burk kinetic plots of the methylcne-H4MPT dehydrogen~e from M. harken. The ~pectrophotometric a s ~ were performed as described under Materials and Methods in the presence of 6.3 ng purified end/me, in (A) reciprocal velocities of the reactions are sho~n versus reciprocal meth~'lene-H a MPT concentrations in the presence of F,~0 ( ~ M ) as indicated. In (C) reciprocal vekg-ities are plotted as a function off reciprocal F4~ concenlrations with meth~tene-H4MPT I p M ) as indicated_ In (B~ and (D) reciprocal relqots of the apparent I/tm;~ti value~ deri~ed from (A~ and (C) versus F~_~ and methylene-H ~MPT. re...l~:Ctivety, are sbar~n.

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/ O-

J

01 711

,/

Z ' 30

!2

i 60

50

2~.

~

70

tet~erat ~ e i "Cl Fig. 4 Teml~erature ,)prima of the m e l i . y l e n e - H ~ M P l dehydrogenase and methylene reductase from M harken, hctn,,itars of the dell~'drol~ ( 0 ) and rcducl,'L~ (.o) were measured ~s des,c~t'~o under Materiah and %4ethods a~ pH 6 ~ ,~nd pH 7, r e s ~ i ~ b , , bolh in I00 mM Bi.~xri~ propane buffer, in ,.Ire reset Arrhenius plots for the dehydrogenas¢ i ~ I and rc~u~,z.:~ (e) are sho~n.

apparent V ~ versus the Fa~ or methylene-H4MPT concentrations agaili yielded straight lines (Fig. 5B and D). This pattern is characteristic of a ternary complex (sequential) mechanism. From the data presented in Fig. 5 the kinetic constants can be calculated {Table IV). By the same prc~'edure it was found that the methylene reductase reaction in the direction of methyt-HaMPT oxidation also proceeds by a ternary complex mechanism. The kinetic constants derived from Lineweaver-Burk plots and replots are given in Table IV. Comparison of our data and those relx~rted b~ Ma and Thauer [131 shows that the V,~, for methylene reduction exceeds the one for methyl oxidation by a factor 11. We also found methylene reduction with F.~r~H: to proceed up to i0 tim~:s faster than the reverse reaction. From the concentn~.tions of substrates and products present at the end ,ff the F4~jH: and

3(X) T A B L E IV

Strt, tt4ral ond kinetic prol)ertics o1 F~,,-d('p(vulcnt 5. I(t-methylenc-H 4 AIPT deh)'drogenase ~.vld 5. lO-metlo'tene-tf 4 M P T reductase .[rom methanogenic ha('t~'ria Organism (strain)

En~me (subunit. kl)a: contigu rat ion )

Kinetic coa.~tants "

Literature

,AT. barkeri (MS)

methylene dehydrogenasc (35: a~,l

Km(Ctt : = ) = 6 Km(l:42.~ = 18

this paper

M. therm,~aut(m'ophicum ( A It t

,ncthylcne dchydrogenasc (36: a , )

Kca t = f 650 K , , ( C H 2 = ) = 33 Kin( F4zo) = 65

M. harken (MS)

methylene reduetasc (38" a~ )

M. harkeri (Fusaro)

methylene reduetase (36: a 4)

M. thermoautotrophicum ( A t ! )

naetllylenc reduct tsc (35: a~ ) methylene reductase (36; a 4)

Vm~,,= 3[~x)

M~ thermoaototrophicum (Marburg)

K,.,,t = 2400 Km(CH 3 ) = 250 Km(F~z. ) = 41) 1~.~,,. = 2iX) K~.,. = 127 Km(CH _. = )-~ 15 Km(Fa2.H 2) = 12 I-~,,,,,= 2 200 h'c,,t = ! 380 nol d e t e r m i n e d Km(('lt_. ~ )= 3(Xl Km(F4:o|t21 = 3 llm,,~ ~ 6(XX) K~. I - 3 6(X)

[5]

this paper

[13]

[8] [9]

" K m vai~es are given in taM: 1.~,,~ is tamol min -i mg 1; K,., 1 is s i. ( C H , = ). 5 . 1 0 - m e t h y l e n e - t l 4 M P T ; ( f l t . ~ ) . m e t h y I - H 4 M P T . The kinetic constants were d e t e r m i n e d a: the following temperatures: 37°C. methylene d e h y d : o g e n a s e and reductase from M. barkeri (MS); 60°C, methylene dehydrogenase from M. thermoautotrophicum (.1|i): 55°C, methylene reductase from M. barkeri (Fusaro) and from M. therrnoautotrophicum (Marburg).

methylene oxidoreduction a K,:q = 11.2 for reaction (3) was estimated. Mon(walent cations did not stimulate methyleneHnMPT dehydrogenasc. Sodium chloride, for instance, tested in concentrations up to 1 M had no effect on the reaction rate. Neither was Na ÷ required for the methylene reductase reaction: with enzyme- and cofactor preparations purified with sodium-free buffer systems. reaction rates measured in the presence and absence of 150 mM sodium chloride were the same. Discussion

In M. barkeri cultured on methanol both methyleneH4MPT dehydrogenase and methylene reductase are present (this paper, Ref. 13). Both enzymes behave as soluble proteins, since their activities were exclusively found in the supernatant after cell rupture and subsequent centrifugation, in the supernatant they are major proteins and comprise each about 1% of the solublc fraction. F~2o-dependent methylene dehydrogenase and -reductase from M, barkeri share many properties with the respective enzymes isolated from M. thermoautotrophicum (Table IV). The dehydrogenase from both

organisms consisted of hexamers of a single 35-36 kDa subunit, In the absence of 1=42o no reaction was observed with the dehydrogenase from M. barkeri, indicating that protons were not used as co-substrate. From M. thermoautotrophicum two different types of methylene dehydrogenase could be isolated, an F.~20dependent enzyme [4-6], and an enzyme which is capable of reducing protons and oxidizing hydrogen [7]. Unlike F42,-dependent dehydrogenase from M. thermoautotrophicum [4,5] the enzyme from M. barkeri was not stimulated by high concentrations of monovalent cations. Except for methylene reductase obtained from the A H strain of M. thermoautotrophicum, which consists of one 35 kDa polypeptide [8], the molecular composition of the enzyme purified from the Marburg strain of the organism and from two strains of /14. barkeri is quite similar, viz. a tetramer of a single 36 to 38 kDa subunit (ReL 9 and 13, this paper). The reductase from the methanogens catalyzed both methyleneH4MPT reduction and methyI-H4MPT oxidation (Ref. 8, this paper). As pointed out before [8], the reaction is in a number of respects an analogon of the NAI~P)Hdependent 5,10-methylenetetrahydrofolate reduction. Methylenctetrahydrofolate reductase was purified as a flavoprotein from a number of sources, but the enzyme

301 only catalyzed methylene reduction [20-22]. The methanogenic methylene reductases as well as the dchydrogenases listed in Table IV all lacked cofactors like flavins and Fe-S centers. They all required F~,, as the co-subs[rate in reactions according to ternary complex mechanisms. Since F4z() acts in hydride transfer [23], the enzyme kinetics and the absence of specific eofactors suggest that the methylene dchydrogenase and reductase reactions proceed by direct hydride transfer between the reactants. The 8-OH adcnylylated F42o ( F 3 , m) did not serve as a substrate (Ref. 5 and 8, this paper). F3,~) is synthesized from ATP and F~_,() when growing cells [24] or cell extracts of M. thermoautotrophicum [16] and M. barkeri [25] are exposed to oxygen. In M. thermoautotrophicum methylene dehydrogenase and reductasc function in the central metabolic pathway of C O , reduction to methane. From the purification factor and the V,,,,,, of the purified dehydrogenase it can be estimated from [51 that cells of the organism may convert m e t h y l e n e - H ~ M P T into methenyI-H.~MPT with a V,,:,~ = 27 U / m g protein; the velocity of the reversed reaction as it occurs during C O , reduction, however, will be about 3-fold lower [4]. A Vm,,~,= 6(1 U / m g cell protein was calculated for methylene-H.~MPT reduction [9]. For methyleneH a M P T oxidation in M. barkeri the I/re,~ = 36 U / r a g cell protein [this paper). M e t h y l e n e - H a M P T reduction in the organism occurs with a I/~,,,~= 17 U / r a g cell protein (at 50°C) [13], whereas for the reversed reaction, methyi-H )MPT oxidation, a L~,,,,~= 2.9 U / r a g cell protein holds at 37°C (this paper). M. thermoautotrophicum and M. barkeri, thus, contain comparable levels of F4,0-dependcnt methylene dehydrogenase and reductase. It should be noted, however, that the various reaction rates and kinetic constants determined for M. barkeri may represent underestimates, since they all have been measured with H 4 M P T and F~zo derivatives isolated from M. thermoautotrophicum. The coenzymes present in M. barkeri both contain additional glutamvl moieties attached to their sidechains [26.27]. From Table Ill, for instance, the effect of the length of the side chain is obvious and it may be noticed that F~.,, derivatives that lack parts of it were less active in the dehydrogenase and reductase reactions. 114. barkeri growing on methanol with a doubling time at 37°C of about 12 h converts its substrate according to Eqn. 2. During growth the organism produces methane with a maximum rate of 0.1-(I.2 #tool rain - t mg -~ protein [28,29]. Hence. C O , must be formed at a maximum rate of 0.035-0.07/a tool per rain per rag. From the data presented above it follows that the organism, thus, contains by far enough methylene reductase and dehydrogenase to allow methanol oxidation to proceed via methyl-H~MPT and mcthylencH4MPT. In fact, the two enzymes from M. harkeri

seem to be more apted to oxidation of the H~MPT derivatives. From Fig. 4 it can bc seen that at 60°C the reductasc from M. barkeh catalyzes mclhyl-H)MPT oxid::.tion at a rate of about 72 l.I/mg, which is nearly l()-fold higher than the reaction rate measured in that direction and at that temperature with the en~'me from M. thermoautotrophicum (7.38 U / r a g , [8]). In addition. K~ values for methylene-H~MPT and F4_~. were 5.5- and 3.5-fold lower for the M. harkeri dehydrogenase (Table IV). in this respect it is remarkable thai in the in vitro assays methyI-H~MPT oxidalion catalyzed by the reductase did not occur unless dehydrogenase was present. The latter enzyme may be required to overcome the untavorable reaction equilibrium (K~.,~ = 11.2) and the high activation energy (35 k j / m o l ) of the reductase by rem~wing methylencH 4 M P T from the reaction.

Acknowledgements The work of J.T. Keltjens was made possible by a senior fellowship of the Royal Netherlands Academy of Arts and Sciences (KNAW). Mr. R. Dijkcrman is grcatfutly acl,~]owlcdgcd for performing HPLC analyses.

Note added in proof: During the publication of this manuscript a paper was published by Enssle. Zirngibl. Linder and Thaucr (Arch. Microbiol. (1991) 155, 483-490) in which the m e t h y l e n e - H t M P T dchvdrogenase from M. harkeri was dcsc~ ibcd.

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