Glyceraldehyde-3-Phosphate Dehydrogenases from Archaea: Objects for Studying Protein Thermoadaptation

Functional and structural properties of the glyceraldehyde-3-phosphate dehydrogenases from the mesophilic archaeum Methanobacterium bryantii and from the hyperthermophilic archaea Methan-othermus fervidus, Pyrococcus woesei and Thermoproteus tenax were compared to characterize the thermophilic phenotype. Site dierected mutagenesis with the M. fervidus enzyme were performed to analyse the structural background of the thermoadaptive features of the archaeal glyceraldehyde-3-phosphate dehydrogenase.     

Nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene from the mesophilic methanogenic archaebacteria Methanobacterium bryantii and Methanobacterium formicicum. Comparison with the respective gene structure of the closely related extreme thermophile Methanothermus fervidus

The genes for glyceraldehyde-3-phosphate dehydrogenase (gap genes) from the mesophilic methanogenic archaebacteria Methanobacterium formicicum and Methanobacterium bryantii were cloned and sequenced. The deduced amino acid sequences show 95% identity to each other and about 70% identity to the glyceraldehyde-3-phosphate dehydrogenase from the thermophilic methanogenic archaebacterium Methanothermus fervidus. Although the sequence similarity between the archaebacterial glyceraldehyde-3-phosphate dehydrogenase and the homologous enzyme of eubacteria and eukaryotes is low, an equivalent secondary-structural arrangement can be deduced from the profiles of the physical parameters hydropathy, chain flexibility and amphipathy. In order to find possible thermophile-specific structural features of the enzyme from M. fervidus, a comparative primary-sequence analysis was performed. Amino acid exchanges leading, to a stabilization of the main-chain conformation, could be found throughout the sequence of the thermophile enzyme. Striking features of the thermophile sequence are the preference for isoleucine, especially in beta-sheets, and a low arginine/lysine ratio of 0.54.     

Properties and primary structure of the L-malate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus

L-Malate dehydrogenase from the extremely thermophilic mathanogen Methanothermus fervidus was isolated and its phenotypic properties were characterized. The primary structure of the protein was deducted from the coding gene. The enzyme is a homomeric dimer with a molecular mass of 70 kDa, possesses low specificity for NAD+ or NADP+ and catalyzes preferentially the reduction of oxalacetate. The temperature dependence of the activity as depicted in the Arrhenius and van't Hoff plots shows discontinuities near 52 degrees C, as was found for glyceraldehyde-3-phosphate dehydrogenase from the same organism. With respect to the primary structure, the archaebacterial L-malate dehydrogenase deviates strikingly from the eubacterial and eukaryotic enzymes. The sequence similarity is even lower than that between the L-malate dehydrogenases and L-lactate dehydrogenases of eubacteria and eukaryotes. The phylogenetic meaning of this relationship is discussed.     

Comparative analysis of thermoadaptation within the archaeal glyceraldehyde-3-phosphate dehydrogenases from mesophilic Methanobacterium bryantii and thermophilic Methanothermus fervidus

To gain insight into the molecular determinants of thermoadaptation within the family of archaeal glyceraldehyde-3-phosphate dehydrogenases (GAPDH), a homology-based 3-D model of the mesophilic GAPDH from Methanobacterium bryantii was built and compared with the crystal structure of the thermophilic GAPDH from Methanothermus fervidus. The homotetrameric model of the holoenzyme was initially assembled from identical subunits completed with NADP molecules. The structure was then refined by energy minimization and simulated-annealing procedures. PROCHECK and the 3-D profile method were used to appraise the model reliability. Striking molecular features underlying the difference in stability between the enzymes were deduced from their structural comparison. First, both the increase in hydrophobic contacts and the decrease in accessibility to the protein core were shown to discriminate in favor of the thermophilic enzyme. Besides, but to a lesser degree, the number of ion pairs involved in cooperative clusters appeared to correlate with thermostability. Finally, the decreased stability of the mesophilic enzyme was also predicted to proceed from both the lack of charge-dipole interactions within alpha-helices and the enhanced entropy of unfolding due to an increase in chain flexibility. Thus, archaeal GAPDHs appear to be governed by thermoadaptation rules that differ in some aspects from those previously observed within their eubacterial counterparts.     

Expression of the glyceraldehyde-3-phosphate dehydrogenase gene from the extremely thermophilic archaebacterium Methanothermus fervidus in E. coli. Enzyme purification, crystallization, and preliminary crystal data

The gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extremely thermophilic archaebacterium Methanothermus fervidus (growth optimum 82 degrees C) was cloned in vector pJF118EH and expressed in E. coli cells. As shown by molecular mass determination, protein sequencing, heat stability, and substrate saturation kinetics, the enzyme synthesized in E. coli is identical to the original enzyme from M. fervidus. The high thermostability of the E. coli-produced M. fervidus GAPDH allows rapid purification to homogeneity. From this enzyme protein crystals were grown which proved to be suitable for X-ray analysis. The crystals are of tetragonal space group P4(1)22 and contain a dimer per asymmetric unit.     

Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli.

The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103 degrees C) was purified to homogeneity. This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus. The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100 degrees C. The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P. woesei was deduced from the nucleotide sequence of the coding gene. Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermus fervidus), the primary structure of the P. woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues. The coding gene of P. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme.     

Primary structure of glyceraldehyde-3-phosphate dehydrogenase deduced from the nucleotide sequence of the thermophilic archaebacterium Methanothermus fervidus

The gene for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the thermophilic methanogenic archaebacterium Methanothermus fervidus (growth optimum at 84 degrees C) was cloned in Escherichia coli and the nucleotide sequence was determined. A striking preference for adenine and thymidine bases was found in the gene, which is in agreement with the low G + C content of the M. fervidus DNA. The deduced amino acid sequence indicates an Mr of 37,500 for the protein subunit. Alignment with the amino acid sequences of GAPDHs from other organisms shows that the archaebacterial GAPDH is homologous to the respective eubacterial and eukaryotic enzymes, but the similarity between the archaebacterial enzyme and the eubacterial or eukaryotic GAPDHs is much less than that between the latter two.     

Selective inhibition of an enzyme in crude cell extracts. The use of subunits modified with a half-of-the-sites reagent

Yeast glyceraldehyde-3-phosphate dehydrogenase carboxymethylated at four active-site cysteine residues was incubated with a crude extract of baker's yeast. This resulted in a loss of the glyceraldehyde-3-phosphate dehydrogenase activity initially present in the extract. The extent of inactivation depended upon the ratio modified enzyme/enzyme present in the extract. Under appropriate conditions 63.1% inactivation of glyceraldehyde-3-phosphate dehydrogenase in crude extract could be achieved. The observed effect is explained in terms of hybridization between the carboxymethylated dimers of the purified enzyme and dimeric species of glyceraldehyde-3-phosphate dehydrogenase present in the crude extract, the inactivation being due to the influence of the half-of-the-sites reagent transmitted via the interdimeric contacts.     

Structural basis for the thermal stability of glyceraldehyde-3-phosphate dehydrogenases

The amino acid sequences of two thermophilic and five mesophilic glyceraldehyde-3-phosphate dehydrogenases have been compared with the known three-dimensional structure of this enzyme to determine the factors responsible for thermal stability. The changes are greatest in the S-loop regions at the center of the tetramer, which show a quantitative increase in hydrophobicity and polarity that can strengthen subunit interactions in a complementary manner. The S-loops also show increases in residue volume and bulk that may indicate a tighter packing at the molecular center. In addition, there are changes in the secondary structural parameters indicating that the helices, in particular, may be more stable in the thermophilic proteins. Increases in the hydrophobicity of domain and subunit contacts for the Thermus aquaticus glyceraldehyde-3-phosphate dehydrogenase may explain why it is the most thermostable protein in this series.     

Characterization of two glyceraldehyde-3-phosphate dehydrogenase isoenzymes from the pentalenolactone producer Streptomyces arenae.

Pentalenolactone (PL) irreversibly inactivates the enzyme glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) and thus is a potent inhibitor of glycolysis in both procaryotic and eucaryotic cells. We showed that PL-producing strain Streptomyces arenae TU469 contains a PL-insensitive glyceraldehyde-3-phosphate dehydrogenase under conditions of PL production. In complex media no PL production was observed, and a PL-sensitive glyceraldehyde-3-phosphate dehydrogenase, rather than the insensitive enzyme, could be detected. The enzymes had the same substrate specificity but different catalytic and molecular properties. The apparent Km values of the PL-insensitive and PL-sensitive enzymes for glyceraldehyde-3-phosphate were 100 and 250 microM, respectively, and the PL-sensitive enzyme was strongly inhibited by PL under conditions in which the PL-insensitive enzyme was not inhibited. The physical properties of the PL-insensitive enzyme suggest that the protein is an octamer, whereas the PL-sensitive enzyme, like other glyceraldehyde-3-phosphate dehydrogenases, appears to be a tetramer.     

Convergence of active center geometries.

Comparisons have been made between the active center geometries of lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, chymotrypsin and papain, and glyceraldehyde-3-phosphate dehydrogenase and papain. In the dehydrogenases, orientation of the nicotinamide ring about the glycosidic bond is determined by the substrate stereochemistry. The proper positioning of the carboxyamide moiety allows for the close approach of the C4 atom on the nicotinamide and the reactive carbon of the substrate. It follows that, once the conformation of the substrate or substrate intermediate has been established with respect to the functional groups in the enzyme, the A- or B-side specificity of the nicotinamide ring is predetermined. Hence, dehydrogenases which are divergently evolving from a common precursor must maintain the nicotinamide specificity if the protein fold of the catalytic domain is conserved. The tetrahedral intermediates produced during acylation of chymotrypsin and papain are found to be of opposite hand, while those of papain and glyceraldehyde-3-phosphate dehydrogenase can be regarded to be of the same hand. Thus the serine proteases, subtilisin and those of the chymotrypsin family, are of one hand while the cysteine enzymes, glyceraldehyde-3-phosphate dehydrogenase and papain, are of the other.     

THE HISTOCHEMICAL DEMONSTRATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ACTIVITY

A histochemical method for demonstration of glyceraldehyde-3-phosphate dehydrogenation by tissues is described. The method utilizes Nitro BT as an indicator, glyceraldehyde-3-phosphate obtained from hydrolysis of commercially obtainable glyceraldehyde-3-phosphate diethylacetal (monobarium salt) as substrate, and (ethylenediamine)tetraacetic acid acid disodium as an activating agent in a medium buffered to pH 7.2 by 0.2 M sodium phosphate. The heat lability, substrate and coenzyme specificity, and sulfhydryl and phosphate dependence of the tissue component catalyzing this reaction indicate that glyceraldehyde-3-phosphate dehydrogenase activity is being demonstrated. The disparity between the known pH optimum of this enzyme and that determined histochemically, and the anomalous histochemical localization to mitochondria of this enzyme which has been found in the soluble fraction by differential centrifugation, are thought to result from the diaphorase dependence of the tetrazolium methods and to emphasize the need for caution in the interpretation of histochemically determined intracellular localization of dehydrogenating enzymes. The evidence gathered by previous workers concerning the feasibility of demonstrating specific dehydrogenases with Nitro BT, and the correspondence of the distribution of glyceraldehyde-3-phosphate dehydrogenase determined histochemically with available quantitative data, suggest that at the cellular level the histochemical results accurately reflect the distribution of this enzyme.     

Microenvironment of the enzyme-bound NADH is different in lobster and pig muscle glyceraldehyde-3-phosphate dehydrogenase microcrystals

Two possible consequences of crystal lattice formation were studied with glyceraldehyde-3-phosphate dehydrogenases isolated from lobster (Palinurus vulgaris) and pig muscle: changes in the microenvironment of the NADH-binding site as detected by fluorescence polarization, and differences in the maximal activities of the microcrystalline enzymes as compared to those in solution. In solution practically no difference was found between the polarization values of the enzyme-NADH and the catalytic intermediate 3-phosphoglyceroyl-enzyme-NADH complexes whether with lobster or with pig enzyme. In microcrystalline state a similar effect was found with the lobster enzyme. However, fluorescence polarization of NADH bound to the pig enzyme was significantly different in the presence and in the absence of the 3-phosphoglyceroyl group. This indicates some change in the microenvironment of the pig enzyme-bound NADH which occurs upon decomposition of the catalytic intermediate. The difference between the microcrystalline lobster and pig muscle glyceraldehyde-3-phosphate dehydrogenases pertains also to their functional properties. Packing of soluble pig muscle enzyme into a crystal lattice stabilizes a unique protein conformation of extremely low activity (about 3% of that measured in solution). The maximal molar activity of the lobster enzyme is identical in crystalline state and in solution, which is an exceptional phenomenon.     

Partial reversible inactivation of enzymes due to binding to the human erythrocyte membrane

Summary Hypotonic human erythrocyte ghosts, devoid of the original glyceraldehyde-3-phosphate dehydrogenase content of the red cell, bind added glyceraldehyde-3-phosphate dehydrogenases, isolated from human erythrocytes, rabbit and pig muscle, as well as rabbit muscle aldolase. There are only slight differences in the affinities towards the various glyceraldehyde-3-phosphate dehydrogenases. On the other hand, glyceraldehyde-3-phosphate dehydrogenases are bound much stronger than aldolase; in an equimolar mixture the former can prevent the binding of the latter, or replace previously bound aldolase at the membrane surface. Binding is always accompanied by the partial inactivation of enzymes, which can be reverted by desorption. Unwashed ghosts rich in hemoglobin seem to have a more pronounced inactivating effect on bound glyceraldehyde-3-phosphate dehydrogenase. In isotonic media ghosts, whether white or unwashed, reseal and do not interact with the enzymes.     

A new chemical mechanism catalyzed by a mutated aldehyde dehydrogenase.

NAD(P) aldehyde dehydrogenases (EC 1.2.1.3) are a family of enzymes that oxidize a wide variety of aldehydes into acid or activated acid compounds. Using site-directed mutagenesis, the essential nucleophilic Cys 149 in the NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Escherichia coli has been replaced by alanine. Not unexpectedly, the resulting mutant no longer shows any oxidoreduction phosphorylating activity. The same mutation, however, endows the enzyme with a novel oxidoreduction nonphosphorylating activity, converting glyceraldehyde 3-phosphate into 3-phosphoglycerate. Our study further provides evidence for an alternative mechanism in which the true substrate is the gem-diol entity instead of the aldehyde form. This implies that no acylenzyme intermediate is formed during the catalytic event. Therefore, the mutant C149A is a new enzyme which catalyzes a distinct reaction with a chemical mechanism different from that of its parent phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This finding demonstrates the possibility of an alternative route for the chemical reaction catalyzed by classical nonphosphorylating aldehyde dehydrogenases.     

Structural studies on glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle

The NH2-terminal amino acid sequence of rat skeletal muscle glyceraldehydephosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NAD+ oxidoreductase(physphorylating), EC 1.2.1.12) was determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Ser-Ser-(-)-(-)--Val-Asx-Ile-Val-Ala-Ile. The presence of Asn instead of Asp in position 6 differentiates this enzyme from other glyceraldehyde-3-phosphate dehydrogenases so far sequenced with the exception of the enzymes isolated from liver. The location of Asn in position 6 has been considered as a specific property of liver glyceraldehyde-3-phosphate dehydrogenase (Kulbe, K.D., Jackson, K.W. and Tang, J. (1975) Biochem. Biophys. Res. Commun. 67, 35--42); this suggestion is not sustained by the results of the present investigation. The amino acid composition of the rat skeletal muscle dehydrogenase demonstrates the unusually low histidine content of this enzyme as compared to other mammalian muscle glyceraldehyde-phosphate dehydrogenases.     

Comparative study of the structure of glyceraldehyde-3-phosphate dehydrogenase from bovine heart muscle

Glyceraldehyde-3-phosphate dehydrogenase with a specific activity of 153 units/mg protein was isolated from bovine heart muscle. Its relative molecular mass was found to be 144,000. The tryptic peptide map and amino acid analysis were obtained. The N-terminal sequence was established as Val-Lys-Val-Gly-Val-Asn-Gly-... and C-terminal as ...-Ala-Ser-Lys-Glu. Fluorescence and optimal rotation dispersion measurements were performed. The data were compared with other glyceraldehyde-3-phosphate dehydrogenases.     

A comparative study of NAD+ binding sites in dehydrogenases by circular polarization of fluorescence.

The spectra of the circular polarization of luminescence of a number of dehydrogenases with the fluorescent coenzyme nicotinamide-1,-N⁶-ethenoadenine dinucleotide were measured. By use of this technique it is demonstrated that there is a difference in structure between the adenine subsite in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase on the one hand and pig heart lactate dehydrogenase, horse liver alcohol dehydrogenase, beef liver glutamate dehydrogenase, and pig heart malate dehydrogenase on the other hand. It is concluded that the non-co-operative dehydrogenases have similar, if not identical, adenine subsites whereas in glyceraldehyde-3-phosphate dehydrogenase, a strongly co-operative enzyme, a different structure of the adenine subsite has evolved.     

Pcal_0632, a phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Pyrobaculum calidifontis

Genome sequence of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0632, annotated as glyceraldehyde-3-phosphate dehydrogenase, which is partially overlapped with phosphoglycerate kinase. In the phylogenetic tree, Pcal_0632 clustered with phosphorylating glyceraldehyde-3-phosphate dehydrogenases characterized from hyperthermophilic archaea and exhibited highest identity of 54% with glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii. To examine biochemical function of the protein, Pcal_0632 gene was expressed in Escherichia coli and the gene product was purified. The recombinant enzyme catalyzed the conversion of glyceraldehyde 3-phosphate and inorganic phosphate into 1,3-bisphosphoglycerate utilizing both NAD and NADP as cofactor with a marked preference for NADP. The enzyme was highly stable against temperature and denaturants. Half-life of the enzyme was 60 min at 100 °C. It retained more than 60% of its activity even after an incubation of 72 h at room temperature in the presence of 6 M urea. High thermostability and resistance against denaturants make Pcal_0632 a novel glyceraldehyde-3-phosphate dehydrogenase.

     

Thermoadaptation of methanogenic bacteria by intracellular ion concentration

Abstract An inter- and intra-species correlation was found between the intracellular potassium concentration and growth temperature within the Methanobacteriales , comprising mesophiles as well as moderate ( Methanobacterium thermoautotrophicum ) and extreme thermophiles ( Methanothermus fervidus, Mt. sociabilis ). Potassium concentrations in different species were determined at optimal growth temperatures and for the same species cultured at different temperatures. The main anionic component was found to be the unusual trianionic cyclic 2,3-diphosphiglycerate. In vitro experiments with the thermolabile enzymes glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase from Mt. fervidus indicated that the potassium salt of the cyclic diphosphoglycerate acts as potent thermostabilizer. Thus it appears that, for the methanogens, changes in the intracellular ion concentration are the basis of thermoadaptation.