Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus, a thermoacidophilic organism isolated from an acidic hot spring (optimal growth conditions: 87 degrees C, pH 3.5) was purified to homogeneity. The enzyme is a dimer (Mr subunit = 53,000) showing microheterogeneity when submitted to chromatofocusing and/or isoelectric focusing analysis (two main bands having pI = 6.8 and 6.3 were observed). The N-terminal sequence (22 residues) does not show any homology with any stretch of known sequence of aspartate aminotransferases from animal and bacterial sources. The apoenzyme can be reconstituted with pyridoxamine 5'-phosphate and/or pyridoxal 5'-phosphate, each subunit binding 1 mol of coenzyme. The absorption maxima of the pyridoxamine and pyridoxal form are centered at 325 and 335 nm, respectively; the shape of the pyridoxal form band does not change with pH. The enzyme has an optimum temperature higher than 95 degrees C, and at 100 degrees C shows a half-inactivation time of 2 h. The above properties seem to be unique even for enzymes from extreme thermophiles (Daniel, R. M. (1986) in Protein Structure, Folding, and Design (Oxender, D. L., ed) pp. 291-296, Alan R. Liss, Inc., New York) and lead to the conclusion that aspartate aminotransferase from S. solfataricus is one of the most thermophilic and thermostable enzymes so far known.     

The active site of the thermophilic CYP119 from Sulfolobus solfataricus

CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 degrees C. UV-visible and resonance Raman show the enzyme is in the low spin state and only modestly shifts to the high spin state at higher temperatures. Styrene only causes a small spin state shift, but T(1) NMR studies confirm that styrene is bound in the active site. CYP119 catalyzes the H(2)O(2)-dependent epoxidation of styrene, cis-beta-methylstyrene, and cis-stilbene with retention of stereochemistry. This catalytic activity is stable to preincubation at 80 degrees C for 90 min. Site-specific mutagenesis shows that Thr-213 is catalytically important and Thr-214 helps to control the iron spin state. Topological analysis by reaction with aryldiazenes shows that Thr-213 lies above pyrrole rings A and B and is close to the iron atom, whereas Thr-214 is some distance away. CYP119 is very slowly reduced by putidaredoxin and putidaredoxin reductase, but these proteins support catalytic turnover of the Thr-214 mutants. Protein melting curves indicate that the thermal stability of CYP119 does not depend on the iron spin state or the active site architecture defined by the threonine residues. Independence of thermal stability from active site structural factors should facilitate the engineering of novel thermostable catalysts.     

Limited proteolysis as a probe of conformational changes in aspartate aminotransferase from Sulfolobus solfataricus.

The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein. Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez-Carrion, M. (1984) J. Biol. Chem. 259, 723-728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323-326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (greater than 60 degrees C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25-75 degrees C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line. Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non-linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of kcat is non-linear as well.     

Cloning and sequencing of the gene coding for aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

The gene coding for aspartate aminotransferase (EC 2.6.1.1) has been cloned from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus strain MT4. Partial sequence data obtained directly from the purified protein and from the two cyanogen-bromide-generated peptides confirm the primary structure of aspartate aminotransferase inferred from the nucleotide sequence of its gene. A comparison of the enzyme with other aminotransferases revealed an interesting similarity with tyrosine aminotransferase from rat liver (EC 2.6.1.5) and allowed some tentative assignments of the residues implied in the catalysis. The aspartate aminotransferase gene-flanking regions were compared to those of other archaebacterial genes already described in the literature with the aim of identifying potential regulatory sites.     

Labeling of the active site of cytoplasmic aspartate aminotransferase by -chloro-L-alanine

Syncatalytic inactivation of pig heart cytoplasmic aspartate aminotransferase by β-chloro-[U-¹⁴C]L-alanine resulted in the incorporation of radioactivity corresponding to one mole of the label per mole of the monomeric unit of the enzyme. A borohydride-reduced and then carboxymethylated preparation of the labeled enzyme was digested by trypsin. A radioactive peptide was isolated and found to contain a covalently linked pyridoxyl derivative which absorbed at 325 nm. The amino acid sequence of this peptide was Tyr-Phe-Val-Ser-Glu-Gly-Phe -Glu-Leu-Phe-Cys-Ala-Gln-Ser-Phe-Ser-Lys-Asn-Phe-Gly-Leu-Tyr-Asn-Glu-Arg. In the peptide the phosphopyridoxyl group seems to be covalently bound via alanyl moiety derived from β-chloro-L-alanine, the β-carbon atom of which is covalently linked to the ?-nitrogen atom of the lysyl residue(Lys). From a comparison with the amino acid composition of the phosphopyridoxyl peptide isolated from the tryptic digest of a borohydride-reduced holoenzyme, it was concluded that the modified lysul residue was identical to that involved in binding pyridoxal phosphate to the apoenzyme.     

Crystal structure at 1.8 A resolution and identification of active site residues of Sulfolobus solfataricus peptidyl-tRNA hydrolase

The 3-D structure of the peptidyl-tRNA hydrolase from the archaea Sulfolobus solfataricus has been solved at 1.8 A resolution. Homologues of this enzyme are found in archaea and eucarya. Bacteria display a different type of peptidyl-tRNA hydrolase that is also encountered in eucarya. In solution, the S. solfataricus hydrolase behaves as a dimer. In agreement, the crystalline structure of this enzyme indicates the formation of a dimer. Each protomer is made of a mixed five-stranded beta-sheet surrounded by two groups of two alpha-helices. The dimer interface is mainly formed by van der Waals interactions between hydrophobic residues belonging to the two N-terminal alpha1 helices contributed by two protomers. Site-directed mutagenesis experiments were designed for probing the basis of specificity of the archaeal hydrolase. Among the strictly conserved residues within the archaeal/eucaryal peptidyl-tRNA hydrolase family, three residues, K18, D86, and T90, appear of utmost importance for activity. They are located in the N-part of alpha1 and in the beta3-beta4 loop. K18 and D86, which form a salt bridge, might play a role in the catalysis thanks to their acid and basic functions, whereas the OH group of T90 could act as a nucleophile. These observations clearly distinguish the active site of the archaeal/eucaryal hydrolases from that of the bacterial/eucaryal ones, where a histidine is believed to serve as the catalytic base.     

Solvent interactions with the active site of the pig heart cytosolic aspartate aminotransferase.

Aqueous solvent interactions with the chromophoric pyridoxal phosphate prosthetic group of aspartate aminotransferase (EC 2.6.1.1) were analyzed quantitatively with ethylene glycol, glycerol, dimethylsulfoxide (DMSO), sucrose, and xylitol as cosolvents. The smaller cosolvents perturb the visible absorption and visible dichroic spectra of the free enzyme, but this solvent perturbation is not observed with the acidic enzymeglutarate complex. Addition of cosolvents caused an increase in the enzyme's affinity for glutarate. This increase in affinity resulted from an increase in the acidic dissociation constant (pK₂) of the enzyme-glutarate complex. The changes in the acidic dissociation constant of the enzyme-glutarate complex, upon addition of cosolvents, correlate well with the changes observed in the pK_a's of carboxylic acids in comparable solvents. Since these solvents have little effect on the pK_a of the enzyme itself, it is concluded that the increase in affinity is due to a specific solvation effect on a carboxyl group of the enzymebound glutarate, rather than resulting from a conformational change in the protein.     

Chemical structure of the active site of pig heart mitochondrial aspartate aminotransferase labeled with beta-chloro-l-alanine

Formate-induced inactivation of pig heart mitochondrial aspartate aminotransferase by beta-chloro-L-alanine resulted in the modification of the epsilon-amino group of the lysyl residue which is involved in the formation of an aldimine bond with 4-formyl group of the coenzyme, pyridoxal 5'-phosphate. The tryptic peptide isolated from the labeled site of the enzyme was composed of 25 residues and exhibited positive circular dichroism at 325 and 254 nm where the pyridoxyl chromophore of the labeled site peptide absorbs, while the phosphopyridoxyl peptide isolated from the boro-hydride-reduced enzyme did not show any ellipticity in this spectral region. Its comparison with the analogous tryptic peptide from the labeled site of the cytosolic isoenzyme revealed a high degree of homology in their primary structures as well as in spectral properties. Structural analysis of the labeled site peptide and mechanistic consideration of the labeling process indicated that with both isoenzymes the phosphopyridoxyl group is covalently bound to the alpha amino group of the alanyl moiety derived from beta-chloro-L-alanine, the beta carbon of which is covalently linked to the epsilon-amino group of the lysyl residue.     

Conformational changes in aspartate aminotransferase. Effect of active site ligands on peptide hydrogen-deuterium exchange

The conformational responses of aspartate aminotransferase (cytosolic isoenzyme from pig) to the binding of the coenzyme and competitive inhibitors and to the bond rearrangement steps during the transamination reaction were probed by the method of peptide hydrogen deuterium exchange. Binding of the coenzyme to the apoenzyme results in a marked retardation of hydrogen exchange; binding of the competitive inhibitor maleate to the pyridoxal enzyme induces a retardation of exchange somewhat exceeding that observed in the presence of the transaminating substrate pair glutamate and 2-oxoglutarate (Pfister, K., K?gi, J.H.R., and Christen, P. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 145-148). On formation of the complex of apoenzyme with N-(5'-phosphopyridoxyl)-L-glutamate or-L-aspartate, analogs of the covalent coenzyme substrate intermediates, a similar exchange retardation occurs. The extent of the exchange retardation in these different functional states of the enzyme correlates with previous results of differential chemical and proteolytic modifications. Apparently, the diverse methods register shifts in one and the same conformational equilibrium. Moreover, the conditions under which peptide hydrogen exchange indicates a pronounced tightening of the protein matrix correspond with those inducing crystallization of the enzyme in the "closed" form. Thus, the transition between the "open" and "closed" form of the enzyme, i.e. the bulk movement of the small domain, as observed and defined by x-ray crystallography (Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525) is the major structural correlate of the conformational changes undergone by the enzyme in solution.     

Peptidyl-tRNA hydrolase from Sulfolobus solfataricus

An enzyme capable of liberating functional tRNA^Lys from Escherichia coli diacetyl-lysyl-tRNA^Lys was purified from the archae Sulfolobus solfataricus. Contrasting with the specificity of peptidyl- tRNA hydrolase (PTH) from E.coli, the S.solfataricus enzyme readily accepts E.coli formyl-methionyl-tRNA^fMet as a substrate. N-terminal sequencing of this enzyme identifies a gene that has homologs in the whole archaeal kingdom. Involvement of this gene (SS00175) in the recycling of peptidyl-tRNA is supported by its capacity to complement an E.coli strain lacking PTH activity. The archaeal gene, the product of which appears markedly different from bacterial PTHs, also has homologs in all the available eukaryal genomes. Since most of the eukaryotes already display a bacterial-like PTH gene, this observation suggests the occurrence in many eukaryotes of two distinct PTH activities, either of a bacterial or of an archaeal type. Indeed, the bacterial- and archaeal-like genes encoding the two full-length PTHs of Saccharomyces cerevisiae, YHR189w and YBL057c, respectively, can each rescue the growth of an E.coli strain lacking endogeneous PTH. In vitro assays confirm that the two enzymes ensure the recycling of tRNA^Lys from diacetyl-lysyl-tRNA^Lys. Finally, the growth of yeast cells in which either YHR189w or YBL057c has been disrupted was compared under various culture conditions. Evidence is presented that YHR189w, the gene encoding a bacterial-like PTH, should be involved in mitochondrial function.     

Appendage-Mediated Surface Adherence of Sulfolobus solfataricus

Attachment of microorganisms to surfaces is a prerequisite for colonization and biofilm formation. The hyperthermophilic crenarchaeote Sulfolobus solfataricus was able to attach to a variety of surfaces, such as glass, mica, pyrite, and carbon-coated gold grids. Deletion mutant analysis showed that for initial attachment the presence of flagella and pili is essential. Attached cells produced extracellular polysaccharides containing mannose, galactose, and N-acetylglucosamine. Genes possibly involved in the production of the extracellular polysaccharides were identified.In microbiology, organisms are isolated from their natural habitats and typically cultivated in the laboratory as planktonic species. Though this method has been essential for understanding the concept of life, it remains unclear how microbial ecosystems operate. For bacteria, it is well known that they are able to form large cellular communities with highly complex cellular interactions and symbioses between different microbial or eukaryotic species. Biofilm formation is an essential component of such communities, and studies have shown that bacteria within biofilms are physiologically different from planktonic ones (20, 21). They can exhibit extensive networks of pili on their surfaces and produce and secrete extracellular polysaccharides (EPS), their growth rate is decreased, and cells are much more resistant to physical stresses and antibiotics (19).The study of surface colonization and cellular communities of archaea is crucial for understanding their ecological properties. The only detailed study showed that the hyperthermophilic organism Archaeoglobus fulgidus produced biofilms when challenged with heavy metals and pentachlorophenol (10). Pyrococcus furiosus was able to adhere to different surfaces, such as mica and carbon-coated gold grids, and cells were connected via cable-like bundles of flagella (12). Methanopyrus kandleri was shown to adhere to glass, but P. furiosus could colonize only by attaching to M. kandleri cells, using flagella and direct cell contacts (16).Here we report on the function of cell surface appendages in initial attachment to surfaces of archaea, using directed gene inactivation mutants. The crenarchaeote Sulfolobus solfataricus P2 is a thermoacidophile which grows optimally at 80°C and pH values of 2 to 4 (22). S. solfataricus possesses cell surface structures such as flagella and UV-induced pili (1, 2). The flagellum operon of S. solfataricus encodes, in addition to the structural subunit FlaB, four proteins of unknown function, the ATPase FlaI, and the only integral membrane protein, FlaJ. Previously, we isolated a ΔflaJ mutant which was nonflagellated and had lost its ability for surface motility on Gelrite plates (17). Recently, we described UV-inducible pili in S. solfataricus that directed cellular aggregation after UV stress (8). Deletion of the central ATPase UpsE, responsible for pilus assembly, rendered cells devoid of pili and defective in cellular aggregation after UV treatment (8). In this study, wild-type cells and deletion strains were tested for the ability to attach to a variety of surfaces and the formed structures and extracellular material were analyzed.     

The chromosome replication machinery of the archaeon Sulfolobus solfataricus

In the three domains of life, the archaea, bacteria, and eukarya, there are two general lineages of DNA replication proteins: the bacterial and the eukaryal/archaeal lineages. The hyperthermophilic archaeon Sulfolobus solfataricus provides an attractive model for biochemical study of DNA replication. Its relative simplicity in both genomic and biochemical contexts, together with high protein thermostability, has already provided insight into the function of the more complex yet homologous molecules of the eukaryotic domain. Here, we provide an overview of recent insights into the functioning of the chromosome replication machinery of S. solfataricus, focusing on some of the relatively well characterized core components that act at the DNA replication fork.     

2-Propanol degradation by Sulfolobus solfataricus

Sulfolobus solfataricus used 2-propanol and 2-propanone (acetone) when grown in static cultures at 78 °C with or without glucose at 10 g l^–1. The presence of 3.92 g 2-propanol l^–1 in both cases inhibited growth. However, acetone accumulation following 2-propanol depletion suggested that 2-propanol was co-metabolized via the acetone metabolic pathway. Glucose at 10 g l^–1 increased 2-propanol and acetone utilization from 0.93 g l^–1 to 1.77 g l^–1 and from 0.11 g l^–1 to 1.62 g l^–1, respectively. Without glucose, immobilized S. solfataricus cells increased the 2-propanol removal rate to 0.035 g l^–1 h^–1, compared to 0.0012 g l^–1 h^–1 by its suspended counterpart. The results suggest the establishment of an immobilized reactor configuration is preferential for the treatment of high temperature solvent waste streams by this acidothermophilic Crenarchaeon.     

An exosome-like complex in Sulfolobus solfataricus

We present the first experimental evidence for the existence of an exosome-like protein complex in Archaea. In Eukarya, the exosome is essential for many pathways of RNA processing and degradation. Co-immunoprecipitation with antibodies directed against the previously predicted Sulfolobus solfataricus orthologue of the exosome subunit ribosomal-RNA-processing protein 41 (Rrp41) led to the purification of a 250-kDa protein complex from S. solfataricus. Approximately half of the complex cosediments with ribosomal subunits. It comprises four previously predicted orthologues of the core exosome subunits from yeast (Rrp41, Rrp42, Rrp4 and Csl4 (cep1 synthetic lethality 4; an RNA-binding protein and exosome subunit)), whereas other predicted subunits were not found. Surprisingly, the archaeal homologue of the bacterial DNA primase DnaG was tightly associated with the complex. This suggests an RNA-related function for the archaeal DnaG-like proteins. Comparison of experimental data from different organisms shows that the minimal core of the exosome consists of at least one phosphate-dependent ribonuclease PH homologue, and of Rrp4 and Csl4. Such a protein complex was probably present in the last common ancestor of Archaea and Eukarya.     

Arginine as a substrate binding site in aspartate aminotransferase.

Modification of one or two arginine residues in pig-heart cytoplasmic aspartate aminotransferase with 1,2-cyclohexanedione nearly abolishes its catalytic activity and abolishes its ability to bind dicarboxylic acids. The modification is competitively inhibited by glutaric acid. Modification of the enzyme causes no change in its ability to transaminate alanine, but causes a tenfold increase in the Michaelis constant and a 10⁴ fold decrease in the rate of transamination of aspartate. These results indicate that the binding site for the β-carboxyl group of aspartic acid is an arginine residue.     

Total reconstitution of active large ribosomal subunits of the thermoacidophilic archaebacterium Sulfolobus solfataricus.

The large ribosomal subunit of the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus has been reconstituted from the completely dissociated RNA and proteins by a two-step incubation procedure at high temperatures. Successful reconstitution requires a preliminary incubation of the ribosomal components for 45 min at 65 degrees C, followed by a second heat-treatment at 80 degrees C for 60 min. Structural reassembly depends upon high concentrations of K+ (300-400 mM) and Mg2+ (20-40 mM) ions. In addition, complete recovery of subunit function stringently requires the presence of a polyamine, thermine (or spermine). The reconstituted archaebacterial subunits are essentially indistinguishable from the native ones by a number of structural and functional criteria.     

Substitution of an arginyl residue for the active site lysyl residue (Lys258) of aspartate aminotransferase

The active site lysyl residue (Lys258) of E. coli aspartate amino transferase was substituted for an arginyl residue by oligonucleotide-directed, site-specific mutagenesis. The mutant enzyme was obviously unable to form an aldimine bond with pyridoxal 5'-phosphate but firmly bound the coenzyme. The finding that the mutation did not lead to entire loss in the enzymic activity suggests that Lys258 may not be essential but auxiliary for enzymic catalysis. It is also conceived that the positive charge provided by Arg258 may contribute to the enzymic catalysis.