Simplified Enzymatic Synthesis of 2-Keto-3-Deoxy-D-Gluconate from D-Gluconate Using the Gluconate Dehydratase from Thermoproteus tenax.

Many research areas, e.g., basic research but also applied fields of biotechnology, biomedicine, and diagnostics often suffer from the unavailability of metabolic compounds. This is mostly due to missing easy and efficient synthesis procedures. We herein describe the biocatalytic/enzymatic production of 2-keto-3-deoxy-D-gluconate, an intermediate of central metabolic pathways in all three domains of life and also of bacterial polysaccharides, lipopolysaccharides, and cell wall components. The method is based on the gluconate dehydratase from the hyperthermophilic crenarchaeon Thermoproteus tenax, which can be easily recombinantly overproduced in Escherichia coli and-due to its intrinsic thermostability-rapidly be purified by two precipitation steps. The enzyme completely converts D-gluconate to solely stereochemically pure KDG, taking benefits from the enol-keto-tautomerism of the primary reaction product. The final product can then easily be separated from the protein by ultrafiltration. The simple one-step procedure, which is suitable at least for the lab-scale/gram-scale production of KDG, replaces lengthy multi-step reactions and is easily scalable. This approach also illustrates the great application potential of Archaea with their unusual metabolic pathways and enzymes for the synthesis of added value products.

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability.

The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications.

Circularization restores signal recognition particle RNA functionality in Thermoproteus.

Signal recognition particles (SRPs) are universal ribonucleoprotein complexes found in all three domains of life that direct the cellular traffic and secretion of proteins. These complexes consist of SRP proteins and a single, highly structured SRP RNA. Canonical SRP RNA genes have not been identified for some Thermoproteus species even though they contain SRP19 and SRP54 proteins. Here, we show that genome rearrangement events in Thermoproteus tenax created a permuted SRP RNA gene. The 5'- and 3'-termini of this SRP RNA are located close to a functionally important loop present in all known SRP RNAs. RNA-Seq analyses revealed that these termini are ligated together to generate circular SRP RNA molecules that can bind to SRP19 and SRP54. The circularization site is processed by the tRNA splicing endonuclease. This moonlighting activity of the tRNA splicing machinery permits the permutation of the SRP RNA and creates highly stable and functional circular RNA molecules.

DNA binding properties of the small cascade subunit Csa5.

CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.

The complete genome sequence of Thermoproteus tenax: a physiologically versatile member of the Crenarchaeota.

Here, we report on the complete genome sequence of the hyperthermophilic Crenarchaeum Thermoproteus tenax (strain Kra1, DSM 2078(T)) a type strain of the crenarchaeotal order Thermoproteales. Its circular 1.84-megabase genome harbors no extrachromosomal elements and 2,051 open reading frames are identified, covering 90.6% of the complete sequence, which represents a high coding density. Derived from the gene content, T. tenax is a representative member of the Crenarchaeota. The organism is strictly anaerobic and sulfur-dependent with optimal growth at 86°C and pH 5.6. One particular feature is the great metabolic versatility, which is not accompanied by a distinct increase of genome size or information density as compared to other Crenarchaeota. T. tenax is able to grow chemolithoautotrophically (CO2/H2) as well as chemoorganoheterotrophically in presence of various organic substrates. All pathways for synthesizing the 20 proteinogenic amino acids are present. In addition, two presumably complete gene sets for NADH:quinone oxidoreductase (complex I) were identified in the genome and there is evidence that either NADH or reduced ferredoxin might serve as electron donor. Beside the typical archaeal A0A1-ATP synthase, a membrane-bound pyrophosphatase is found, which might contribute to energy conservation. Surprisingly, all genes required for dissimilatory sulfate reduction are present, which is confirmed by growth experiments. Mentionable is furthermore, the presence of two proteins (ParA family ATPase, actin-like protein) that might be involved in cell division in Thermoproteales, where the ESCRT system is absent, and of genes involved in genetic competence (DprA, ComF) that is so far unique within Archaea.

Regulation of autotrophic CO2 fixation in the archaeon Thermoproteus neutrophilus.

Thermoproteus neutrophilus, a hyperthermophilic, chemolithoautotrophic, anaerobic crenarchaeon, uses a novel autotrophic CO(2) fixation pathway, the dicarboxylate/hydroxybutyrate cycle. The regulation of the central carbon metabolism was studied on the level of whole cells, enzyme activity, the proteome, transcription, and gene organization. The organism proved to be a facultative autotroph, which prefers organic acids as carbon sources that can easily feed into the metabolite pools of this cycle. Addition of the preferred carbon sources acetate, pyruvate, succinate, and 4-hydroxybutyrate to cultures resulted in stimulation of the growth rate and a diauxic growth response. The characteristic enzyme activities of the carbon fixation cycle, fumarate hydratase, fumarate reductase, succinyl coenzyme A (CoA) synthetase, and enzymes catalyzing the conversion of succinyl-CoA to crotonyl-CoA, were differentially downregulated in the presence of acetate and, to a lesser extent, in the presence of other organic substrates. This regulation pattern correlated well with the differential expression profile of the proteome as well as with the transcription of the encoding genes. The genes encoding phosphoenolpyruvate (PEP) carboxylase, fumarate reductase, and four enzymes catalyzing the conversion of succinyl-CoA to crotonyl-CoA are clustered. Two putative operons, one comprising succinyl-CoA reductase plus 4-hydroxybutyrate-CoA ligase genes and the other comprising 4-hydroxybutyryl-CoA dehydratase plus fumarate reductase genes, were divergently transcribed into leaderless mRNAs. The promoter regions were characterized and used for isolating DNA binding proteins. Besides an Alba protein, a 18-kDa protein characteristic for autotrophic Thermoproteales that bound specifically to the promoter region was identified. This system may be suitable for molecular analysis of the transcriptional regulation of autotrophy-related genes.

DNA microarray analysis of central carbohydrate metabolism: glycolytic/gluconeogenic carbon switch in the hyperthermophilic crenarchaeum Thermoproteus tenax.

In order to unravel the role of regulation on transcript level in central carbohydrate metabolism (CCM) of Thermoproteus tenax, a focused DNA microarray was constructed by using 85 open reading frames involved in CCM. A transcriptional analysis comparing heterotrophic growth on glucose versus autotrophic growth on CO2-H2 was performed.

CC1, a novel crenarchaeal DNA binding protein.

The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.

Embden-Meyerhof-Parnas and Entner-Doudoroff pathways in Thermoproteus tenax: metabolic parallelism or specific adaptation?

Genome data as well as biochemical studies have indicated that--as a peculiarity within hyperthermophilic Archaea--Thermoproteus tenax uses three different pathways for glucose metabolism, a variant of the reversible EMP (Embden-Meyerhof-Parnas) pathway and two different modifications of the ED (Entner-Doudoroff) pathway, a non-phosphorylative and a semi-phosphorylative version. An overview of the three different pathways is presented and the physiological function of the variants is discussed.