Characterization, cloning, and in vitro expression of the extremely thermostable glutamate dehydrogenase from the hyperthermophilic Archaeon, ES4.

Glutamate dehydrogenase (GDH) from the hyperthermophilic Archaeon ES4 (optimal growth temperature 98 degrees C and maximum growth temperature 110 degrees C) was purified to homogeneity. The purified native enzyme had an M(r) of 270,000 +/- 5,000 and was shown by gel filtration and SDS-polyacrylamide gel electrophoresis to be a hexamer with identical subunits of M(r) = 46,000 +/- 3,000. The hexameric subunit composition was also evident from electron micrographs, which show a triangular antiprism structure very similar to that of bovine GDH. The enzyme is exceptionally thermostable, with a half-time of inactivation of 3.5 h at 105 degrees C. Differential scanning calorimetry revealed a tm for denaturation of 113 degrees C, and a tm for activation at 60 degrees C. Antigenic cross-reaction with ES4 GDH was observed with the purified GDH from the thermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis as well as with bovine and yeast GDHs. The genome of ES4 was shown to contain a single copy of the gdhA gene, and this was cloned and sequenced. The deduced amino acid sequence of the GDH from ES4 corresponded to the NH2-terminal amino acid sequence obtained from the pure protein. From the nucleotide sequence the ES4 protein is composed of 420 residues. It has a relatively high hydrophobicity and a low number of sulfur-containing residues compared with mesophilic GDHs. Relatively high homology (52%) exists between the deduced amino acid sequence of ES4 GDH and Clostridium difficile GDH. Of the two distinct families of GDH sequences known, ES4 GDH belongs to the same family as vertebrates, C. difficile, and other Archaea. The gdhA gene of ES4 was expressed in vitro in a rabbit reticulocyte cell-free lysate, thus providing a system for structural studies of the mechanisms of thermostability in hyper-thermophilic proteins.

observed with the purified GDH from the thermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralia as well as with bovine and yeast GDHs. The genome of ES4 was shown to contain a single copy of the gdhA gene, and this was cloned and sequenced. The deduced amino acid sequence of the GDH from ES4 corresponded to the NHderminal amino acid sequence obtained from the pure protein. From the nucleotide sequence the ES4 protein is composed of 420 residues. It has a relatively high hydrophobicity and a low number of sulfur-containing residues compared with mesophilic GDHs. Relatively high homology (52%) exists between the deduced amino acid sequence of ES4 GDH and Clostridium difficile GDH. Of the two distinct families of GDH sequences known, ES4 GDH beIongs to the same family as vertebrates, C. difficile, and other Archaea. The gdhA gene of ES4 was expressed in vitro in a rabbit reticulocyte cell-free lysate, thus providing a system for structural studies of the mechanisms of thermostability in hyper-thermophilic proteins. This article must therefore he hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequencels) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) L12408.
ll To whom correspondence should be addressed. A group of microorganisms have recently been discovered that have the remarkable property of growing optimally a t or above 100 "C. These so-called hyperthermophiles are all members of the Archaea (1, 2). The as yet unidentified archeon, ES4, was isolated by Pledger and Baross (3). It is an anaerobic heterotroph that grows by the fermentation of peptides and carbohydrates to produce organic acids, COz and Hz. ES4 is unusual among known hyperthermophiles as it is able to grow in a defined medium, albeit slowly, using L-glutamate as the sole carbon and nitrogen source. The optimal temperature and maximal temperature for growth are 98 and 110 "C, respectively (3).
In order to investigate some of the metabolic properties of hyperthermophiles and mechanisms of protein "hyperthermostability," we have focused on glutamate dehydrogenase (EC 1.4.1.3,GDH),' which utilizes the nicotinamide-dependent oxidation of glutamate to a-oxoglutarate. This enzyme has been proposed by us to play a major role in amino acid utilization by Pyrococcus furiosus, another hyperthermophilic Archeon (4,5). GDH is also an important enzyme because of its pivotal position between carbon and nitrogen metabolism. Recently, a number of other enzymes have been isolated from hyperthermophiles, and with the exception of the Pyrodictium brockii hydrogenase (6), these studies have been applied to P. furiosus (4,5,(7)(8)(9)(10)(11)(12)(13), or the very closely related isolate Pyrococcus woesei (14). However, GDH from P. furiosus is among the most thermostable of these enzymes (5, 13). ES4 GDH was of interest because its maximal growth temperature is 7 "C higher than that of P. furiosus.
We describe here the characterization of ES4 GDH and the molecular cloning of the gene encoding this exceptionally thermostable enzyme, as well as its expression in uitru. ES4 GDH is the most thermostabIe dehydrogenase reported so far.
Comparison of the ES4 GDH sequence with sequences from thermophilic and mesophilic organisms provides information on the structural adaptation to thermostability of these enzymes and on the molecular evolution of two families of GDH encoding genes.

MATERIALS AND METHODS
NADP(H), NAD(H), and a-oxoglutarate were from Boehringer Mannheim. All other chemicals were of the highest available purity and were used without further purification.
Purification of GDH-GDH was purified from 600 g of cells (wet weight) at 23 "C. The cell-free extract was prepared by sonication of a 20% (w/v) suspension in 50 mM Tris-HC1, pH 8.0, for 16 h in a Branson sonifying bath. The subsequent purification procedure was the same as for P. furiosus GDH (4), up to and including the Q-Sepharose column. A gel filtration step using a Superdex 200 column (6 X 60 cm), equilibrated at 4 ml/min with 50 mM Tris, pH 8, containing 2 mM sodium dithionite, 2 mM dithiothreitol, 1 mM MgCl,, 200 mM NaCl, and glycerol (lo%, w/v) was employed instead of DEAE-Sephadex. These and all subsequent columns were controlled by a Pharmacia fast protein liquid chromatography system. The active fractions from the Superdex 200 column were combined, concentrated to about 20 ml using an Amicon ultrafiltration cell fitted with a PM30 membrane (Amicon, Beverly, MA), and applied to a column (5 cm X 1 cm) of Cibacron blue F3GA (Bio-Rad), in the presence of 5 mM of L-glutamate, at 24 "C. The enzyme was eluted with a 3-ml pulse of 1 mM NADP' (5). Homogeneity of the pure GDH was established by SDS-gel electrophoresis. The M, of GDH was estimated by gel filtration usinga 9 X 1.5-cm column of Sephacryl S-200HR (Pharmacia LKB Biotechnologies Inc.), operated by a Bio-Rad Econo system, with 50 mM Tris-HC1 buffer, pH 7.5, containing 50 mM NaCl as the eluent. The protein standards used were obtained Enzyme Assay-GDH activity was measured by the glutamatedependent reduction of NADP+ at 85 "C as described previously (4). One unit of activity of GDH is defined as 1 pmol of NADPH formed per minute/mg protein (4).
Electron Microscopy-A 4-pl sample of a 100 mg/ml solution of pure GDH was applied to a carbon-coated collodion-covered grid for 30 s and removed by capillary action of filter paper. The grid was negatively stained with 1% aqueous uranyl acetate. Preparations were examined in a JEOL 100-CX electron microscope operating at 80 kV. Micrographs at nominal magnifications of X 58,000 were recorded on Kodak emulsion S0163. The nomenclature for describing different views of the enzyme are those used by Josephs (17) for bovine liver GDH.
Microcalorimetry and Thermostability Determination-For determination of the thermostability of ES4 GDH at high temperatures, 1 mg/mI of the purified enzyme in 20 mM Tris-HC1 buffer, pH 7.6, containing 1 mM dithiothreitol, in the presence or absence of 250 mM potassium citrate, was placed in microcentrifuge tubes with O-ring sealed caps. Duplicate tubes were placed in a Van Waters and Rogers heat block maintained at the indicated temperatures. Control experiments in which the buffer was incubated for similar time periods resulted in no change in the pH, indicating that the buffer system was stable under these conditions. Tubes were removed at hourly intervals, chilled on ice, centrifuged briefly, and sampled for enzyme assays.
For the temperature dependence of the heat capacity, the purified enzyme was scanned over a range of temperatures from 40 to 130 "C using the differential adiabatic scanning calorimeter DASM-4 (18). The area under the curve represents the enthalpy change due to the temperature-induced activation of 1.0 mg (1.5 mg/ml) of the enzyme dialyzed against 15 mM glycyl glycine buffer, pH 8.25. The heating rate applied was 1 K . min". The absorbance of pure GDH at 280 nm was recorded with a Pye Unicam model 1800 spectrophotometer using a thermostatted and pressurized cuvette compartment and a Hellma quartz cuvette with an optical path length of 1 cm.
Protein Sequencing-Protein microsequencing was carried out on enzyme resolved by SDS-PAGE in 20 mM Tricine buffer, pH 8.2, and electroblotted onto a polyvinylidene difluoride membrane (19).
Western Blot-Pure GDH protein samples from ES4, P. furiosus, T. litoralis, bovine, and yeast were resolved by 12% SDS-PAGE and electroblotted onto polyvinylidene difluoride Immobilon-P membrane (Millipore, MA). Immunodetection was performed with a rabbit antiserum directed against pure GDH. Anti-rabbit alkaline phosphatase conjugate was used for detection.
Nucleic Acid Extraction-Cells (1.5 liter of culture) were collected by centrifugation 4,300 x g for 20 min at 4 "C, and the pellet was washed twice with 2 ml of TE buffer (10 mM Tris-HC1, pH 7.6,1 mM EDTA). DNA was extracted using the hexadecytri-methyl ammonium bromide/cesium chloride method (20). Isolation of plasmid DNA was performed by standard methods (21). Plasmid DNA for in uitro transcription was prepared by incubation of pelleted cells in 25% sucrose, 50 mM Tris-HCI, pH 8.0, 5 mg/ml lysozyme, on ice for 10 min, followed by addition of EDTA and Brij 58 to give a final concentration of 25 mM and 0.5%, respectively. Plasmid DNA was isolated in a 13,000 X g supernatant followed by CsCl density gradient fractionation and chromatography on an A15M columm (25 X 1.5 cm). Cloning and Sequencing-Genomic DNA from ES4 was partially digested with BamHI and the restriction fragments, with a size range of 14-20 kb, were cloned into the lambda Fix vector (Stratagene, CA). The lambda Fix library of ES4 was screened using a 0.9-kb polymerase chain reaction fragment of the ES4 gdhA gene obtained from the genomic DNA. The primers used to obtain this product were designed from the NHz-terminal amino acid sequence of the ES4 GDH protein and from conserved GDH sequences. The polymerase chain reaction probe was labeled with [(u-~'P]~ATP by random priming. A clone with a 17-kb insert and containing the gdhA gene was then subcloned into a pBluescript plasmid (Stratagene). The positive clones were sequenced using the dideoxy chain termination method (22).
Southern Hydridization-Genomic DNA from ES4 and P. furiosw were digested with EcoRI, EcoRV, Sau3A, and BamHI. The fragments were separated on a 1% agarose gel and transferred to a nylon membrane (Magnagraph, MSI, MA). The hybridization was carried out with the 0.9-kb fragment of the ES4 gdhA following labeling by random-primed [o-~'P]~ATP incorporation.
Expression of the GDH Gene-Polymerase chain reaction products with NcoI and BamHI sites at the 5' and 3' ends, respectively, were inserted into pTMl (23) between the NcoI and BamHI restriction sites. The T7 promoter-containing vector, pTM1, puts the 5'-untranslated region of encephalomyocarditis virus mRNA upstream of the initiation codon of gdhA, allowing efficient translation of mRNA without capping (23). RNA was produced in uitro using T7 RNA polymerase and NTPs each at 4 mM. A tracer amount of [o-~'P]UTP was included to allow quantification of mRNA transcribed. The mRNA was translated in an mRNA-dependent cell-free system, at 30 "C, containing either 1 mCi/ml [35S]methionine, or non-radioactive methionine, and other components as described (24). The specific activity of [35S]methionine in the translation system was 760 disintegrations/min/pmol, taking into consideration an endogenous methionine pool of 11.5 p~. The products were analyzed by SDS-PAGE, fluorography, and Western blot. The sizes of the GDH translation products were determined by chromatography on a calibrated Sephacryl S-200 column (0.5 X 12 cm) (Pharmacia) and scintillation counting. The chromatography fraction containing the 270-kDa protein was assayed for activity after concentration with a Centricon-100 filtration device (Amicon, MA).
Computer analysis of the sequences was done with the GENEPRO program (Riverside Scientific Enterprises, Seattle, WA). The multiple alignment was constructed using the Treealign program (25) based on the Mutation Data Matrix (26,27).

Enzyme Purification, Molecular Weight, and Enzymatic
Properties of GDH-ES4 GDH was purified 75-fold from a cell-free extract and was homogeneous when tested by polyacrylamide gel electrophoresis (data not shown). The enzyme had an apparent molecular weight of 270,000 & 5,000 M, as estimated by gel filtration on a calibrated Sephacryl S-20OHR column, and a subunit molecular weight of 46,000 +-3,000 M , as estimated by SDS-PAGE. These results suggest that it was isolated as a hexameric species like P . furiosus GDH (4). However, in contrast ES4 GDH utilizes NADP' exclusively as cofactor, whereas P . furiosus utilizes both NAD+ and NADP+ (4). Fig. 1 is an electron micrograph of a typical field of ES4 GDH as seen after staining with uranyl acetate. The hexam- Thermstability"ES4 GDH displayed total stability at room temperature or at 4 "C over a period of a t least 6 months and did not require anaerobic conditions for stability. Fig. 2 shows the remarkable resistance to thermal denaturation exhibited by the pure enzyme. At a final concentration of 1.0 mg/ml, the half-life was 10.5 h at 100 "C, and 3.5 h at 105 "C. Addition of 250 mM potassium citrate improved the thermostability marginally (Fig. 2). The apparent half-life of the enzyme at 90 "C was 20 h.   where the bold letters denote conserved amino acid residues.
The enzymes from ES4, P. furiosus, and T. litoralis do not have a methionine in the first position.
Western Blot"ES4 and P. furiosus enzymes have a hexameric structure with 46,000 & 3,000 M, subunits; the bands detected on the Western blot (Fig. 4) corresponded to the GDH subunits visible on duplicate gels. The GDHs from ES4, T. litoralis, and P. furiosus all reacted strongly. Detectable, but much weaker, antigenic cross-reaction occurred with bovine and yeast GDHs.
Cloning and Sequencing of the ES4 gdhA Gene-Several clones carrying an identical 17-kb BamHI insert were identified by plaque hybridization of a lambda Fix library of ES4 genomic DNA. Southern hybridization of a digested X clone showed an XbaI, an EcoRI, and Hind111 fragments containing the partial or complete gdhA gene. The fragments were subcloned into Bluescript KS+.The ES4gdhA gene was sequenced using these subclones (Fig. 5). Hybridization of the digested genomic DNA from ES4 and P. furiosus with an ES4 gdhA clone showed a single band using several restriction enzymes (data not shown), corresponding to a single copy of the gdhA gene in the genomes of these two Archaea.
The complete nucleotide sequence of the ES4 gdhA gene, consisting of a single open reading frame of 1260 nucleotides, is shown in Fig. 6. The open reading frame is preceded by AT-rich regions in which a putative ribosome-binding site GAGGTG, at position -7, and a putative promoter consensus TTTATATA, at position -51, were found. The deduced amino acid sequence of the ES4 GDH consists of 420 residues and confirms the NH2-terminal amino acid sequence obtained from the pure protein, except that methionine is encoded at made per milliliter of reticulocyte lysate as calculated from the methionine content of GDH (13 mol methionine/mol GDH), the calculated final specific activity of [35S]methionine and the trichloroacetic acid-precipitable radioactivity. These results were consistent with the quantitation by Western blot analysis. The translation product was analyzed by SDS-PAGE and fluorography. Fig. 7 shows the accumulation of a 46-kDa polypeptide with maximum level obtained after 90 min of incubation. Gel filtration analysis was used to determine the molecular weight of the translation product. The hexameric form accounted for 12% of the total GDH accumulated, whereas the remaining 88% occurred as monomers with a molecular weight of 46,000. Apparently complete molecular assembly is not achieved under these conditions (see "Discussion"). Despite the ability of the subunits to form hexamers, the hexameric fraction did not show any enzyme activity when assayed as described for the native enzyme. Western blot analysis of the translation products (Fig. 8) showed a strong reaction of the 46-kDa polypeptide with the antiserum to P. furiosus GDH, and the in uitro product migrated identically to the subunits of the ES4 protein.
Comparison of ES4 GDH with Protein Sequences from Other Organisms-A sequence alignment of GDHs from ES4, other archaea, bacteria, and eucaryotes is presented in Fig. 9. The proposed alignment has been constructed with the Treealign program based on a Dayhoff matrix. The primary sequence of ES4 GDH is 40-52% similar to a group of organisms including Clostridium difficile (52%), Halobacterium salinarium (48%), Sulfolobus solfataricus (47%) and bovine (40%), and only 33% similar to another group containing Clostridium symbwsum, Escherichia coli, and the yeast Saccharomyces cereuisiue. A comparative analysis of amino acid composition among GDHs from mesophilic and thermophilic organisms revealed preferential changes of amino acid residues in the thermophiles, as follows: decreases were found in cysteine, methionine, and asparagine residues and increases in isoleucine and aspartic acid residues.

DISCUSSION
This paper describes the purification, cloning, and expression for the first time of a thermostable GDH from a hyperthermophile with a maximal growth temperature of 110 "C (3). We also describe the first enzyme to be characterized from this organism and the largest enzyme to be characterized and cloned from an organism that can grow above 100 "C. GDH is a major protein in the cytoplasm of ES4 as the yield of this enzyme during purification is comparable to that reported for the GDH (4), hydrogenase (8), and glyceraldehyde ferredoxin oxidoreductase (12) from P. furiosus. Our previous work with the GDH from P. furiosus established that it had a half-life of 1.8 h at 103 "C (51, the tmax for growth of the organism (28). The present study indicates that ES4 GDH has a half-life of 3.5 h at 105 "C and that it retained 16% activity after incubation for 23 h at 105 "C. It is therefore the most thermostable dehydrogenase reported to date. This extreme thermostability may relate to the maximal temperature for growth of the organism.
ES4 GDH has a hexameric structure with a molecular weight of 270,000. Microcalorimetry studies showed a denaturation temperature of 113 "C and an activation temperature of 60 "C. These values are very similar to those reported previously for P. furiosus GDH (5). The post-transitional rise reflects the exposure of nonpolar hydrophobic amino acids to the polar aqueous solvent during the unfolding process, indicating the contribution of hydrophobic interactions to protein stability (5). TTGCCAAAATATTTGCTATAGTTTTGCAATTCAATCGAATTGAATTTCTATTGAGAACTCAATAAACAA AAGGATTTCCACCCTTATTTACCGAG~GCTTATTGTCCAAATTTGTATCGCCAATCGACTAAATTGAGGTGATGAAC *******  The NHz-terminal amino acid sequence of ES4 GDH was determined and showed strong homology with sequences from two other hyperthermophiles, P. furiosus (4) and T. litoralis (29). The GDHs from these three hyperthermophiles do not possess methionine at the NH*-terminal, although methionine is encoded in the ES4 GDH gene. The absence of methionine could allow the NH2-terminal of the protein to be locked by hydrogen bonding to internal &sheet structures to prevent it from "unzipping" at high temperature (32). This has been described in the case of P. furiosus rubredoxin, where a three-dimensional structure has been determined (31, 32). Sulfolobus solfataricus GDH possesses an acetylated me-thionine at the NH2-terminal (30). This emphasizes the importance of obtaining direct amino acid sequencing data in addition to DNA deduced sequences, since the latter do not provide information about post-translational modification. The homology among thermophilic GDH amino acid sequences was confirmed by the Western blot analysis (Fig. 4) with ES4, P. furiosus, and T. litoralis enzymes. Bovine and yeast GDHs showed weak cross-reaction with the ES4 protein. This attests to the presence of highly conserved regions among GDHs (33), which confirms that GDH is an excellent model for studies of phylogeny (34).

GAC AAG AAG ATG ACC AAG GCA TTC TAC GAT GTC TAC AAC ACC GCG AAG GAG AAG AAC ATC CAC ATG 1188
The ES4 gdhA gene encodes 420 amino acids (Fig. 6). The control region shows an AT-rich region in which a putative promoter can be detected at position -51. The putative promoter sequence TTTATATA, referred to as Box A, resembles the eucaryotic TATA box and the consensus TTTAA/T A that is also found among most of the archaeal promoter regions (35,36), although it is not as strongly conserved in the sulfurdependent thermophiles as it is in the methanogens (37). This finding correlates with the observation that the archaeal RNA polymerase is more closely related to the eucaryotic RNA polymerase 11 than to bacterial RNA polymerases (36,37).
The Box B sequence, described by Wich et al. (38) in methanogens, including the motif TGCA located at the transcription start site, and later modified by Reiter et al. (36), has not been found in the ES4 GDH control region or in several thermophile genes. Immediately upstream of the open reading frame, at position -7, a putative ribosome-binding site, GAGGTGA, has been found, which corresponds to Shine-Dalgarno sequences for Archaea (37).
The G+C content of the gdhA gene sequence (48.7%) is lower than the value given by Pledger and Baross (3) for the genomic DNA (55%). However, a G+C content of 47% was obtained in our laboratory by microcalorimetric determination and hyperchromic shift? In ES4 and other sulfur-dependent hyperthermophiles whose genomic G+C content is less than 50%, G and C are rarely used in the third positions of codons, and a strong bias against the CG dinucleotide is observed.' This bias is reflected in arginine codon usage; with six possibilities, AGG and AGA are strongly preferred and the other four codons are rarely found. The opposite bias occurs in E. coli, where the codons CGA, CGT, CGG, and CGC are used in preference to AGG and AGA. The bias against CG is also found for alanine, proline, and serine codons in hyperthermophiles, but to a smaller extent than for arginine codons. The CG dinucleotide-containing codons are frequently used for alanine, proline, and serine in E. coli (39).
The codon usage of vertebrates, such as rabbits, does not show a bias against codons preferentially used by ES4 and other hyperthermophilic Archaea, and the rabbit reticulocyte H. Klump, unpublished data. ' J. DiRuggiero, unpublished results. system was therefore a good candidate for the expression of the ES4 GDH. The ES4 gdhA gene was first transcribed in uitro, and the mRNA produced was then translated in a rabbit reticulocyte cell-free system. Most of the protein produced by in uitro translation occurred as monomers, suggesting that the system did not provide the conditions required for proper assembly of the hexamer. In ES4 cells the enzyme is always produced at temperatures higher than 80 "C since the organism does not grow below this temperature. Also, the cytoplasm of the hyperthermophile P. furiosus, which is closely related to ES4, contains 700 mM KC1 (14). Therefore, extreme physicochemical conditions such as heat and high ionic strength may be required during molecular assembly of hyperthermostable enzymes. The participation of other factors such as chaperonins may also be required. Chaperone-like particles have been described from several thermophiles, for example Thermus thermophilus (40), Sulfolobus shibatae (41) and Pyrodictium occultum (42,43). All possess ATPase activity and are induced by heat stress. These molecules resemble procaryotic (GroEL) and eucaryotic (Hsp6O and ribulosebisphosphate carboxylase/oxygenase) chaperonins and are able to promote the refolding of several guanidine-HC1 denatured enzymes from thermophilic bacteria in uitro (40). We are proceeding with further studies that may permit us to produce an active enzyme using the eucaryotic in uitro system.
A sequence alignment of GDHs from the three domains, Eucarya, Bacteria, and Archaea (1) revealed strong homology among thermophilic enzymes. It is interesting to note that ES4 GDH has 56% homology with GDH from the bacterium C. difficik, and 40% homology with bovine GDH.
The fact that ES4 GDH is closer to a bacterial GDH (C. difficile) than it is to S. solfataricus, a sulfur-dependent thermophile, or to H. salinarium, bespeaks the enormous phylogenetic distances within the Archaea. These results are in agreement with a GDH phylogenetic tree recently proposed by Forterre et al. (34), but do not agree with the universal trees of life previously presented by Woese et al. (1) or Rivera and Lake (44). The existence of two families of genes encoding hexameric GDHs has been deduced from the alignment of primary sequences and the use of percentage similarity between each pair of proteins (34). The appearance of these two gene families predates the divergence of the Bacteria, Eucarya, and the Archaea, since there are representatives of GDH from both families (I and 11) in the Bacteria and the Eucaryotes. Thus, we find no evidence that the Archaea have a polyphyletic origin as suggested by Rivera and Lake (44). So far, only one family of GDH molecules has been found in the Archaea, so that it is possible that the ancestral split between the families occurred after the divergence of the Archaea. This would imply that the Archaea diverged very early in the time span of evolution. This must be treated with caution, however, since only three GDH sequences are available from Archaea, and none of these is from a methanogen.

--Q I P D F L C N A G G V T V S Y F E~Q N I T G~T L E~R E K L ----D K~T K A F Y D~~A -K -------------LTPDILTNAGGVTVSYFEWVQNLYG~SEEEVEQKE----EIRMVKAFESIWKI--K-------------VIPDILANAGGVTVSYFEWLQDINRRAWS-LE--RVNDE-LEAEMQAAWRAV-----KD----------
Proteins from hyperthermophiles, including the multisubunit enzymes that we have described, display an extraordinary level of intrinsic thermostability in vitro (4,5, 14, this work). There is no evidence for additional stabilizing factors to account for their function during growth at or near to 100 "C. Processes causing irreversible inactivation of enzymes at high temperatures include deamination of asparagine residues, hydrolysis of peptide bonds at aspartic acid residues, destruction of disulfide bonds, and subsequent formation of incorrectly folded structures (48). ES4 GDH shows a decrease in the number of the sulfur containing residues, methionine, and cysteine, which is in agreement with several studies on hyperthermophilic enzymes (14, 49, 50). This characteristic might serve to stabilize covalent protein structures at high temperatures. The absence of initiating methionines in enzymes from several hyperthermophiles (4, 31, this work) may represent an extension of this adaptive feature. Zwickl et al. (14) found that the thermostable glyceraldehyde-3-phosphate dehydrogenase molecule had a "striking increase in the aromatic residue phenylalanine and a respective decrease in aspartic acid residues" through comparison with homologous mesophilic enzymes. In contrast, we find that GDH from ES4 exhibits a decrease in phenylalanine when compared with S. solfaturicus (50) and C. dificile (51). Conversely, aspartic acid is indeed increased with increasing thermostability when GDH from these organisms is compared. It is therefore clear that the consideration of amino acid composition alone is insufficient to allow prediction of thermostability. Hensel et al. (49) show that most of the Asn residues that are hydrolyzed in M. feruidus GAPDH at 85 "C are replaced by more stable residues in P. woesei GAPDH. The conserved Asn residues are involved in catalytic sites and are probably protected by the rigid conformation of the molecule (49). Compared with mesophilic GDHs, ES4 GDH has an increased overall hydrophobicity. Similar tendencies have been reported for P. woesei GAPDH (14). Enhanced hydrophobic interactions therefore represent mechanisms for structural thermoadaptation.
Further findings concerning the thermostability of this extremely stable enzyme must await the elucidation of its three-dimensional structure, which is in progress.