Formylmethanofuran: Tetrahydromethanopterin
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Research Article 635 Formylmethanofuran: tetrahydromethanopterin formyltransferase from Methanopyrus kandleri — new insights into salt-dependence and thermostability U Ermler1*, MC Merckel1, RK Thauer2 and S Shima2 Background: Formylmethanofuran: tetrahydromethanopterin formyltransferase Addresses: 1Max-Planck-Institut für Biophysik, (Ftr) from the methanogenic Archaeon Methanopyrus kandleri (optimum growth Heinrich-Hoffmann-Straße 7, 60528 Frankfurt, Germany and 2Max-Planck-Institut für terrestrische temperature 98°C) is a hyperthermophilic enzyme that is absolutely dependent Mikrobiologie and Laboratorium für Mikrobiologie, on the presence of lyotropic salts for activity and thermostability. The enzyme is Philipps-Universität, Karl-von-Frisch-Straße, 35043 involved in the pathway of carbon dioxide reduction to methane and catalyzes Marburg, Germany. the transfer of formyl from formylmethanofuran to tetrahydromethanopterin. *Corresponding author. E-mail: [email protected] Results: The crystal structure of Ftr, determined to a resolution of 1.73 Å, reveals a homotetramer composed essentially of two dimers. Each subunit is Key words: crystal structure, formyltransferase, subdivided into two tightly associated lobes both consisting of a predominantly halophilic enzymes, hyperthermophilic enzymes, antiparallel b sheet flanked by a helices forming an a/b sandwich structure. The methanogenic Archaea, Methanopyrus kandleri approximate location of the active site was detected in a region close to the Received: 1 January 1997 dimer interface. Revisions requested: 29 January 1997 Revisions received: 1 April 1997 Conclusions: The adaptation of Ftr against high lyotropic salt concentrations is Accepted: 1 April 1997 structurally reflected by a large number of negatively charged residues and their Structure 15 May 1997, 5:635–646 high local concentration on the surface of the protein. The salt-dependent http://biomednet.com/elecref/0969212600500635 thermostability of Ftr might be explained on a molecular basis by ionic inter- actions at the protein surface, involving both protein and inorganic salt ions, and © Current Biology Ltd ISSN 0969-2126 the mainly hydrophobic interactions between the subunits and within the core. Introduction methanogenesis [7]. This is a unique biological pathway Formylmethanofuran: tetrahydro-methanopterin formyl- involving novel coenzymes and enzymes [8,9]. The struc- transferase (Ftr) from Methanopyrus kandleri (a hyperther- tures of all the coenzymes have previously been eluci- mophilic Archaeon with an optimum growth temperature dated [10] but the study presented here reports the first of 98°C) is a soluble enzyme composed of only one type of structural analysis of an enyzme of the methanogenic subunit with an apparent molecular mass of 35kDa [1]. At pathway. low concentrations (<0.1M) of lyotropic salts (K2HPO4 or (NH4)2SO4) the protein is present in a catalytically The crystal structures of only four enzymes from hyper- inactive, monomeric state and denatures rapidly at thermophilic organisms (growth temperature optimum temperatures above 50°C. At high salt concentrations Ftr above 80°C) have, until now, been published: aldehyde assembles into a homotetramer, is active and stable to ferredoxin oxidoreductase from Pyrococcus furiosus (100°C) nearly 130°C (SS, UE, H Fukada, K Takahashi and RKT, at 2.3Å [11]; glutamate dehydrogenase from P. furiosus at unpublished results). The gene encoding Ftr from M. kan- 2.2Å [12]; glyceraldehyde-3-phosphate dehydrogenase dleri has been cloned, sequenced and functionally overex- from Thermotoga maritima (80°C) at 2.5Å [13]; and indol-3- pressed in Escherichia coli [2]. The enzyme, which is stable glycerol phosphate synthase from Sulfolobus solfataricus under oxic conditions, is devoid of a chromophoric pros- (87°C) at 2.0Å resolution [14]. We report here the crystal thetic group. Ftr catalyzes the transfer of a formyl group structure of Ftr from M. kandleri at 1.73Å resolution. Ftr from formylmethanofuran to tetrahydromethanopterin via differs from the other four enzymes studied in not only a ternary complex type catalytic mechanism [1]. For com- being adapted to high temperatures but also to high con- parison the primary structures of Ftr from Methanosarcina centrations (>1M) of lyotropic salts present in the barkeri (optimum growth temperature 37°C) [3], Methano- methanogenic Archaeon [15–17]. The tolerance of Ftr to bacterium thermoautotrophicum (65°C) [4], Methanothermus lyotropic salts, like K2HPO4 or (NH4)2SO4, provides a fervidus (83°C) [5] and Methanococcus jannashii (85°C) [6] close relationship to halophilic proteins although those are are available. optimally adapted to high salt concentrations of NaCl, KCl or MgCl2. The observed high salt adaptation and salt- In M. kandleri, which grows on H2 and CO2 with the dependent thermostability are interpretated on the basis formation of methane, Ftr catalyzes a reaction involved in of the crystal structure. 636 Structure 1997, Vol 5 No 5 Results coordinate error was determined to be 0.11Å, as obtained Structure determination from the diffraction data precision indicator sw(x) [19]. The crystal form P of Ftr was grown at a salt concentration The Ramachandran plot [20] indicated that all non- of 0.3M (NH4)2SO4 [18], where the protein in solution is glycine residues have favourable dihedral angles. The present partly in the monomeric form (see Introduction). average per residue real-space correlation [21] between However, the biologically relevant tetrameric form was the model and the 2|Fo|–|Fc| electron density is 0.86; no observed in the crystal structure. Obviously crystallization residue has a value below 0.7. in 0.3M (NH4)2SO4, pH7.0 at high polyethylene glycol concentrations also favours oligomeric assembly. An overlay of the four monomers in the asymmetric unit reveals a nearly identical backbone conformation reflected The structure of Ftr in the tetrameric form has been by a low overall rms deviation of 0.25Å, using only the Ca determined by the multiple isomorphous replacement atoms for superposition [21]. (MIR) method. After improving the initial map by solvent flattening, histogram matching and fourfold molecular Monomer structure averaging the polypeptide chain could be traced. The A single subunit of Ftr has a size of 30Å×40Å×70Å and model has been refined to a conventional and free R factor can be subdivided into two tightly associated lobes with a of 19.2% and 24.3%, respectively, in the resolution range small intermediate crevice (Fig. 2). Both the N- and the 1.73–10.0Å. The model is composed of all 4×296 residues C-terminal ends of the polypeptide chain are located in (8884 atoms) and 188, 150, 181, and 163 water molecules the same lobe, which is therefore named the proximal (present in subunits 1–4, respectively). An alternative lobe. The other lobe is consequently designated the distal conformation was only found for Cys58. The root mean lobe. The proximal lobe consists of residues 1–17 and square (rms) deviation of the polypeptide model from the 163–296, the distal lobe of residues 18–162. Both lobes ideal stereochemistry is 0.008Å for bond lengths and 0.97° belong to the class of a+b structures. The proximal lobe for bond angles. The average temperature factor for the is basically built upon a four-stranded antiparallel b sheet protein is 22Å2. The quality of the final electron-density enlarged by two small strands (1 and 2) close to the N-ter- map is documented in Figure 1. There are no chain minal end. The central b sheet is surrounded by two breaks. An alternative conformation was only found for helices (D and E) at the front which pack against the Cys58. Poor electron density was observed for several distal lobe and the so-called insertion region, a region charged solvent-exposed sidechains: Glu111, Glu133, which is only loosely attached to the back of the central Glu136, Glu232, Asp233, Glu238, Lys241 and Glu254. A b sheet. This highly unusual insertion region extends structure obtained from a high salt crystal form (not between residues 198–241 and contains only two short, available yet) could distinguish, whether the high temper- antiparallel oriented b strands (13 and 14) as secondary ature factors are a consequence of their solvent-exposed structure elements (Figs 2,3). The distal lobe comprises a positions or of the low salt concentration. The average seven-stranded b sheet composed of a four-stranded Figure 1 Representative section of the final 2Fo–Fc electron-density map at 1.73 Å resolution, contoured at a 2s level. The region portrays a 71 71 portion of b strand 5 and helix A of the distal lobe (see Fig. 2). 32 32 7743 77 43 Research Article Structure of a formyltransferase Ermler et al. 637 Figure 2 (a) (b) 1 240 1 240 2 1 16 280 296 280 296 15 13 14 180 180 17 260 260 D 220 220 E 200 200 11 12 80 80 4 20 20 3 60 60 5 40 40 160 160 A 7 B 120 120 100 100 6 140 140 C 10 8 9 Structure of the Ftr monomer from M. kandleri. (a) ‘Paint ramp’ are shown as red arrows, a helices as green spirals and loops are in coloured stereoview of a Ca trace of Ftr labelled at every twentieth yellow. (The figure was drawn with the program MOLSCRIPT [53] and residue. (Figure generated with the program O [21].) (b) Ribbon the secondary structure elements were calculated with the program diagram of Ftr showing the secondary structure elements. b Strands DSSP [54].) antiparallel b sheet combined with a b meander motif which are isoleucines. A preference for isoleucines was consisting of a three-stranded antiparallel b sheet. The also described for a thermostable glutamate dehydroge- whole b sheet is flanked by three a helices (A, B and C) nase [12]. The hydrophobic core in the distal lobe is on its backside and partly by the proximal lobe on its formed by residues of the b strands 3, 5, 6, 9 and 10 and by frontside (Fig.