Thermoacidophilic Crenarchaeon Sulfolobus Acidocaldarius Research Articles

Functional diversity in archaeal Hsp60: a molecular mosaic of Group I and Group II chaperonin.

External stress disrupts the balance of protein homeostasis, necessitating the involvement of heat shock proteins (Hsps) in restoring equilibrium andensuring cellular survival. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius, lacks the conventional Hsp100, Hsp90, and Hsp70, relying solely on a single ATP-dependent Group II chaperonin, Hsp60, comprising three distinct subunits (α, β, and γ) to refold unfolded substrates and maintain protein homeostasis. Hsp60 forms three different complexes, namely Hsp60αβγ, Hsp60αβ, and Hsp60β, at temperatures of 60 °C, 75 °C, and 90 °C, respectively. This study delves into the intricacies of Hsp60 complexes in S. acidocaldarius, uncovering their ability to form oligomeric structures in the presence of ATP. The recognition of substrates by Hsp60 involves hydrophobic interactions, and the subsequent refolding process occurs in an ATP-dependent manner through charge-driven interactions. Furthermore, the Hsp60β homo-oligomeric complex can protect the archaeal and eukaryotic membrane from stress-induced damage. Hsp60 demonstrates nested cooperativity in ATP hydrolysis activity, where MWC-type cooperativity is nested within KNF-type cooperativity. Remarkably, during ATP hydrolysis, Hsp60β, and Hsp60αβ complexes exhibit a mosaic behavior, aligning with characteristics observed in both Group I and Group II chaperonins, adding a layer of complexity to their functionality.

Exopolysaccharide composition and size in Sulfolobus acidocaldarius biofilms.

Extracellular polymeric substances (EPS) comprise mainly carbohydrates, proteins and extracellular DNA (eDNA) in biofilms formed by the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius. However, detailed information on the carbohydrates in the S. acidocaldarius biofilm EPS, i.e., the exopolysaccharides (PS), in terms of identity, composition and size were missing. In this study, a set of methods was developed and applied to study the PS in S. acidocaldarius biofilms. It was initially shown that addition of sugars, most significantly of glucose, to the basal N-Z-amine-based growth medium enhanced biofilm formation. For the generation of sufficient amounts of biomass suitable for chemical analyses, biofilm growth was established and optimized on the surface of membrane filters. EPS were isolated and the contents of carbohydrates, proteins and eDNA were determined. PS purification was achieved by enzymatic digestion of other EPS components (nucleic acids and proteins). After trifluoroacetic acid-mediated hydrolysis of the PS fraction, the monosaccharide composition was analyzed by reversed-phase liquid chromatography (RP-LC) coupled to mass spectrometry (MS). Main sugar constituents detected were mannose, glucose and ribose, as well as minor proportions of rhamnose, N-acetylglucosamine, glucosamine and galactosamine. Size exclusion chromatography (SEC) revealed the presence of one single PS fraction with a molecular mass of 4-9 × 104 Da. This study provides detailed information on the PS composition and size of S. acidocaldarius MW001 biofilms and methodological tools for future studies on PS biosynthesis and secretion.

Archaeal Hsp14 drives substrate shuttling between small heat shock proteins and thermosome: insights into a novel substrate transfer pathway.

Heat shock proteins maintain protein homeostasis and facilitate the survival of an organism under stress. Archaeal heat shock machinery usually consists of only sHsps, Hsp70, and Hsp60. Moreover, Hsp70 is absent in thermophilic and hyperthermophilic archaea. In the absence of Hsp70, how aggregating protein substrates are transferred to Hsp60 for refolding remains elusive. Here, we investigated the crosstalk in the heat shock response pathway of thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. In the present study, we biophysically and biochemically characterized one of the small heat shock proteins, Hsp14, of S. acidocaldarius. Moreover, we investigated its ability to interact with Hsp20 and Hsp60 to facilitate the substrate proteins' folding under stress conditions. Like Hsp20, we demonstrated that the dimer is the active form of Hsp14, and it forms an oligomeric storage form at a higher temperature. More importantly, the dynamics of the Hsp14 oligomer are maintained by rapid subunit exchange between the dimeric states, and the rate of subunit exchange increases with increasing temperature. We also tested the ability of Hsp14 to form hetero-oligomers via subunit exchange with Hsp20. We observed hetero-oligomer formation only at higher temperatures (50 °C-70 °C). Furthermore, experiments were performed to investigate the interaction between small heat shock proteins and Hsp60. We demonstrated an enthalpy-driven direct physical interaction between Hsp14 and Hsp60. Our results revealed that Hsp14 could transfer sHsp-captured substrate proteins to Hsp60, which then refolds them back to their active form.

High-Temperature Live-Cell Imaging of Cytokinesis, Cell Motility, and Cell-Cell Interactions in the Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius.

Significant technical challenges have limited the study of extremophile cell biology. Here we describe a system for imaging samples at 75°C using high numerical aperture, oil-immersion lenses. With this system we observed and quantified the dynamics of cell division in the model thermoacidophilic crenarchaeon Sulfolobus acidocaldarius with unprecedented resolution. In addition, we observed previously undescribed dynamic cell shape changes, cell motility, and cell-cell interactions, shedding significant new light on the high-temperature lifestyle of this organism.

YtrASa, a GntR-Family Transcription Factor, Represses Two Genetic Loci Encoding Membrane Proteins in Sulfolobus acidocaldarius.

In bacteria, the GntR family is a widespread family of transcription factors responsible for the regulation of a myriad of biological processes. In contrast, despite their occurrence in archaea only a little information is available on the function of GntR-like transcription factors in this domain of life. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius harbors a GntR-like regulator belonging to the YtrA subfamily, encoded as the first gene in an operon with a second gene encoding a putative membrane protein. Here, we present a detailed characterization of this regulator, named YtrASa, with a focus on regulon determination and mechanistic analysis with regards to DNA binding. Genome-wide chromatin immunoprecipitation and transcriptome experiments, the latter employing a ytrASa overexpression strain, demonstrate that the regulator acts as a repressor on a very restricted regulon, consisting of only two targets including the operon encoding its own gene and a distinct genetic locus encoding another putative membrane protein. For both targets, a conserved 14-bp semi-palindromic binding motif was delineated that covers the transcriptional start site and that is surrounded by additional half-site motifs. The crystallographic structure of YtrASa was determined, revealing a compact dimeric structure in which the DNA-binding motifs are oriented ideally to enable a specific high-affinity interaction with the core binding motif. This study provides new insights into the functioning of a YtrA-like regulator in the archaeal domain of life.

A defined cultivation medium for Sulfolobus acidocaldarius

The thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius is an important model organism for Archaea and genetic systems are well established. To date, the organism is routinely cultivated on complex media based on protein hydrolysates and no common defined medium is established. In this work we address this lack of a standardized defined medium and replaced the complex protein hydrolysate with sodium glutamate as primary substrate. Starting from an existing medium formulation we stepwise managed to improve the medium regarding formation of precipitates, buffer capacity, concentration of basal salts and trace elements, and optimized growth rates. The differences on the cellular level between the original medium and our new formulation, called VD Medium, were investigated by comparative gene expression analysis and significant differences were discussed. The final formulation of the VD Medium contains 1.75 g/L Na-glutamate, 3 g/L D-glucose and 0.5 g/L citric acid as carbon sources. Using the described medium for the cultivation of S. acidocaldarius DSM 639 in shake flasks yields 1.1 g/L dry cell weight (OD600 = 1.7) after a typical incubation time of 95 h with an overall biomass yield of 0.33 gDCW/gsubstrate.

Two membrane-bound transcription factors regulate expression of various type-IV-pili surface structures in Sulfolobus acidocaldarius.

In Archaea and Bacteria, gene expression is tightly regulated in response to environmental stimuli. In the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius nutrient limitation induces expression of the archaellum, the archaeal motility structure. This expression is orchestrated by a complex hierarchical network of positive and negative regulators—the archaellum regulatory network (arn). The membrane-bound one-component system ArnR and its paralog ArnR1 were recently described as main activators of archaellum expression in S. acidocaldarius. They regulate gene expression of the archaellum operon by targeting the promoter of flaB, encoding the archaellum filament protein. Here we describe a strategy for the isolation and biochemical characterization of these two archaellum regulators. Both regulators are capable of forming oligomers and are phosphorylated by the Ser/Thr kinase ArnC. Apart from binding to pflaB, ArnR but not ArnR1 bound to promoter sequences of aapF and upsX, which encode components of the archaeal adhesive pilus and UV-inducible pili system, demonstrating a regulatory connection between different surface appendages of S. acidocaldarius.

Early Response of Sulfolobus acidocaldarius to Nutrient Limitation.

In natural environments microorganisms encounter extreme changes in temperature, pH, osmolarities and nutrient availability. The stress response of many bacterial species has been described in detail, however, knowledge in Archaea is limited. Here, we describe the cellular response triggered by nutrient limitation in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. We measured changes in gene transcription and protein abundance upon nutrient depletion up to 4 h after initiation of nutrient depletion. Transcript levels of 1118 of 2223 protein coding genes and abundance of approximately 500 proteins with functions in almost all cellular processes were affected by nutrient depletion. Our study reveals a significant rerouting of the metabolism with respect to degradation of internal as well as extracellular-bound organic carbon and degradation of proteins. Moreover, changes in membrane lipid composition were observed in order to access alternative sources of energy and to maintain pH homeostasis. At transcript level, the cellular response to nutrient depletion in S. acidocaldarius seems to be controlled by the general transcription factors TFB2 and TFEβ. In addition, ribosome biogenesis is reduced, while an increased protein degradation is accompanied with a loss of protein quality control. This study provides first insights into the early cellular response of Sulfolobus to organic carbon and organic nitrogen depletion.

The oligomeric plasticity of Hsp20 of Sulfolobus acidocaldarius protects environment-induced protein aggregation and membrane destabilization

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that rescue misfolded proteins from irreversible aggregation during cellular stress. Many such sHsps exist as large polydisperse species in solution, and a rapid dynamic subunit exchange between oligomeric and dissociated forms modulates their function under a variety of stress conditions. Here, we investigated the structural and functional properties of Hsp20 from thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. To provide a framework for investigating the structure-function relationship of Hsp20 and understanding its dynamic nature, we employed several biophysical and biochemical techniques. Our data suggested the existence of a ~24-mer of Hsp20 at room temperature (25 °C) and a higher oligomeric form at higher temperature (50 °C–70 °C) and lower pH (3.0–5.0). To our surprise, we identified a dimeric form of protein as the functional conformation in the presence of aggregating substrate proteins. The hydrophobic microenvironment mainly regulates the oligomeric plasticity of Hsp20, and it plays a key role in the protection of stress-induced protein aggregation. In Sulfolobus sp., Hsp20, despite being a non-secreted protein, has been reported to be present in secretory vesicles and it is still unclear whether it stabilizes substrate proteins or membrane lipids within the secreted vesicles. To address such an issue, we tested the ability of Hsp20 to interact with membrane lipids along with its ability to modulate membrane fluidity. Our data revealed that Hsp20 interacts with membrane lipids via a hydrophobic interaction and it lowers the propensity of in vitro phase transition of bacterial and archaeal lipids.

Effect of UV irradiation onSulfolobus acidocaldarius and involvement of the general transcription factor TFB3 in the early UV response.

Exposure to UV light can result in severe DNA damage. The alternative general transcription factor (GTF) TFB3 has been proposed to play a key role in the UV stress response in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Reporter gene assays confirmed that tfb3 is upregulated 90–180 min after UV treatment. In vivo tagging and immunodetection of TFB3 confirmed the induced expression at 90 min. Analysis of a tfb3 insertion mutant showed that genes encoding proteins of the Ups pili and the Ced DNA importer are no longer induced in a tfb3 insertion mutant after UV treatment, which was confirmed by aggregation assays. Thus, TFB3 plays a crucial role in the activation of these genes. Genome wide transcriptome analysis allowed a differentiation between a TFB3-dependent and a TFB3-independent early UV response. The TFB3-dependent UV response is characterized by the early induction of TFB3, followed by TFB3-dependent expression of genes involved in e.g. Ups pili formation and the Ced DNA importer. Many genes were downregulated in the tfb3 insertion mutant confirming the hypothesis that TFB3 acts as an activator of transcription. The TFB3-independent UV response includes the repression of nucleotide metabolism, replication and cell cycle progression in order to allow DNA repair.

Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius.

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D100), IV (F220) and V (F264) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

PLFE as a Liposomal Stabilizing Agent: A Shear Stress Study

Cholesterol has been commonly used as a stabilizing agent within liposome formulations. However, cholesterol can have a negative impact in patients with cardiovascular diseases. Therefore, there is a need for a better stabilizing agent that can achieve liposome stability and has minimal effect on patient health. Bipolar tetraether lipids isolated from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius, such aspolar lipid fraction E (PLFE), have been shown to make very stable liposomes. Shear rates within the body range from 40 s−1 to 2000 s−1 during physiological conditions. This study examined the effect of shear rates on dye leakage on 100 mol% DPPC, 100 mol% PLFE, 70% DPPC/30% PLFE and 70% DPPC/30% cholesterol liposomes. Liposomes with entrapped 5(6)-carboxyfluorescein (5(6)-CF) exhibit spontaneous release of the dye. Changes in fluorescence intensity were measured to monitor the leakage of 5(6)-CF. Liposomes were subjected to a constant shear rate for 3 hours through an IBIDI perfusion and pump system. Dye leakage rate constants of 5(6) CF from liposomes were determined under 0, 1000 s−1, and 7600 s−1 shear flow rates at 37°C. The data demonstrate that addition of PLFE in liposomes greatly reduces 5(6)-CF release and minimizes the effect of shear stress when compared to cholesterol. This study demonstrates that the 70% DPPC/30% PLFE liposomes were the most stable under shear stress, with 100% DPPC exhibiting a 4-fold decrease in leakage rate constant and 100% PLFE exhibiting a 2-fold decrease. Inserting PLFE into liposomal systems in place of cholesterol increases the stability of liposomes under various rates of shear stress. PLFE can potentially be utilized to improve the broad range of liposome formulations and improve the lifetime of drug release. (NSF DMR1105277 and CBET1350841, NIH/NINDS NS086570-01, The Shriners Hospitals for Children 85110-PHI-14 and NIH-NIDA T32 DA007237).

Dissection of key determinants of cleavage activity in signal peptidase III (SPaseIII) PibD.

In Archaea, type IV prepilins and prearchaellins are processed by designated signal peptidase III (SPaseIII) prior to their incorporation into pili and the archaellum, respectively. These peptidases belong to the family of integral membrane aspartic acid proteases that contain two essential aspartate residues of which the second aspartate is located in a conserved GxGD motif. To this group also bacterial type IV prepilin peptidases, Alzheimer disease-related secretases, signal peptide peptidases and signal peptide peptidase-like proteases in humans belong. Here we have performed detailed in vivo analyses to understand the cleavage activity of PibD, SPaseIII from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Using an already established in vivo heterologous system cleavage assay, we could successfully identify the key amino acid residues essential for catalysis of PibD. Furthermore, in trans complementation of a pibD S. acidocaldarius deletion mutant with PibD variants having substituted key amino acids has consolidated our observations of the importance of these residues in catalysis. Based on our data, we propose to re-define class III peptidases/type IV prepilin/prearchaellin peptidases as GxHyD group (rather than GxGD) of proteases [Hy-hydrophobic amino acid].

The archaellum: a rotating type IV pilus

Microbes have evolved sophisticated mechanisms of motility allowing them to respond to changing environmental conditions. While this cellular process is well characterized in bacteria, the mode and mechanisms of motility are poorly understood in archaea. This study examines the motility of individual cells of the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Specifically, we investigated motility of cells producing exclusively the archaeal swimming organelle, the archaellum. Archaella are structurally and in sequence similar to bacterial type IV pili involved in surface motility via pilus extension-retraction cycles and not to rotating bacterial flagella. Unexpectedly, our studies reveal a novel type of behaviour for type IV pilus like structures: archaella rotate and their rotation drives swimming motility. Moreover, we demonstrate that temperature has a direct effect on rotation velocity explaining temperature-dependent swimming velocity.

The one‐component system ArnR: a membrane‐bound activator of the crenarchaeal archaellum

Linking the motility apparatus to signal transduction systems enables microbes to precisely control their swimming behaviour according to environmental conditions. Bacteria have therefore evolved a complex chemotaxis machinery, which has presumably spread through lateral gene transfer into the euryarchaeal subkingdom. By contrast Crenarchaeota encode no chemotaxis-like proteins but are nevertheless able to connect external stimuli to archaellar derived motility. This raises fundamental questions about the underlying regulatory mechanisms. Recently, we reported that the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius becomes motile upon nutrient starvation by promoting transcription of flaB encoding the filament forming subunits. Here we describe two transcriptional activators as paralogous one-component-systems Saci_1180 and Saci_1171 (ArnR and ArnR1). Deletions of arnR and arnR1 resulted in diminished flaB expression and accordingly the deletion mutants revealed impaired swimming motility. We further identified two inverted repeat sequences located upstream of the flaB core promoter of S. acidocaldarius. These cis-regulatory elements were shown to be critical for ArnR and ArnR1 mediated flaB gene expression in vivo. Finally, bioinformatic analysis revealed ArnR to be conserved not only in Sulfolobales but also in the crenarchaeal order of Desulfurococcales and thus might represent a more general control mechanism of archaeal motility.

Biochemical and structural characterization of recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius highly enantioselective on diaryl diketone benzil

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh2 gene was heterologously overexpressed in Escherichia coli, and the resulting protein (SaADH2) was purified to homogeneity and both biochemically and structurally characterized. The crystal structure of the SaADH2 NADH-bound form reveals that the enzyme is a tetramer consisting of identical 27,024-Da subunits, each composed of 255 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 80 °C and a 30-min half-inactivation temperature of ∼88 °C. It also shows good tolerance to common organic solvents and a strict requirement for NAD(H) as the coenzyme. SaADH2 displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, showing no activity on aldehydes. Interestingly, the enzyme catalyses the asymmetric reduction of benzil to (R)-benzoin with both excellent conversion (98 %) and optical purity (98 %) by way of an efficient in situ NADH-recycling system involving a second thermophilic ADH. The crystal structure of the binary complex SaADH2-NADH, determined at 1.75 Å resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.

Compressibilities and Volume Fluctuations of Archaeal Tetraether Liposomes

Bipolar tetraether lipids (BTLs) are abundant in crenarchaeota, which thrive in both thermophilic and nonthermophilic environments, with wide-ranging growth temperatures (4–108°C). BTL liposomes can serve as membrane models to explore the role of BTLs in the thermal stability of the plasma membrane of crenarchaeota. In this study, we focus on the liposomes made of the polar lipid fraction E (PLFE). PLFE is one of the main BTLs isolated from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Using molecular acoustics (ultrasound velocimetry and densimetry), pressure perturbation calorimetry, and differential scanning calorimetry, we have determined partial specific adiabatic and isothermal compressibility, their respective compressibility coefficients, partial specific volume, and relative volume fluctuations of PLFE large unilamellar vesicles (LUVs) over a wide range of temperatures (20–85°C). The results are compared with those obtained from liposomes made of dipalmitoyl-L- α-phosphatidylcholine (DPPC), a conventional monopolar diester lipid. We found that, in the entire temperature range examined, compressibilities of PLFE LUVs are low, comparable to those found in gel state of DPPC. Relative volume fluctuations of PLFE LUVs at any given temperature examined are 1.6–2.2 times more damped than those found in DPPC LUVs. Both compressibilities and relative volume fluctuations in PLFE LUVs are much less temperature-sensitive than those in DPPC liposomes. The isothermal compressibility coefficient ( β T lipid) of PLFE LUVs changes from 3.59 × 10 −10 Pa −1 at 25°C to 4.08 × 10 −10 Pa −1 at 78°C. Volume fluctuations of PLFE LUVs change only 0.25% from 30°C to 80°C. The highly damped volume fluctuations and their low temperature sensitivity, echo that PLFE liposomes are rigid and tightly packed. To our knowledge, the data provide a deeper understanding of lipid packing in PLFE liposomes than has been previously reported, as well as a molecular explanation for the low solute permeation and limited membrane lateral motion. The obtained results may help to establish new strategies for rational design of stable BTL-based liposomes for drug/vaccine delivery.

Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

Evidence for an operative glyoxylate cycle in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

Both key enzymes for the glyoxylate cycle, isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), were purified and characterized from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Whereas the former enzyme was copurified with the aconitase, the latter enzyme could be enriched to apparent homogeneity. Amino acid sequencing of three internal peptides of the isocitrate lyase revealed the presence of highly conserved residues. With respect to cofactor requirement and quarternary structure the crenarchaeal malate synthase might represent a novel type of this enzyme family. High activities of both glyoxylate cycle enzymes could already be detected in extracts of glucose grown cells and both increased about two-fold in extracts of acetate grown cells.