Purification, characterization, and molecular modeling of pyrolysin and other extracellular thermostable serine proteases from hyperthermophilic microorganisms.
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Amino Acid Sequence↗
Biomedical subjects
W G Voorhorst
Publications and source records attributed to W G Voorhorst.
Genetic and biochemical characterization of a short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.
The gene encoding a short-chain alcohol dehydrogenase, AdhA, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus, as part of an operon that encodes two glycosyl hydrolases, the beta-glucosidase CelB and the endoglucanase LamA. The adhA gene was functionally expressed in Escherichia coli, and AdhA was subsequently purified to homogeneity. The quaternary structure of AdhA is a dimer of identical 26-kDa subunits. AdhA is an NADPH-dependent oxidoreductase that converts alcohols to the corresponding aldehydes/ketones and vice versa, with a rather broad substrate specificity. Maximal specific activities were observed with 2-pentanol (46 U x mg(-1)) and pyruvaldehyde (32 U x mg(-1)) in the oxidative and reductive reaction, respectively. AdhA has an optimal activity at 90 degrees C, at which temperature it has a half life of 22.5 h. The expression of the adhA gene in P. furiosus was demonstrated by activity measurements and immunoblot analysis of cell extracts. A role of this novel type of archaeal alcohol dehydrogenase in carbohydrate fermentation is discussed.
Alcohol Dehydrogenase↗
Transcriptional regulation in the hyperthermophilic archaeon Pyrococcus furiosus: coordinated expression of divergently oriented genes in response to beta-linked glucose polymers.
The genetic organization, expression, and regulation of the celB locus of the hyperthermophilic archaeon Pyrococcus furiosus were analyzed. This locus includes the celB gene, which codes for an intracellular beta-glucosidase, and a divergently orientated gene cluster, adhA-adhB-lamA, which codes for two alcohol dehydrogenases and an extracellular beta-1,3-endoglucanase that is transcribed as a polycistronic messenger (the lamA operon). During growth of P. furiosus on either the beta-1,4-linked glucose dimer cellobiose or the beta-1,3-linked glucose polymer laminarin, the activities of both beta-glucosidase and endoglucanase were increased at least fivefold compared with levels during growth on maltose or pyruvate. Northern blot analysis revealed an enhanced transcription of both the celB gene and the lamA operon in the presence of these glucose-containing substrates. The in vivo and in vitro transcription initiation sites of both the celB gene and the lamA operon were identified 25 nucleotides downstream of conserved TATA box motifs. A number of repeating sequences have been recognized in the celB-adhA intergenic region, some of which might be part of a transcriptional regulator-binding site.
Alcohol Dehydrogenase↗
Sugar utilization and its control in hyperthermophiles.
Many hyperthermophilic microorganisms show heterotrophic growth on a variety of carbohydrates. There has been considerable fundamental and applied interest in the utilization of glucose and its alpha- and beta-polymers by hyperthermophiles. While glycolysis by Bacteria at high temperatures shows conventional characteristics, it has been found that glucose catabolism by hyperthermophilic Archaea differs from the canonical glycolytic pathways, involves novel enzymes, and shows a unique control. This review addresses these aspects with specific attention to Pyrococcus furiosus, which is one of the best studied hyperthermophilic Archaea, has the capacity to grow on a variety of sugars including the marine beta-(1,3)-linked glucose polymer laminarin, and has been found to contain three novel glycolytic enzymes, two ADP-dependent kinases, and a ferredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase.
Carbohydrate Metabolism↗
Molecular and biochemical characterization of an endo-beta-1,3- glucanase of the hyperthermophilic archaeon Pyrococcus furiosus.
We report here the first molecular characterization of an endo-beta-1,3-glucanase from an archaeon. Pyrococcus furiosus is a hyperthermophilic archaeon that is capable of saccharolytic growth. The isolated lamA gene encodes an extracellular enzyme that shares homology with both endo-beta-1,3- and endo-beta-1,3-1,4-glucanases of the glycosyl hydrolase family 16. After deletion of the N-terminal leader sequence, a lamA fragment encoding an active endo-beta-1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system. The purified P. furiosus endoglucanase has highest hydrolytic activity on the beta-1,3-glucose polymer laminarin and has some hydrolytic activity on the beta-1,3-1,4 glucose polymers lichenan and barley beta-glucan. The enzyme is the most thermostable endo-beta-1,3-glucanase described up to now; it has optimal activity at 100-105 degrees C. In the predicted active site of glycosyl hydrolases of family 16 that show predominantly endo-beta-1,3-glucanase activity, an additional methionine residue is present. Deletion of this methionine did not change the substrate specificity of the endoglucanase, but it did cause a severe reduction in its catalytic activity, suggesting a structural role of this residue in constituting the active site. High performance liquid chromatography analysis showed in vitro hydrolysis of laminarin by the endo-beta-1,3-glucanase proceeds more efficiently in combination with an exo-beta-glycosidase from P. furiosus (CelB). This most probably reflects the physiological role of these enzymes: cooperation during growth of P. furiosus on beta-glucans.
Amino Acid Sequence↗
Homology modelling of two subtilisin-like proteases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus stetteri.
The hyperthermophilic archaeon Pyrococcus furiosus produces an extracellular, glycosylated hyperthermostable subtilisin-like serine protease, termed pyrolysin (Voorhorst,W.G.B., Eggen,R.I.L., Geerling,A.C.M., Platteeuw,C., Siezen,R.J. and de Vos,W.M. (1996) J. Biol. Chem., 271, 20426-20431). Based on the pyrolysin coding sequence, a pyrolysin-like gene fragment was cloned and characterized from the extreme thermophilic archaeon Thermococcus stetteri. Like pyrolysin, the deduced sequence of this serine protease, designated stetterlysin, contains a catalytic domain with high homology with other subtilases, allowing homology modelling starting from known crystal structures. Comparison of the predicted three-dimensional models of the catalytic domain of stetterlysin and pyrolysin with the crystal structure of subtilases from mesophilic and thermophilic origin, i.e. subtilisin BPN' and thermitase, and the homology model of subtilisin S41 from psychrophilic origin, led to the identification of features that could be related to protein stabilization. Higher thermostability was found to be correlated with an increased number of residues involved in pairs and networks of charge-charge and aromatic-aromatic interactions. These highly thermostable proteases have several extra surface loops and inserts with a relatively high frequency of aromatic residues and Asn residues. The latter are often present in putative N-glycosylation sites. Results from modelling of known substrates in the substrate-binding region support the broad substrate range and the autocatalytic activation previously suggested for pyrolysin.
Amino Acid Sequence↗
Isolation and characterization of the hyperthermostable serine protease, pyrolysin, and its gene from the hyperthermophilic archaeon Pyrococcus furiosus.
The hyperthermostable serine protease pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus was purified from membrane fractions. Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunological cross-reaction and identical NH2-terminal sequences in which the third residue could be glycosylated. The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin at 95 degrees C resulted in the formation of LMW pyrolysin, probably as a consequence of COOH-terminal autoproteolysis. The 4194-base pair pls gene encoding pyrolysin was isolated and characterized, and its transcription initiation site was identified. The deduced pyrolysin sequence indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerable homology to subtilisin-like serine proteases and a COOH-terminal domain that contained most of the 32 possible N-glycosylation sites. The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl peptidases II on the amino acid level but a different cleavage specificity as shown by its endopeptidase activity toward caseins, casein fragments including alphaS1-casein and synthetic peptides.
Amino Acid Sequence↗
Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli.
The celB gene encoding the cellobiose-hydrolyzing enzyme beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus has been identified, cloned, and sequenced. The transcription and translation gene was overexpressed in Escherichia coli, resulting in high-level (up to 20% of total protein) production of beta-glucosidase that could be purified by a two-step purification procedure. The beta-glucosidase produced by E. coli had kinetic and stability properties similar to those of the beta-glucosidase purified from P. furiosus. The deduced amino acid sequence of CelB showed high similarity with those of beta-glycosidases that belong to glycosyl hydrolase family 1, implicating a conserved structure. Replacement of the conserved glutamate 372 in the P. furiosus beta-glucosidase by an aspartate or a glutamine led to a high reduction in specific activity (200- or 1,000-fold, respectively), indicating that this residue is the active site nucleophile involved in catalysis above 100 degrees C.
Amino Acid Sequence↗
Axial coordination and reduction potentials of the sixteen hemes in high-molecular-mass cytochrome c from Desulfovibrio vulgaris (Hildenborough).
A spectroelectrochemical study is described of the sixteen hemes in the high-molecular-mass, monomeric cytochrome c (Hmc) from the periplasmic space of Desulfovibrio vulgaris, strain Hildenborough. One of the hemes has special properties. In the oxidized state at pH 7 it is predominantly high-spin, S = 5/2, with a g perpendicular value of less than 6 indicative of quantum-mechanical mixing with a low-lying (800 cm-1) S = 3/2 state; the balance is probably a low-spin derivative. The high-spin heme has an Em.7.5 value of +61 mV. The fifteen other hemes are low-spin bis-histidine coordinated with Em.7.5 values of approximately -0.20 V. Two of these hemes exhibit very anisotropic EPR spectra with a g1 value of 3.65 characteristic for strained bis-histidine coordination. A previous proposal, namely that methionine is coordinated to one of the hemes [Pollock, W.B.R., Loufti, M. Bruschi, M. Rapp-Giles, B.J., Wall, J. & Voordouw, G. (1991) J. Bacteriol. 173, 220] is disproved using spectroscopic evidence. Contrasting electrochemical data sets from two previous studies [Tan, J. & Cowan, J.A. (1990) Biochemistry 29, 4886; Bruschi, M., Bertrand, P., More, C., Leroy, G., Bonicel, J., Haladjian, J., Chottard, G., Pollock, W.B.R. & Voordouw, G. (1992) Biochemistry 31, 3281] are not consistent with our EPR titration results and are not reproducible. Hmc can be reduced by D. vulgaris Fe-hydrogenase in the presence of molecular hydrogen.
Cytochrome c Group↗
On the two iron centers of desulfoferrodoxin.
Desulfoferrodoxin from Desulfovibrio vulgaris, strain Hildenborough, is a homodimer of 28 kDa; it contains two Fe atoms per 14.0 kDa subunit. The N-terminal amino-acid sequence is homogeneous and corresponds to the previously described Rho gene, which encodes a highly charged 14 kDa polypeptide without a leader sequence. Although one of the two iron centers, FeA, has previously been described as a 'strained rubredoxin-like' site, EPR of the ferric form proves very similar to that of the pentagonal bipyramidally coordinated iron in ferric complexes of DTPA, diethylenetriaminepentaacetic acid: both systems have spin S = 5/2 and rhombicity E/D = 0.08. Unlike the Fe site in rubredoxin the FeA site in desulfoferrodoxin has a pH dependent midpoint potential with pKox = 9.2 and pKred = 5.3. Upon reduction (Em,7.5 = +2 mV) FeA exhibits an unusually sharp S = 2 resonance in parallel-mode EPR. The second iron, FeB, has S = 5/2 and E/D = 0.33; upon reduction (Em,7.5 = +90 mV) FeB turns EPR-silent.
Amino Acid Sequence↗
A regulatory role for the 32K protein in proteolytic processing of cowpea mosaic virus polyproteins.
We have studied the regulation of proteolytic processing of the polyproteins encoded by cowpea mosaic virus M-RNA and B-RNA. For that purpose mutations were introduced in full-length cDNA clones of these RNAs. RNA transcripts were translated in rabbit reticulocyte lysate and the effect of mutations on the processing was analysed. These studies revealed that the 32K protein is released from the 200K B-polyprotein by an intramolecular cleavage and remains associated with the 170K protein, probably by interaction with the 58K domain of the 170K protein. In this complex the conformation of the 170K protein is such that further cleavages are very slow. This complex carries out the processing of the Gln/Met site in the M-polyprotein. The 170K protein produced by a B-RNA mutant that lacks the 32K coding region was efficiently processed into 110K, 87K, 84K, 60K, 58K and 24K cleavage products. Thus, the 32K protein regulates the B-polyprotein processing by slowing it down and, on the other hand, enhances trans cleavage of M-polyproteins at a Gln/Met site.
Base Sequence↗
Processing of VPg-containing polyproteins encoded by the B-RNA from cowpea mosaic virus.
To study the processing of putative VPg precursors the expression of specific mutant transcripts derived from a full-length cDNA clone of cowpea mosaic virus (CPMV) B-RNA was examined in a rabbit reticulocyte lysate system. This study revealed that the 170K protein produced by a B-RNA mutant that lacks the 32K coding region was efficiently processed by mainly intramolecular cleavages at three different sites into three sets of proteins of 60K + 110K, 84K + 87K, and 58K + 112K. Further cleavage of the 60K protein into 58K and VPg has not been observed in this in vitro system. The 84K protein can be further processed by an intramolecular cleavage reaction via two alternative pathways, either into 26K (VPg + 24K) and 58K proteins or into 24K and 60K proteins. VPg can be released from the 112K (VPg + 110K) precursor either directly or via the 26K intermediate. Immunoblot analysis showed that the 112K protein is present in CPMV-infected plant cells indicating that the in vitro observations may hold true in vivo.
Base Sequence↗