Thermoadaptation of a mesophilic hygromycin B phosphotransferase by directed evolution in hyperthermophilic Archaea: selection of a stable genetic marker for DNA transfer into Sulfolobus solfataricus

A mutated version of the hygromycin B phosphotransferase (hph(mut)) gene from Escherichia coli, isolated by directed evolution at 75 degrees C in transformants of a thermophilic strain of Sulfolobus solfataricus, was characterized with respect to its genetic stability in both the original mesophilic and the new thermophilic hosts. This gene was demonstrated to be able to express the hygromycin B resistance phenotype and to be steadily maintained and propagated also in other, more thermophilic strains of S. solfataricus, i.e., up to 82 degrees C. Furthermore, it may be transferred to S. solfataricus cells by cotransformation with pKMSD48, another extrachromosomal element derived from the virus SSV1 of Sulfolobus shibatae, without any loss of stability and without affecting the replication and infectivity of this viral DNA. The hph(mut) and the wild-type gene products were expressed at higher levels in E. coli and purified by specific affinity chromatography on immobilized hygromycin B. Comparative characterization revealed that the mutant enzyme had acquired significant thermoresistance and displayed higher thermal activity with augmented catalytic efficiency.     

3-hydroxy-3-methylglutaryl coenzyme A reductase of Sulfolobus solfataricus: DNA sequence, phylogeny, expression in Escherichia coli of the hmgA gene, and purification and kinetic characterization of the gene product.

The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii. Phylogenetic analyses of HMG-CoA reductase protein sequences suggested that the two archaeal genes are distant homologs of eukaryotic genes. The only known bacterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseudomonas mevalonii, is highly diverged from archaeal and eukaryotic HMG-CoA reductases. The S. solfataricus hmgA gene encodes a true biosynthetic HMG-CoA reductase. Expression of hmgA in Escherichia coli generated a protein that both converted HMG-CoA to mevalonate and cross-reacted with antibodies raised against rat liver HMG-CoA reductase. S. solfataricus HMG-CoA reductase was purified in 40% yield to a specific activity of 17.5 microU per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate was (S)- not (R)-HMG-CoA; the reductant was NADPH not NADH. The Km values for HMG-CoA (17 microM) and NADPH (23 microM) were similar in magnitude to those of other biosynthetic HMG-CoA reductases. Unlike other HMG-CoA reductases, the enzyme was stable at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C.     

beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: structure and activity in the presence of alcohols.

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a tetrameric protein with a molecular mass of 240 kDa, stable in the presence of detergents, and with a maximal activity at temperatures above 95 degrees C. Understanding the structure-activity relationships of the enzyme under different conditions is of fundamental importance for both theoretical and applicative purposes. In this paper we report the effect of methanol, ethanol, 1-propanol, and 1-butanol on the activity of S. solfataricus beta-glycosidase expressed in Escherichia coli. The alcohols stimulated the enzyme activity, with 1-butanol producing its maximum effect at a lower concentration than the other alcohols. The structure of the enzyme was studied in the presence of 1-butanol by circular dichroism, and Fourier-transform infrared and fluorescence spectroscopies. Circular dichroism and steady-state fluorescence measurements revealed that at low temperatures the presence of the alcohol produced no significant changes in the tertiary structure of the enzyme. However, time-resolved fluorescence data showed that the alcohol modifies the protein microenvironment, leading to a more flexible enzyme structure, which is probably responsible for the enhanced enzymatic activity.     

Heterologous expression of 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability.

The gene for the extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus was expressed at a high level in Escherichia coli thus providing a basis for detailed structural and functional studies of the enzyme. The recombinant enzyme was purified to homogeneity by means of a heat treatment (10 min at 100 degrees C) and by a single affinity chromatography step. The appropriate expression vector and host strain were selected and the culture conditions were determined that would ensure a consistent yield of 6 mg of pure enzyme per liter of culture. The heterologously expressed enzyme is identical to the original S. solfataricus 5'-methylthioadenosine phosphorylase regarding molecular weight, substrate specificity, and the presence of intersubunit disulfide bonds. On the other hand, the recombinant 5'-methylthioadenosine phosphorylase is less thermophilic and thermostable than the S. solfataricus enzyme, since an incorrect positioning of disulfide bonds within the molecule generates structures less stable to thermal unfolding.     

A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein

A genomic library of the hyperthermophilic archaeon Sulfolobus solfataricus strain MT4 was constructed in Escherichia coli using a cloning vector not designed for heterologous gene expression. One positive clone exhibiting acquired thermophilic acetylesterase activity was directly detected by an in situ plate assay using a colony staining procedure with the chromogenic substrate beta-naphthyl acetate. The plasmid isolated from the clone contained a 3.3 kb genomic fragment from S. solfataricus and a full-length esterase coding sequence could be identified. Expression of the active thermostable esterase in E. coli was independent of isopropyl-beta-D-thiogalactopyranoside and of the kind of vector, suggesting that the archaeal esterase gene was controlled by fortuitous bacterial-like sequences present in its own 5' flanking region, not by the bacterial lac promoter or other serendipitous vector-located sequences. The protein, partially purified by thermoprecipitation of the host proteins at high temperature and gel exclusion chromatography, showed a homo-tetrameric structure with a subunit of molecular mass of 32 kDa which was in perfect agreement with that deduced from the cloned gene. The same protein was revealed in S. solfataricus cell extracts, thus demonstrating its functional occurrence in vivo under the cell culture conditions tested. The recombinant enzyme exhibited high thermal activity and thermostability with optimal activity between pH 6.5 and 7.0. The hydrolysis of p-nitrophenyl esters of fatty acids (from C(2) to C(8)) allowed the enzyme to be classified as a short length acyl esterase.     

The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro.

We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 degrees C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920-kDa oligomer of 57-kDa subunits, displays a potassium-dependent ATPase activity with an optimum temperature at 80 degrees C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride-denatured enzymes from thermophilic and mesophilic sources. At a molar ratio of chaperonin oligomer to single polypeptide chain of 1:1, S. solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes. S. solfataricus chaperonin prevents the irreversible inactivations at 90 degrees C of several thermophilic enzymes by the binding of the denaturation intermediate; the time-courses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP. S. solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 degrees C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measurements provide evidence that S. solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change. The ATP/Mg-induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites. This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated.     

S-adenosylmethionine decarboxylase from the thermophilic archaebacterium Sulfolobus solfataricus. Purification, molecular properties and studies on the covalently bound pyruvate.

S-Adenosylmethionine decarboxylase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium optimally growing at 87 degrees C, has been purified to homogeneity. The specific activity of the homogeneous enzyme is 12 nmol CO2 formed min-1 (mg protein)-1 and the overall yield 8%. The enzyme is thermophilic with an optimum at 75 degrees C, is thermostable, and does not require divalent cations or putrescine for activity. It has a molecular mass of 32 kDa, and appears to be a monomeric protein. S-Adenosylmethionine decarboxylase from S. solfataricus contains covalently linked pyruvate as prosthetic group and is inactivated in a time-dependent process by NaCNBH3, in the presence of both the substrate and the product. Incubation with decarboxylated S-adenosyl[Me-3H]methionine and NaCNBH3 resulted in the labeling of the protein at the active site.     

Sulfolobus aconitase,a regulator of iron metabolism?

The aconitase of Sulfolobus solfataricus, a hyperthermophilic crenarchaeon, was cloned and heterologously expressed in Escherichia coli. Enzymic analyses and EPR measurements indicated clearly that the iron-sulphur cluster of the thermophilic aconitase was already inserted in the mesophilic host. The enzyme was purified to a specific activity of approx. 44 units/mg and to 90% homogeneity. The enzymic parameters of the recombinant aconitase turned out to be in the same range as the respective values for the previously characterized native enzyme from the closely related S. acidocaldarius. Based on its primary sequence, the recombinant aconitase is closely related to bacterial A-like and to eukaryotic iron regulatory protein-like proteins. Specific aconitase activities in cytosolic extracts of S. acidocaldarius were found to be decreased markedly in iron-starved compared with iron-repleted cells. However, no differences in aconitase levels between iron-starved and iron-supplemented cells could be detected by immunostaining.     

The Sulfolobus solfataricus electron donor partners of thermophilic CYP119: an unusual non-NAD(P)H-dependent cytochrome P450 system

CYP119 from Sulfolobus solfataricus is the first well-characterized thermophilic cytochrome P450 enzyme. The endogenous substrate for this enzyme is not known but it hydroxylates lauric acid in a reaction supported by surrogate mesophilic electron donors. However, reconstitution of a high-temperature catalytic system requires identification of the normal thermophilic electron donor partners of CYP119. Here, we describe cloning, expression in Escherichia coli, and characterization of the requisite electron donor partners from S. solfataricus. One is a thermostable ferredoxin and the second a 2-oxoacid-ferredoxin oxidoreductase that utilizes pyruvic acid rather than NAD(P)H as the source of reducing equivalents. CYP119 is the only cytochrome P450 to date known to obtain electrons from a non-NAD(P)H-dependent protein. The two thermophilic partners have been used to reconstitute a catalytic system that hydroxylates lauric acid at 70 degrees C, and the optimal conditions for this system have been defined. This first high-temperature in vitro catalytic system represents an important step in the development of industrially relevant catalysts.     

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.     

Expression, purification, and characterization of recombinant S-adenosylhomocysteine hydrolase from the thermophilic archaeon Sulfolobus solfataricus

S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus was expressed in Escherichia coli by inserting the genomic fragment containing the gene encoding for S-adenosylhomocysteine hydrolase downstream the isopropyl-beta-d-thiogalactoside-inducible promoter of pTrc99A expression vector. An ATG positioned 25 bp upstream of the gene which is in frame with a stop codon was utilized as the initiation codon. This construct was used to transform E. coli RB791 and E. coli JM105 strains. The recombinant protein, purified by a fast and efficient two-step procedure (yield of 0.4 mg of enzyme per gram of cells), does not appear homogeneous on SDS-PAGE because of the presence of a protein contaminant corresponding to a "truncated" S-adenosylhomocysteine hydrolase subunit lacking the first 24 amino acid residues. The recombinant enzyme shows the same molecular mass, optimum temperature, and kinetic features of S-adenosylhomocysteine hydrolase isolated from S. solfataricus but it is less thermostable. To construct a vector which presents a correct distance between the ribosome-binding site and the start codon of S-adenosylhomocysteine hydrolase gene, a NcoI site was created at the translation initiation codon using site-directed mutagenesis. The expression of the homogeneous mutant S-adenosylhomocysteine hydrolase was achieved at high level (1.7 mg of mutant protein per gram of cells). The mutant S-adenosylhomocysteine hydrolase and the native one were indistinguishable in all physicochemical and kinetic properties including thermostability, indicating that the interactions involving the NH(2)-terminal sequence of the protein play a role in the thermal stability of S. solfataricus S-adenosylhomocysteine hydrolase.     

Characterization of ribonuclease P from the archaebacterium Sulfolobus solfataricus

Ribonuclease P is the endonuclease that removes the leader fragments from the 5'-ends of precursor tRNAs. The enzyme isolated from eubacteria contains a catalytic RNA subunit. RNAs also copurify with eukaryotic RNase P, although catalysis by those RNAs has not been demonstrated. This paper reports the isolation and characterization of ribonuclease P from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Archaebacteria are a primary evolutionary lineage, distinct from both eukaryotes and eubacteria. Ribonuclease P of S. solfataricus has reaction component requirements and a Km for substrate tRNA (2.5 X 10(-7) M) that are roughly similar to those reported for eubacterial and eukaryotic ribonuclease P. The temperature optimum for the reaction is 77 degrees C, reflecting the thermophilic character of the organism. The enzyme activity is not affected by treatment with micrococcal nuclease, suggesting that there is no RNA subunit or that it is protected from nuclease action. The density of the enzyme in cesium sulfate equilibrium density gradients is 1.27 g/ml, which is similar to that of protein. However, several RNAs between 200 and 400 nucleotides in size copurify with the enzyme activity on the density gradients, and one of them remains after micrococcal nuclease treatment. These properties of the S. solfataricus enzyme are compared with those of ribonuclease P from eukaryotes and eubacteria.     

A novel hyperthermostable 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus

We report herein the first molecular characterization of 5'-deoxy-5'-methylthio-adenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII). The isolated gene of SsMTAPII was overexpressed in Escherichia coli BL21. Purified recombinant SsMTAPII is a homohexamer of 180 kDa with an extremely low Km (0.7 microm) for 5'-deoxy-5'-methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120 degrees C and extremely thermostable with an apparent Tm of 112 degrees C that increases in the presence of substrates. The enzyme is characterized by high kinetic stability and remarkable SDS resistance and is also resistant to guanidinium chloride-induced unfolding with a transition midpoint of 3.3 m after 22-h incubation. Limited proteolysis experiments indicated that the only one proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. Moreover, the binding of 5'-deoxy-5'-methylthioadenosine induces a conformational transition that protected the enzyme against protease inactivation. By site-directed mutagenesis we demonstrated that Cys259, Cys261 and Cys262 play an important role in the enzyme stability since the mutants C259S/C261S and C262S show thermophilicity and thermostability features significantly lower than those of the wild-type enzyme. In order to get insight into the physiological role of SsMTAPII a comparative kinetic analysis with the homologous 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was carried out. Finally, the alignment of the protein sequence of SsMTAPII with those of SsMTAP and human 5'-deoxy-5'-methylthioadenosine phosphorylase (hMTAP) shows several key residue changes that may account why SsMTAPII, unlike hMTAP, is able to recognize adenosine as substrate.     

Isolation and sequencing of a new beta-galactosidase-encoding archaebacterial gene

The gene lacS coding for a beta-galactosidase (beta Gal; EC 3.2.1.23) has been cloned from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. It encodes a polypeptide chain of 489 amino acids (aa) (56,764 Da) in good agreement with the value directly measured for the enzyme (60 +/- 2 kDa per subunit). The aa composition of the enzyme and, in particular, its peculiarly low cysteine content (one Cys per subunit) has been confirmed; at the same time, it has been observed that the very low G + C content of the S. solfataricus genome strongly influences the codon usage preferences in the lacS sequence. There appears to be no evident similarity between this and the Escherichia coli lacZ sequence, thus suggesting that the two enzymes have analogous function, but are not homologous. By comparison with the published sequences of archaebacterial promoters, terminators and ribosome-binding sites, potential regulatory sites have been identified in the flanking regions of the S. solfataricus lacS gene.     

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.     

Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.     

Cellulose degradation by Sulfolobus solfataricus requires a cell-anchored endo-β-1-4-glucanase

A sequence encoding a putative extracellular endoglucanase (sso1354) was identified in the complete genome sequence of Sulfolobus solfataricus. The encoded protein shares signature motifs with members of glycoside hydrolases family 12. After an unsuccessful first attempt at cloning the full-length coding sequences in Escherichia coli, an active but unstable recombinant enzyme lacking a 27-residue N-terminal sequence was generated. This 27-amino-acid sequence shows significant similarity with corresponding regions in the sugar binding proteins AraS, GlcS, and TreS of S. solfataricus that are responsible for anchoring them to the plasma membrane. A strategy based on an effective vector/host genetic system for Sulfolobus and on expression control by the promoter of the S. solfataricus gene which encodes the glucose binding protein allowed production of the enzyme in sufficient quantities for study. In fact, the enzyme expressed in S. solfataricus was stable and highly thermoresistant and showed optimal activity at low pH and high temperature. The protein was detected mainly in the plasma membrane fraction, confirming the structural similarity to the sugar binding proteins. The results of the protein expression in the two different hosts showed that the SSO1354 enzyme is endowed with an endo-β-1-4-glucanase activity and specifically hydrolyzes cellulose. Moreover, it also shows significant but distinguishable specificity toward several other sugar polymers, such as lichenan, xylan, debranched arabinan, pachyman, and curdlan.     

Expression and characterization of the HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus

The thermostable class I HMG-CoA reductase of Sulfolobus solfataricus offers potential for industrial applications and for the initiation of crystallization trials of a biosynthetic HMG-CoA reductase. However, of the 15 arginine codons of the hmgA gene that encodes S. solfataricus HMG-CoA reductase, 14 (93%) are AGA or AGG, the arginine codons used least frequently by Escherichia coli. The presence of these rare codons in tandem or in the first 20 codons of a gene can complicate expression of that gene in E. coli. Problems include premature chain termination and misincorporation of lysine for arginine. We therefore sought to improve the expression and subsequent yield of S. solfataricus HMG-CoA reductase by expanding the pool size of tRNA(AGA,AGG), the tRNA that recognizes these two rare codons. Coexpression of the S. solfataricus hmgA gene with the argU gene that encodes tRNA(AGA,AGG) resulted in an over 10-fold increase in enzyme yield. This has provided significantly greater quantities of purified enzyme for potential industrial applications and for crystallographic characterization of a stable class I HMG-CoA reductase. It has, in addition, facilitated determination of kinetic parameters and of pH optima for all four catalyzed reactions, for determination of the K(i) for inhibition by the statin drug mevinolin, and for comparison of the properties of the HMG-CoA reductase of this thermophilic archaeon to those of other class I HMG-CoA reductases.