Hyperthermophiles are recently discovered microorganisms which are able to grow optimally above 85 °C. Most hyperthermophiles belong to the Archaea, the third domain of life. One of the main interests in hyperthermophiles to deepen the insight in the way their proteins are stabilized and how to apply this knowledge to improve the stability of biotechnologically relevant enzymes. In this thesis attention has been focused on hydrolytic enzymes from hyperthermophilic archaea to provide insight in the way these microorganisms stabilize their proteins and are able to perform catalysis around the normal boiling point of water as well as to functionally produce these enzymes in a mesophilic heterologous host. Additionally, the organization and expression of a number of genes in a hyperthermophilic archaeon were studied. Members of two different classes of hydrolytic enzymes, that represent key enzymes in the metabolism of hyperthermophilic archaea, have been characterized at the molecular level: (i) glycosyl hydrolases that are required for growth on beta-linked sugars, and (ii) serine proteases that are involved in the growth on proteins and peptides. The most extensively studied hyperthermophilic archaeon Pyrococcus furiosus was used as model organism for the work described in this thesis.Chapter 1 gives a brief introduction into different aspects of hyperthermophilic archaea, including an overview of the metabolism of polymeric substrates and the hydrolytic enzymes involved, a listing of the mechanisms by which hydrolases and other enzymes from hyperthermophiles are stabilized, and the main characteristics of their molecular biology. The main part of the thesis deals with the celB locus (Fig. 1) of P.furiosus and serine proteases of P.furiosus and the related Thermococcus stetteri.Figure 1 : Genetic and transcriptional organization of the P.furiosus celB locus.In chapter 2 the isolation and characterization of the extremely thermostable beta-glucosidase (half-life of 3 days at 100 °C) and its celB gene of P.furiosus is described. The transcriptional organization of the celB gene was analyzed and indicated a single transcriptional unit, which was sustained by Northern blot analysis (see below) (Fig. 1). The deduced amino sequence of the beta-glucosidase showed that it is a member of the glycosyl hydrolase family 1. The pyrococcal celB gene was overexpressed in the mesophilic host Escherichia coli using the tac promoter, which resulted in a high production level of beta-glucosidase (up to 20% of the total soluble proteins). The P.furiosus beta-glucosidase was produced in an active form by E.coli with kinetic and stability characteristics identical to that of the native pyrococcal enzyme. The production of the hyperthermostable beta-glucosidase in a mesophilic host allowed for a simple purification procedure consisting of a heat-treatment of the cell-extracts followed by a single column chromatography. The high production level of the beta-glucosidase in the genetically well-accessible host E.coli allowed for protein engineering to gain insight in structure-stability and function relations. Mutational analysis of the active site of the beta-glucosidase from P.furiosus showed that the mechanisms for catalysis near the boiling point of water does not differ from that used by homologous enzymes from the family 1 of glycosyl hydrolases optimally active at lower temperatures.Over the last years, the potential of the extremely thermostable P.furiosus beta-glucosidase as a biocatalyst has been studied. Due to its broad substrate specificity and high chemical and thermal stability a wide variety of products could be in glucoconjugation and transglycosylation reactions in water and organic solvents at temperatures in between 75 to 95 °C (Fischer et al., 1996; Trincone et al., 1997). The beta-glucosidase from P.furiosus has also been analysed for its potential as biocatalyst in the production of beta-galacto-oligosaccharides from lactose (Jansen et al., 1997). Furthermore, the celB gene has been developed into a genetic marker to study plant-bacterium interactions and in competition experiments with differently marked Rhizobium strains (Sessitsch et al ., 1996). The applicability of this system is exemplified by the development of the CelB Gene Marking Kit (FAO/IAEA).Due to the high production level of the pyrococcal beta-glucosidase in E.coli and its simple purification procedure, sufficient pure protein has been generated to initiate crystalization experiments in collaboration with the group of Prof. G.E. Schulz (University of Freiburg) and resulted in crystals that diffract to 3.5 Ångstrom resolution (Schulz, 1997). Efforts are under way to elucidate a high resolution three-dimensional structure of beta-glucosidase from P.furiosus guided by the recently determined structure of the homologous Sulfolobus solfataricus beta-glycosidase LacS in conjunction with a mutational approach to improve the packaging of the pyrococcal crystals and resolve uncertain regions.Analysis of the genomic region preceding the celB gene of P.furiosus , revealed the presence of two tandem genes, oppositely orientated of celB (Fig. 1). These have been designated adhA and adhB , since their predicted products showed high homology to short-chain and iron-containing NADP(H)-dependent alcohol dehydrogenases (ADHs), respectively. In chapter 3 these two distinct types of ADHs are studied. The AdhA of P.furiosus showed a high degree of conservation in its primary structure with bacterial and eucaryal short-chain ADHs, suggesting that a short-chain ADH was present in the last common ancestor. The AdhB is a member of the group of iron-containing ADHs that is characterized by the presence of four conserved histidine residues. The adhA and adhB genes were overexpressed in E.coli resulting in functional proteins.AdhB showed NADP(H)-dependent activity with methanol as substrate, but this activity was rapidly lost preventing a detailed characterization. The purified AdhA showed a stable NADP(H)-dependent activity towards a broad range of primary and secondary alcohols. AdhA revealed an optimum activity for substrates with a carbon chain of 5 residues, with a preference for the oxidation of secondary over primary alcohols. A higher affinity for aldehydes than for alcohols was observed for AdhA and the pH optimum of this reaction is near the optimum pH for growth of P.furiosus . Therefore, it is most likely that the physiological role of AdhA is the reduction of the aldehyde to the corresponding alcohol thereby removing reduction equivalents and regenerating the cofactor. Given the localization of the adhA and adhB genes, in the vicinity of the lamA and celB genes, it tempting to speculate that AdhA and AdhB both have a function in the metabolism of beta-linked glucose polymers.In chapter 4 the molecular characterization of an endo-beta-1,3-glucanase and its lamA gene from P.furiosus is described. The lamA gene was found to be located downstream of the tandem adhA-adhB genes within the celB locus (Fig. 1). The endo-beta-1,3-glucanase is the first archaeal member of the glycosyl hydrolases family 16 that is composed of endo-beta-1,3 and endo-beta-1,3-1,4-glucanases. The lamA gene, without the coding sequence for its N-terminal leader, was cloned behind the T7-promoter in E.coli . Using this expression system a functional and extremely stable endo-beta-1,3-glucanase was produced in E.coli up to 15 % of the total soluble protein. The purified endoglucanase showed the highest activity on the beta-1,3-glucose polymer laminarin, but has also activity on the beta-1,3-1,4-glucose polymers lichenan and beta-glucan. The pyrococcal endoglucanase showed optimal activity at pH 6-6.5 and the temperature for maximum activity was 100-105 °C, while at 100 °C it has a half-life of 19 h. Amino acid sequence alignment of the glycosyl hydrolases of family 16 showed two subgroups; one with endo-beta-1,3-1,4-glucanase activity and another with predominantly endo-beta-1,3-glucanase activity. The latter group is characterized by an additional methionine residue in the predicted active site. Removal of this methionine in the pyrococcal endo-beta-1,3-glucanase by protein engineering did not alter its substrate specificity, but only the catalytic activity.It was found that P.furiosus was able to grown on laminarin and that the endoglucanase in vitro hydrolysed laminarin into oligomers and glucose. However, the hydrolysis proceeded more efficiently in combination with the beta-glucosidase of P.furiosus . These observations suggest a key role for the extracellular endoglucanase as well as the intracellular beta-glucosidase in the utilization of beta-1,3-linked glucose polymer laminarin.Chapter 5 describes the regulation of transcription of the divergent celB gene and the adhA-adhB-lamA gene cluster in P.furiosus . Northern blot showed that the adhA-adhB-lamA gene cluster form a 2.8-kb operon, designated the lamA operon. This operon is flanked upstream by the celB gene in opposite orientation and downstream by the birA gene, with the celB gene being transcribed as monocistronic messengers (Fig. 1). The expression of the enzymes encoded by the celB gene and the lamA operon of P.furiosus was found to be largely dependent on the carbon source present and was highest when the pyrococcal cells were grown on the beta-linked glucose polymers cellobiose and laminarin. The celB gene and the divergently orientated lamA operon were found to be controlled at the transcriptional level. Moreover, the transcripts were co-regulated and induced by growth on beta-linked glucose polymers. The transcription initiation sites of the celB gene and the lamA operon were found to be separated by a relatively small intergenic spacer of 142 nucleotides that included the back-to-back promoters, that showed a high degree of conservation, including identical archaeal TATA-box sequences. Transcriptional analysis using an in vitro transcription system for P.furiosus revealed that both transcripts are initiated from their in vivo initiation site. However, the efficiency of transcription initiation was significantly lower than that of the gdh gene of P.furiosus , suggesting that a positive regulator may contribute to increase the efficiency of transcription of the divergent celB gene and lamA operon in P.furiosus . If so, this would be a bacterial type of regulation. A putative regulatory binding-site could be identified upstream of the promoters of the celB gene and lamA operon. These findings open the way use the cell-free transcription system of P.furiosus for the identification of the regulator that is likely to be involved in regulation of the divergently orientated genes of the celB locus. Alternatively, in vivo analysis of the transcriptional control of the celB locus may become feasible with the recent development of a transformation system in P.furiosus (Aagaard et al., 1996).A combination of the current knowledge and ideas of the utilization of beta-linked glucose polymers and the transcriptional regulation in the hyperthermophilic archaeon P.furiosus led to the following working model (Fig. 2). Large polymeric substrates containing beta-1,3-linked glucose residues, such as laminarin are depolymerized to smaller fragments by the extracellular endo-beta-1,3-glucanase (LamA). After uptake these fragments can be degraded by the intracellular beta-glucosidase activity of CelB. The complementary activity of these two hydrolases has been shown in vitro . The co-regulation of transcription of the genes encoding these hydrolases support their concerted action in polymere degradation.Figure 2. : Model for the utilization of beta-linked glucose polymers, like laminarin. 1, the extracellular endo-beta-1,3-glucanase (LamA); 2, unknown uptake system for oligosaccharides; 3, beta-glucosidase (CelB); 4, ADP-dependent hexokinase.The two remaining experimental chapters deal with the characterization and molecular modelling of proteases involved in the growth on proteinaceous substrates. In chapter 6 the isolation and characterization of the hyperthermostable serine protease, pyrolysin, and its gene from P.furiosus are presented. The extracellular pyrolysin was found to be associated to the cell membrane of P.furiosus . The major purification step for pyrolysin was obtained via an autoincubation of the membrane fraction in 6 M urea at 95 °C, during which a 100-fold purification was obtained. The purification procedure resulted in two proteolytically activity fractions, HMW and LMW pyrolysin that were found to have identical NH 2 -termini and were glycosylated to a similar extent. Additionally, autoincubation of the HMW pyrolysin resulted in a proteolytically active fraction with the size of LMW pyrolysin. Together, these data indicate that the LMW pyrolysin is generated from the HMW pyrolysin by the autoproteolytic removal of its COOH-terminal part.Via reversed genetics the pls gene, coding for the pyrolysin of P.furiosus , was cloned and characterized. The deduced pyrolysin amino acid sequence consists of 1398 residues and is synthesized as prepro-enzyme, with the mature part of 1249 residues that showed the highest homology with eucaryal tripeptidyl-peptidases, that form a distinct subgroup of the subtilisin-like serine proteases (also referred to as subtilases). The NH 2 -terminal catalytic domain (approximately 500 residues) showed considerable homology to subtilisin-like serine protease and contained a large insert of more than 150 residues between the aspartate and histidine active site residues. The COOH-terminal domain of pyrolysin together with the large insert in the catalytic domain contains almost all (29 of 32) of the possible N -glycosylation sites present in pyrolysin. Substrate specificity studies indicated that pyrolysin is a true endopeptidase with a rather broad substrate specificity and may autocatalytically remove its own propeptide.The amino acid sequence homology in the catalytic domain of pyrolysin with other subtilases was sufficiently high to allow for homology modelling to gain insight in how the structure of the enzyme is stabilized. However, the identification of features as being important for protein stabilization based on only a single enzyme may lead to misinterpretations. Therefore, we screened a number of hyperthermophiles for the presence of subtilases using a PCR approach. Degenerated oligonucleotides were deduced based on the amino acid sequence of pyrolysin as well as on that surrounding the active site residues which are highly conserved among most of the subtilisin-like serine proteases. Chapter 7 describes the results of such a PCR approach that led to the identification of a DNA fragment in the genome of the extreme thermophilic archaeon Thermococcus stetteri , which could encode a serine protease, designated stetterlysin. The deduced sequence of stetterlysin showed high homology with pyrolysin and contained a similar sized insert between the aspartate and histidine active site residues. The high homology between the two subtilases, stetterlysin and pyrolysin, and subtilases with an identified three dimensional structure allowed for homology modelling.Three-dimensional structure models for stetterlysin and pyrolysin were constructed based on the crystal structures of subtilases from mesophilic and thermophilic origin, respectively subtilisin BPN' and thermitase, and on the sequence alignment of the core residues of stetterlysin and pyrolysin with subtilisin and thermitase. The predicted model of subtilisin-S41 from psychrophilic origin was also included in the comparisons. The alignment and the predicted three-dimensional models were used to the analyze amino acid composition and structural features of the catalytic domain that could be related to thermostability. The higher thermostability of the subtilases, especially stetterlysin and pyrolysin, was found to be correlated with an increased number of residues involved in pairs and networks of charge-charge and aromatic-aromatic interactions. For stetterlysin and pyrolysin, most of the aromatic residues were located on the surface of the catalytic core and present in inserts within this domain, suggesting that for the overall structure of the proteases aromatic-aromatic interactions may have a even larger impact on the stability of the structure.Analysis of the location of N-glycosylation sites in highly thermostable subtilases, including stetterlysin and pyrolysin, showed that most of these sites are located in surface loops. The modelling of the substrate binding region with known substrates was in good agreement with the observed broad substrate range for pyrolysin and the proposed autocatalytic activation (chapter 6).The PCR approach with deduced oligonucleotides for subtilisin-like serine proteases also resulted in products with the expected size with DNA from the hyperthermophilic archaeon Pyrodictium abyssi and the hyperthermophilic bacterium Fervidobacterium pennavorans , that hybridized with a pyrolysin-derived probe and had the expected size. The PCR products were cloned and characterized and their deduced amino acid sequences showed significant homology with the catalytic domain of subtilases. However, unlike pyrolysin and stetterlysin, they did not contain the large inserts between the first two active site residues. Using this approach a F.pennavorans gene for a subtilisin-like serine has been isolated and is currently being characterized.The hydrolases described in this thesis have been used as models to study different aspects of enzymes from hyperthermophiles, also referred to as thermozymes. Industry has screened hyperthermophiles for enzymes that can be applied as industrial biocatalysts to replace less stable homologous in current processes or to initiate new processes. The industrial use of thermozymes is hampered by the cost-ineffective fermentation properties of the hyperthermophilic organisms, in which they reside. Therefore, functional overproduction of thermozymes in heterologous hosts may have considerable impact on the development of these enzymes as biocatalyst. This thesis describes the functional overproduction of number of thermozymes including a beta-glucosidase an short-chain alcohol dehydrogenase and an endo-beta-1,3-glucanase. Moreover, this work has contributed to the insight hydrolytic enzymes from hyperthermophilic archaea in the relation between their structure-function and structure-stability.
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