Volume 190, number 1 FEBS 2962 October 1985

Sirohydrochlorin, a precursor of factor F,,, biosynthesis in Methanobacterium thermoautotrophicum

Helmut Mucha, Eberhard Keller*, Hans Weber, Franz Lingens+ and Walter Triisch

Fraunhofer-Institut fiir Grenzftichen- und Bioverfahrenstechnik, Nobelstrasse 12, D-7000 Stuttgart 80, FRG

Received 1 August 1985

Factor F,, is a nickel-containing coenzyme of methanogenic bacteria with porphinoid structure which is derived from III. It is shown that sirohydrochlorin is metabolized by cell free extracts of Methanobacterium thermoautotrophicum to factor F, demonstrating that this compound, or a reduced form of it, is an intermediate in the biosynthesis of F,, and not only of vitamin B,, and .

Factor F,30 Sirohydrochlorin &AminolevuIinic acid S-Adenosylmethionine Methanobacterium thermoautotrophicum

1. INTRODUCTION

Factor F430, a yellow non-fluorescent nickel- Uroporphyrrngen IJI containing chromophore, is the coenzyme of the 2 x methylatlon derarboxylotlon / methylcoenzyme M reductase [ 11. The structure of \ SAM this compound has been previously shown to con- Protoporphyrln Ix Sirohydrochlorln sist of an uroporphinoid (type III) ligand skeleton with an attached carbocyclic ring. The chromophore system is a tetrahydro derivative of Swoheme Cvtochromes the corphin system which combines the structural ‘a pJ pJ elements of both and . Such a lngl system has not been previously encountered among Fig. 1. Biosynthetic pathways of natural . natural porphinoids [2,3]. It has been shown that factor F430, like all naturally occurring por- Our intention was to investigate the role of phinoids (chlorophylls, hemes, cytochromes, sirohydrochlorin in the biosynthesis of F430 by sirohemes and corrinoids), is synthesized via studying the incorporation of labeled sirohydro- uroporphyrinogen III (fig.1) [4]. It is also known (3H, 14C) into F430 by cell-free extracts of that factor F430 contains 2 methyl groups derived Methanobacterium thermoautotrophicum. from methionine [5] and that methanogenic bacteria contain corrinoids. From this information 2. MATERIALS AND METHODS it has been suggested, that F430 is synthesised via sirohydrochlorin or a dihydrosirohydrochlorin. 2.1. Culture conditions and preparation of cell free extracts * Present address: BASF AG Landwirtschaftliche Ver- M. thermoautotrophicum AH, DSM 1053, was suchsstation, D-6703 Limburgerhof, FRG grown in medium 1 described by Balch et al. [6]. + Institut fiir Mikrobiologie der Universitlt Hohen- This medium was supplemented with 6 mg/l heim, Garbenstrasse 30, D-7000 Stuttgart 70, FRG NiClz - 6H2O, which was omitted in the last scale-

Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/85/$3.30 0 1985 Federation of European Biochemical Societies 169 Volume 190, number 1 FEBS LETTERS October 1985

up step of the cultivation to suppress the feed-back After 24 h the resulting methyl esters were ex- inhibition of the F430 biosynthesis. The bacteria tracted with chloroform. After evaporation of the were cultivated either in 1 1 bottles containing chloroform, the residue was dissolved in 0.5 ml 200 ml medium or in a 20 1 fermenter with Hz/C02 methanol, applied on silica gel 60 TLC plates (80: 20, v/v) as carbon and energy sources. After (Merck) and developed with methanol/ethyl centrifugation and washing the cells were suspend- acetate/chloroform (1: 2 : 3, v/v). The UV-Vis ed in an incubation buffer (pH 6.8), containing spectra and the radioactivities were measured. The 0.01 M potassium phosphate, 0.06 M NaHCO3, fractions absorbing at 430 nm were separated by 0.01 M MgCl2.6Hz0, 20 mg penicillin, 1OOyg HPLC (RP-18) and eluted either with a gradient of mercaptoethanesulfonate, 1 mg DNase (pancreas) 0.01 K-phosphate buffer, pH 6.8, and 80% from Merck (Darmstadt), and disrupted by 2 methanol in water, as described by Livingston et subsequent anaerobic passages through a French al. [3], or with methanol. The absorbance of the pressure cell. The cell debris was separated by cen- eluate was recorded at 206 and 430 nm. The trifugation (Beckman JA-20 rotor, 18000 rpm, radioactivity of the fractions containing F430 M 5”C, 20 min) under an Nz atmosphere and the were measured with a beta-counter. supernatant (crude extract) was used for the studies. 2.5. Analytical methods The UV-Vis spectra were recorded on an 2.2. Metabolization of 5- by HP 8450 A spectrophotometer. Molar extinction crude extracts coefficients C30(H20) or 23 100 cm2/mmol and 2 mg 5-amino[4-r4C]levulinic acid (ALA) &o(CH~OH) of 21900 cm2/mmol [3] were taken (7860 Bq) were added to 40 ml crude extract. The as a basis for the calculations. The radioactivities reaction was performed in 120 ml serum vials (gas were determined with a rackbeta II (LKB, Wallac) atmosphere H2/C02 = 80:20 (v/v)) at 55°C at and the Unisolve 100 scintillator system (Zinsser, 120 rpm. Frankfurt).

2.3. ~et~bo~iz~tion of siro~ydroc~~ori~ by crude 3. RESULTS AND DISCUSSION extracts 10.43 pmol [2,7-methyf-3H, 4,5,9,10,14,15,16, Thauer and co-workers [2-51 used growing and 20-‘4C]sirohydrochlorin were added to 44 ml crude resting cells for biosynthetic studies to produce extract. The specific radioactivity was 77.7 Bq/ factor F430 and analyze its structure. For the pre- pmol consisting of 515 Bq 3H and 295 Bq 14C and sent experiments a cell-free system was prepared in some experiments additionally 8300 Bq 63NiC12. because it is unlikely that sirohydrochlorin is incor- Sirohydrochlorin was isolated and purified as the porated by intact cells. In a first experiment we octamethyl ester [7]. The reaction was performed proved the operability of the cell-free system and as described in section 2.2. investigated the incorporation rate of ALA into factor F430. The methylated factor (F430 M) was 2.4. rso~~t~on of the metabolic products purified by subsequent TLC and HPLC An equivalent volume of cold methanoi (4°C) chromatographic~ methods to approximately con- was added to the incubation mixture. After cen- stant specific radioactivity. However, the working trifugation (18000 rpm, 5’C, 20 min), the sedi- up and purification procedure was terminated ment was extracted with several batches of 20 ml after a second HPLC run because of appreciable methanol by heating for 1 h at 80°C. The extracts losses of factor F430 (table 1). About 5% of added were recovered by evaporating the solvent, and ALA was incorporated into F430. After the second then redissolved in 5 ml of 25 mM K-phosphate HPLC step, however, only 1.2% of the initial buffer (pH 7.0) and adsorbed on DEAE-Sephadex radioactivity applied as [14C]ALA could be found A-25 [S]. The column was washed with the same in the F430 M fraction. This loss of radioactivity buffer and dried overnight. Thereafter, the bound can be accounted for by the loss of sample during metabolites were esterified (methanol/H$S04 = the working up procedure. The isolated F430 M did 95 : 5 (v/v)) to yield the F430methyl esters (F430 M). not contain any detectable impurity as confirmed

170 Volume 190, number 1 FEBS LETTERS October 1985 by UV-Vis, TLC and HPLC. As with [14C]ALA, chlorin by the crude enzyme extract of M. thermo- radioactivity was incorporated with either L-S- autotrophicum. The reaction mixture containing [methyl-3H]adenosylmethionine (the methylating cell-free extract, sirohydrochlorin and NiClz (fig.2, agent) or with 63Ni (not shown). plot 1) was incubated anaerobically. UV-Vis spec- Approximately the same amount (5%) of the tra were recorded as indicated. Sirohydrochlorin labeled sirohydrochlorin was incorporated into concentration decreased and a novel absorption F430 (table 2). The observed large decrease in band at 420 nm appeared. As sirohydrochlorin is a specific radioactivity after the second purification rather unstable compound, a control experiment step reflects the removal of other sirohydrochlorin- was performed. The UV-Vis spectra demonstrate derived substances. After further purification steps that sirohydrochlorin is degraded in the absence of (2 TLC and 2 HPLC runs) the specific radioac- the enzyme fraction within the incubation period. tivities remained nearly constant. These data show However, the redox state of the true biological in- that sirohydrochlorin, or a derivative of termediate could not be identified from these data, sirohydrochlorin (i.e. a reduced form) is incor- because no spectrophotometric data are available porated into F430. for reduced sirohydrochlorins. To distinguish between the incorporation of Our experiments show strong evidence that sirohydrochlorin or a reduced form we studied the sirohydrochlorin, or a reduced form of it, is, as effect of crude extract on sirohydrochlorin with suggested by Gilles and Thauer [4], an in- spectrophotometric methods. Fig.2 shows the termediate of F430-biosynthesis. time-dependent metabolisation of sirohydro- ACKNOWLEDGEMENTS 0.40 We are indebted to Dr G. Mtiller, Institut fur Organische Chemie, Biochemie und Isotopen- 0.350 forschung, Universitat Stuttgart, for his generous contributions of labeled sirohydrochlorin and to Professor H. Chmiel, Fh-IGB, for supporting this study.

0.250 REFERENCES l? Ellefson, W.L., Whitman, W.B. and Wolfe, R.S. f VI P 0.20 (1982) Proc. Natl. Acad. Sci. USA 79, 3707-3710. B Pfaltz, A., Jaun, B., Ftissler, A., Eschenmoser, A., 2 121 Jaenchen, R., Gilles, H.H., Diekert, G. and Thauer, 0.150 R.K. (1982) Helv. Chim. Acta 65, 828-865. 131 Livingston, D.A., Pfaltz, A., Schreiber, J., Eschenmoser, A., Ankel-Fuchs, D., Moll, J., Jaenchen, R. and Thauer, R.K. Helv. Chim. Acta 67, 334-351. 141Gilles, H. and Thauer, R.K. (1983) Eur. J. Biochem. 135, 109-112. 151 Jaenchen, R., Diekert, G. and Thauer, R.K. (1981) FEBS Lett. 130, 133-136. 161 Balch, W.E., Fox, G.E., Magrum, L.J., Woese, C.R. and Wolfe, R.S. (1979) Microbial. Rev. 43, 260-296.

WAVELENGTH (nm) [71 Mtiller, G., Gneul3, K.D., Kriemler, H.-P., Scott, A.I. and Irwin, A.J. (1979) J. Am. Chem. Sot. 101, Fig.2. Difference spectra of anaerobic incubations of 3655-3657. sirohydrochlorin in cell-free extracts of M. thermo- PI Keltjens, J.T., Caerteling, C.G., Van Dijk, H.F. autotrophicum after 5 min (plot 1) and 4 h (plot 2). and Vogels, G.D. (1983) Arch. Biochem. Biophys. 223, 235-253. 171