Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature

The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60-95 degrees C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH-dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques. At pH 7.0 all three techniques show the presence of two species, in about equal amounts, which is compatible with the existence of a dimeric and an octameric form. In decreasing pH, the dimers formed the octameric species and in increasing pH, the octameric species was converted to dimers. Above pH 8.0, only dimers were present, below pH 3.0 only octamers were present. The association of dimers into octamers decreased in non-polar solvents and increased with temperature. A mutant (Y41C) was obtained that did not show this behavior.     

Glucose Transport in the Extremely Thermoacidophilic Sulfolobus solfataricus Involves a High-Affinity Membrane-Integrated Binding Protein

The archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 2.5 to 3.5 on carbon sources such as yeast extracts, tryptone, and various sugars. Cells rapidly accumulate glucose. This transport activity involves a membrane-bound glucose-binding protein that interacts with its substrate with very high affinity (Kd of 0. 43 microM) and retains high glucose affinity at very low pH values (as low as pH 0.6). The binding protein was extracted with detergent and purified to homogeneity as a 65-kDa glycoprotein. The gene coding for the binding protein was identified in the S. solfataricus P2 genome by means of the amino-terminal amino acid sequence of the purified protein. Sequence analysis suggests that the protein is anchored to the membrane via an amino-terminal transmembrane segment. Neighboring genes encode two membrane proteins and an ATP-binding subunit that are transcribed in the reverse direction, whereas a homologous gene cluster in Pyrococcus horikoshii OT3 was found to be organized in an operon. These data indicate that S. solfataricus utilizes a binding-protein-dependent ATP-binding cassette transporter for the uptake of glucose.     

Molecular and biochemical characterization of the recombinant amidase from hyperthermophilic archaeon Sulfolobus solfataricus

We have cloned, sequenced, and overexpressed in Escherichia coli the amidase gene from the hyperthermophilic archaeon Sulfolobus solfataricus (strain MT4). The recombinant thermophilic protein was expressed as a fusion protein with an N-terminus six-histidine-residue affinity tag. The enzyme, the first characterized archaeal amidase, is a monomer of 55,784 daltons, enantioselective, and active on 2- to 6-carbon aliphatic amides and on many aromatic amides, over the pH range 4-9 and at temperatures from 60 degrees to 95 degrees C. The S. solfataricus amidase belongs to the class of amidases that share a characteristic signature, GGSS(S/ G)GS, located in the central region of the protein, and which show remarkable variability in their individual substrate specificities, can hydrolyze aliphatic or aromatic substrates, and share a large invariance of their primary structure.     

Plastid-associated Porphobilinogen Synthase from Toxoplasma gondii: KINETIC AND STRUCTURAL PROPERTIES VALIDATE THERAPEUTIC POTENTIAL*

Apicomplexan parasites (including Plasmodium spp. and Toxoplasma gondii) employ a four-carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast, a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin. The PBGS family exhibits a complex use of metal ions (Zn²⁺ and Mg²⁺) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ∼320-kDa octamer, and low levels of dimers but no hexamers were also observed. The enzyme displays a broad activity peak (pH 7–8.5), with a K_m for aminolevulinic acid of ∼150 μm and specific activity of ∼24 μmol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg²⁺ but not Zn²⁺ and shows two Mg²⁺ affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg²⁺-binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic, and structural features of apicomplexan PBGS offer scope for developing selective inhibitors of the parasite enzyme based on its quaternary structure characteristics.     

Influence of glycosylation on the oligomeric structure of yeast acid phosphatase

Secreted yeast acid phosphatase is found to be an octamer under physiological conditions rather than a dimer, as previously believed. The octameric form of the enzyme dissociates rapidly into dimers at pH below 3 and above 5, or by treatment with guanidine hydrochloride or urea, without further dissociation of dimers. Crosslinking experiments revealed that the dissociation of the octamer occurs through very unstable hexamers and tetramers, showing that the octamer is built of dimeric units. Dissociation to dimer was in all cases accompanied with a loss of most of the enzyme activity. The underglycosylated acid phosphatase, with less than eight carbohydrate chains per subunit, secreted from cells treated with moderate tunicamycin concentrations, contained besides octamers a high proportion of the dimers. With decreasing levels of enzyme glycosylation, the proportion of dimers increases and the amount of octamers correspondingly decreases. Furthermore, underglycosylated octamers were found to be significantly less stable than the fully glycosylated ones. This showed that carbohydrate chains play a significant role in the octamer formation in vivo, and in stabilization of the enzyme octameric form.     

Dissociation of the Octameric Enolase from S. Pyogenes - One Interface Stabilizes Another

Most enolases are homodimers. There are a few that are octamers, with the eight subunits arranged as a tetramer of dimers. These dimers have the same basic fold and same subunit interactions as are found in the dimeric enolases. The dissociation of the octameric enolase from S. pyogenes was examined, using NaClO₄, a weak chaotrope, to perturb the quaternary structure. Dissociation was monitored by sedimentation velocity. NaClO₄ dissociated the octamer into inactive monomers. There was no indication that dissociation of the octamer into monomers proceeded via formation of significant amounts of dimer or any other intermediate species. Two mutations at the dimer-dimer interface, F137L and E363G, were introduced in order to destabilize the octameric structure. The double mutant was more easily dissociated than was the wild type. Dissociation could also be produced by other salts, including tetramethylammonium chloride (TMACl) or by increasing pH. In all cases, no significant amounts of dimers or other intermediates were formed. Weakening one interface in this protein weakened the other interface as well. Although enolases from most organisms are dimers, the dimeric form of the S. pyogenes enzyme appears to be unstable.     

The signature amidase from Sulfolobus solfataricus belongs to the CX3C subgroup of enzymes cleaving both amides and nitriles. Ser195 and Cys145 are predicted to be the active site nucleophiles

The signature amidase from the extremophile archeum Sulfolobus solfataricus is an enantioselective enzyme that cleaves S-amides. We report here that this enzyme also converts nitriles in the corresponding organic acid, similarly to the well characterized amidase from Rhodococcus rhodochrous J1. The archaeal and rhodococcal enzymes belong to the signature amidases and contain the typical serine-glycine rich motif. They work at different optimal temperature, share a high sequence similarity and both contain an additional CX3C motif. To explain their dual specificity, we built a 3D model of the structure of the S. solfataricus enzyme, which suggests that, in addition to the classical catalytic Ser-cisSer-Lys, a putative additional Cys-cisSer-Lys catalytic site, likely to be responsible for nitrile hydrolysis, is present in these proteins. The results of random and site-directed mutagenesis experiments, as well as inhibition studies support our hypothesis.     

Over-expression, purification and characterization of the oligomerization dynamics of an invertebrate mitochondrial creatine kinase

Mitochondrial creatine kinase (MtCK) plays a central role in energy homeostasis within cells that display high and variable rates of ATP turnover. Vertebrate MtCKs exist primarily as octamers but readily dissociate into constituent dimers under a variety of circumstances. MtCK is an ancient protein that is also found in invertebrates including sponges, the most primitive of all multi-cellular animals. We have cloned, expressed, and purified one of these invertebrate MtCKs from a marine polychaete worm, Chaetopterus variopedatus (CVMtCK). Size exclusion chromatography and dynamic light scattering (DLS) were used to characterize oligomeric state in comparison with that of octameric chicken sarcomeric isoform (SarMtCK). At protein concentrations >1 mg/ml, CVMtCK was predominantly octameric (>90%). When diluted to 0.1 mg/ml, CVMtCK dissociated into dimers much more rapidly than SarMtCK when observed under identical conditions. The rate of dissociation for both MtCKs increased as temperature rose from 2 to 28 degrees C, and in CVMtCK, fell at higher incubation temperatures. The fraction of octameric CVMtCK at equilibrium increased with temperature and then fell. Temperature transition studies showed that octamers and dimers were rapidly interconvertible on a similar time scale. Importantly, when CVMtCK was converted to the transition state analog complex (TSAC), both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. These results clearly show distinct differences in octamer stability between CVMtCK and SarMtCK, which could impact function under physiological circumstances. Furthermore, the large yield of recombinant protein and high stability of CVMtCK in the TSAC suggest that this protein might be a good target for crystallization efforts.     

Properties of a novel thermostable glucoamylase from the hyperthermophilic archaeon Sulfolobus solfataricus in relation to starch processing

A gene (ssg) encoding a putative glucoamylase in a hyperthermophilic archaeon, Sulfolobus solfataricus, was cloned and expressed in Escherichia coli, and the properties of the recombinant protein were examined in relation to the glucose production process. The recombinant glucoamylase was extremely thermostable, with an optimal temperature at 90 degrees C. The enzyme was most active in the pH range from 5.5 to 6.0. The enzyme liberated beta-d-glucose from the substrate maltotriose, and the substrate preference for maltotriose distinguished this enzyme from fungal glucoamylases. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation analysis revealed that the enzyme exists as a tetramer. The reverse reaction of the glucoamylase from S. solfataricus produced significantly less isomaltose than did that of industrial fungal glucoamylase. The glucoamylase from S. solfataricus has excellent potential for improving industrial starch processing by eliminating the need to adjust both pH and temperature.     

Structural basis of the destabilization produced by an amino-terminal tag in the beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus

We have previously shown that the major ion-pairs network of the tetrameric beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus involves more than 16 ion-pairs and hydrogen bonds between several residues from the four subunits and protects the protein from thermal unfolding by sewing the carboxy-termini of the enzyme. We show here that the amino-terminal of the enzyme also plays a relevant role in the thermostabilization of the protein. In fact, the addition of four extra amino acids at the amino-terminal of the beta-glycosidase, though not affecting the catalytic machinery of the enzyme and its thermophilicity, produced a faster enzyme inactivation in the temperature range 85-95 degrees C and decreased the Tm of the protein of 6 degrees C, measured by infrared spectroscopy. In addition, detailed two-dimensional IR correlation analysis revealed that the quaternary structure of the tagged enzyme is destabilized at 85 degrees C whilst that of the wild type enzyme is stable up to 98 degrees C. Molecular models allowed the rationalization of the experimental data indicating that the longer amino-terminal tail may destabilize the beta-glycosidase by enhancing the molecular fraying of the polypeptide and loosening the dimeric interfaces. The data support the hypothesis that fraying of the polypeptide chain termini is a relevant event in protein unfolding.     

Isolation, characterization and microsequence analysis of a small basic methylated DNA-binding protein from the Archaebacterium, Sulfolobus solfataricus

DNA-binding proteins have been extracted from the thermoacidophilic archaebacterium Sulfolobus solfataricus strain P1, grown at 86 degrees C and pH 4.5. These proteins, which may have a histone-like function, were isolated and purified under standard, non-denaturing conditions, and can be grouped into three molecular mass classes of 7, 8 and 10 kDa. We have purified to homogenity the main 7 kDa protein and determined its DNA-binding affinity by filter binding assays and electron microscopy. The Stokes radius of gyration indicates that the protein occurs as a monomer. The complete amino-acid sequence of this protein contains 14 lysine residues out of 63 amino acids and the calculated Mr is 7149. Five of the lysine residues are partially monomethylated to varying extents and the methylated residues are located exclusively in the N-terminal (positions 4 and 6) and the C-terminal (positions 60, 62 and 63) regions only. The protein is strongly homologous to the 7 kDa proteins of Sulfolobus acidocaldarius with the highest homology to protein 7d. Accordingly, the name of this protein from S. solfataricus was assigned as DNA-binding protein Sso7d.     

Identification of a novel alpha-galactosidase from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable alpha-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for alpha-linked galactosides, which are optimally hydrolyzed at pH 5 and 90 degrees C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable alpha-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.     

Driving forces of protein association: the dimer-octamer equilibrium in arylsulfatase A

The enzyme arylsulfatase A (ASA) occurs in solution as dimer (alpha(2)) above pH 6 and associates to octamers (alpha(2))(4) below pH 6. The crystal structure of ASA suggests that the (alpha(2))-(alpha(2))(4) equilibrium is regulated by protonation/deprotonation of Glu-424 located at the interface between (alpha(2)) dimers in the octamer. The reason for this assumption is that Glu-424 can be in two different conformers where it forms an intra or intermolecular hydrogen bond, respectively. In the present study we investigate this protein association process theoretically. The electrostatic energies are evaluated by solving the Poisson-Boltzmann equation for the inhomogeneous dielectric of the protein-water system for the dimer and octamer configurations. If a conventional surface energy term is used for the nonelectrostatic interactions, the absolute value of free energy of association fails to agree with experiment. A more detailed treatment that explicitly accounts for hydrophilic and hydrophobic character of the amino acids in the dimer-dimer interface of the octamer can explain this discrepancy qualitatively. The pH dependence of the computed association energy clearly demonstrates that the octamer is more stable at low pH if Glu-424 becomes protonated and forms an intermolecular hydrogen bond. We found a slight preference of Glu-424 to be in a conformation where its acidic group is fully solvent-exposed in the dimer state to form hydrogen bonds with water molecules. Application of the proton linkage model to calculate the association energy from the simulated data yielded results identical to the one obtained from the corresponding direct method.     

Dynamic metabolic adjustments and genome plasticity are implicated in the heat shock response of the extremely thermoacidophilic archaeon Sulfolobus solfataricus

Approximately one-third of the open reading frames encoded in the Sulfolobus solfataricus genome were differentially expressed within 5 min following an 80 to 90 degrees C temperature shift at pH 4.0. This included many toxin-antitoxin loci and insertion elements, implicating a connection between genome plasticity and metabolic regulation in the early stages of stress response.     

Evidence that the xylanase activity from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> Oα is encoded by the endoglucanase precursor gene (<Emphasis Type="Italic">sso1354</Emphasis>) and characterization of the associated cellulase activity

Sulfolobus solfataricus strain Oalpha was previously isolated for its ability to grow on minimal medium supplemented with xylan as a carbon source. The strain exhibited thermostable xylanase activity but several attempts to identify the gene encoding for the activity failed. Further studies showed that the xylanase displayed activity on carboxymethylcellulose (CMC) and the new activity was characterized. It exhibited an optimal temperature and pH of 95 degrees C and 3.5, respectively, and a half-life of 53 min at 95 degrees C. The enzyme, which was demonstrated to be glycosylated, hydrolyzed CMC in an endo-manner releasing cellobiose and other cello-oligomers. Analysis of the tryptic fragments by tandem mass spectrometry led to identification of the endoglucanase precursor, encoded by the sso1354 gene, as the protein possessing dual activity. The efficiency of the SSO1354 protein in degrading cellulosic and hemicellulosic fractions contained in agronomic residues was tested at low pH and high temperature. Cellulose and xylan were degraded to glucose and xylose at 90 degrees C, pH 4 by an enzyme mix consisting of SSO1354 and additional glycosyl hydrolases from S. solfataricus Oalpha. Given its role in saccharification processes requiring high temperatures and acidic environments, SSO1354 represents an interesting candidate for the utilization of agro-industrial waste for fuel production.     

The role of an absolutely conserved tryptophan residue in octamer formation and stability in mitochondrial creatine kinases

In most organisms, mitochondrial creatine kinase (MtCK) is present as dimers and octamers with the latter predominating under physiological conditions. An absolutely conserved tryptophan residue (Trp-264 in chicken sarcomeric MtCK) appears to play a key role in octamer stability. Recently, it has been shown that the sponge Tethya aurantia, a member of the most ancient group of living multi-cellular animals, expresses an obligate, dimeric MtCK that lacks this absolutely conserved tryptophan residue, instead possessing a tyrosine in this position. In the present study we confirm that the absolutely conserved tryptophan residue is lacking in other sponge MtCKs where it is instead substituted by histidine or asparagine. Site directed mutations of the Trp-264 in expression constructs of chicken sarcomeric MtCK and the octameric MtCK from the marine worm Chaetopterus destabilized the octameric quaternary structure producing only dimers. A Tyr-->Trp mutation in an expression construct of the Tethya MtCK construct failed to produce octamerization; Tyr-->His and Tyr-->Asn mutations also yielded dimers. These results, in conjunction with analysis of homology models of Chaetopterus and Tethya MtCKs, strongly support the view that while the absolutely conserved tryptophan residue is important in octamer stability, octamer formation involves a complex suite of interactions between a variety of residues.     

Comparative analysis of the catechol 2,3-dioxygenase gene locus in thermoacidophilic archaeon Sulfolobus solfataricus strain 98/2

A catechol 2,3-dioxygenase (C23O) gene was found from Sulfolobus solfataricus strain 98/2. Heterologous thermophilic C23O expressed in Escherichia coli showed the highest activity against catechol and 4-chlorocatechol, and at neutral pH. The C23O gene located with a putative multicomponent monooxygenase (MM) gene cluster that exactly matched with the homologous region of S. solfataricus strain P2. Primary sequence comparison identified an insertion sequence (IS) element inserted into a putative MM protein A N-terminal fragment gene in strain 98/2. Both ends of the transposase gene in the IS element, ISC1234, were flanked by 19 bp inverted repeat and 4 bp direct repeat sequences which are typical features of mobile elements. Our analysis and the two geographically distant origins of strains 98/2 and P2 (USA and Italy, respectively) suggest that the two strains have evolved from a common ancestor.     

Capillary electrophoresis is a sensitive monitor of the hairpin-random coil transition in DNA oligomers

Capillary electrophoresis (CE) has been used to characterize the hairpin-random coil transition of four octamers in the GCxxxxGC minihairpin family, where xxxx is GAAA, TTTC, TTTT, or AAAA. The transition can be monitored by CE because differences in the frictional coefficients of the hairpin and coil forms of each octamer lead to a difference of approximately 9% in the free solution mobilities of the two conformations. The GAAA octamer is unusually stable, with a melting temperature of 65 degrees C. The TTTT octamer forms a minihairpin with a melting temperature of 29 degrees C, the TTTC octamer has a melting temperature of 16 degrees C, and the AAAA octamer has a melting temperature below 0 degrees C. The thermal transitions of the TTTT, TTTC, and AAAA octamers are well fitted by a structure prediction algorithm; however, the GAAA minihairpin is considerably more stable than predicted. The melting temperature of the GAAA minihairpin is reduced to 47 degrees C in aqueous buffers containing 7.2M urea and to 33 degrees C in buffers containing 7.2M urea plus 40% (v/v) formamide. The combined results indicate that CE is a sensitive technique for monitoring conformational transitions in small DNA molecules.