Thermophilus HB8 Research Articles

Three-Dimensional Structure of Recombinant Adenine Phosphoribosyltransferase from Thermophilic Bacterial Strain Thermus thermophilus HB27

Adenine phosphoribosyltransferase (APRT) from a thermophilic bacterial strain Thermus thermophilus НВ27 (TthHB27APRT) belongs to the family of type I phosphoribosyltransferases and catalyzes the magnesium-dependent transfer of 5'-phosphoribosyl group from 5'-phosphoribosylpyrophosphate to N9 adenine nitrogen with formation of adenosine-5'-monophosphate and pyrophosphate. The crystals of the recombinant enzyme suitable for X-ray study were grown in a capillary using the counter-diffusion technique. Crystals with unit-cell parameters α = 69.860 A, b = 82.160 A, c = 91.390 A, α = 90.00°, β = 102.58°, and γ = 90.00° belong to the space group Р21 and contain six enzyme monomers in the asymmetric unit. The set of X-ray data from grown crystals was collected on a Spring-8 synchrotron radiation facility (Japan) and three-dimensional structure of the enzyme was solved at 2.7-A resolution by molecular replacement method using the BALBES software. The polypeptide fold in the enzyme monomer and the structure of biologically active dimer were described. Based on the comparison with structures of homologous APRTs from a thermophilic strain ThtHB8 and Homo sapiens, positions of active site and a number of functionally important amino acids were located.

Characterization of a promiscuous cadmium and arsenic resistance mechanism in Thermus thermophilus HB27 and potential application of a novel bioreporter system

BackgroundThe characterization of the molecular determinants of metal resistance has potential biotechnological application in biosensing and bioremediation. In this context, the bacterium Thermus thermophilus HB27 is a metal tolerant thermophile containing a set of genes involved in arsenic resistance which, differently from other microbes, are not organized into a single operon. They encode the proteins: arsenate reductase, TtArsC, arsenic efflux membrane transporter, TtArsX, and transcriptional repressor, TtSmtB.ResultsIn this work we show that the arsenic efflux protein TtArsX and the arsenic responsive transcriptional repressor TtSmtB are required to provide resistance to cadmium. We analyzed the sensitivity to Cd(II) of mutants lacking TtArsX, finding that they are more sensitive to this metal than the wild type strain. In addition, using promoter probe reporter plasmids, we show that the transcription of TtarsX is also stimulated by the presence of Cd(II) in a TtSmtB-dependent way. Actually, a regulatory circuit composed of TtSmtB and a reporter gene expressed from the TtarsX promoter responds to variation in Cd(II), As(III) and As(V) concentrations.ConclusionsOur results demonstrate that the system composed by TtSmtB and TtArsX is responsible for both the arsenic and cadmium resistance in T. thermophilus. The data also support the use of T. thermophilus as a suitable chassis for the design and development of As-Cd biosensors.

Characterization of two 2-isopropylmalate synthase homologs from Thermus thermophilus HB27

2-Isopropylmalate synthase (IPMS) catalyzes the first step of leucine biosynthesis and is regulated via feedback inhibition by leucine. The thermophilic bacterium, Thermus thermophilus HB27, has two IPMS homologous genes: TTC0847 and TTC0849, both of which are in the branched-chain amino acid biosynthetic gene cluster. Since enzymes involved in the leucine biosynthetic pathway are evolutionarily related to those in isoleucine biosynthesis, TTC0847 and TTC0849 are expected to function as IPMS or citramalate synthase, which is the first enzyme in the isoleucine biosynthetic pathway from pyruvate. We characterized these proteins in vitro and in vivo, and revealed that TTC0849 plays a key role in the biosynthesis of leucine and isoleucine, whereas TTC0847 is only involved in that of isoleucine.

Improvement of Trehalose Production by Immobilized Trehalose Synthase from Thermus thermophilus HB27.

Trehalose is a non-reducing disaccharide with a wide range of applications in the fields of food, cosmetics, and pharmaceuticals. In this study, trehalose synthase derived from Thermus thermophilus HB27 (TtTreS) was immobilized on silicalite-1-based material for trehalose production. The activity and the stability of TtTreS against pH and temperature were significantly improved by immobilization. Enzyme immobilization also led to a lower concentration of byproduct glucose, which reduces byproduct inhibition of TtTreS. The immobilized TtTreS still retained 81% of its initial trehalose yield after 22 cycles of enzymatic reactions. The immobilized TtTreS exhibited high operational stability and remarkable reusability, indicating that it is promising for industrial applications.

One-Pot, One-Step Production of Dietary Nucleotides by Magnetic Biocatalysts

The enzymatic synthesis of nucleotides offers several advantages over traditional multistep chemical methods, such as stereoselectivity, regioselectivity, enantioselectivity, simple downstream processing, and the use of mild reaction conditions. However, in order to scale up these bioprocesses, several drawbacks, such as the low enzyme stability and recycling, must be considered. Enzyme immobilization may overcome these cost-related problems by enhancing protein stability and facilitating the separation of products. In this regard, tetrameric hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRT) from Thermus thermophilus HB8 was covalently immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles (MTtHGXPRT). In this context, two different strategies were followed: (a) an enzyme immobilization through its N-terminus residues at pH 8.5 (derivatives MTtHGXPRT1-3); and (b) a multipoint covalent immobilization through the surface lysine residues at pH 10 (derivatives MTtHGXPRT4-5). The immobilized derivatives of MTtHGXPRT3 (activity 1581 international units per gram of support, IU/g; retained activity 29%) and MTtHGXPRT5 (activity 1108 IU/g; retained activity 23%) displayed the best wet biocatalyst activity, and retained activity values in the enzymatic synthesis of inosine-5′-monophosphate (IMP). In addition, the dependence of the activities and stabilities of both derivatives on pH and temperature was tested, as well as their reusability potential. Taking these results into account, MTtHGXPRT3 was chosen as the best biocatalyst (negligible loss of activity at 60 °C during 24 h; reusable up to seven cycles). Finally, as proof of concept, the enzymatic production of dietary nucleotides from high concentrations of low soluble bases was achieved.

Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5′ to 3′ exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol.

Optimization of the production of an extracellular and thermostable amylolytic enzyme by Thermus thermophilus HB8 and basic characterization.

The objective of this study was to determine the potential of Thermus thermophilus HB8 for accumulating a high level of extracellular, thermostable amylolytic enzyme. Initial production tests indicated clearly that only very low levels of amylolytic activity could be detected, solely from cells after extraction using the mild, non-ionic detergent Triton X-100. A sequential optimization strategy, based on statistical designs, was used to enhance greatly the production of extracellular amylolytic activity to achieve industrially attractive enzyme titers. Focus was placed on the optimal level of initial biomass concentration, culture medium composition and temperature for maximizing extracellular amylolytic enzyme accumulation. Empirical models were then developed describing the effects of the experimental parameters and their interactions on extracellular amylolytic enzyme production. Following such efforts, extracellular amylolytic enzyme accumulation was increased more than 70-fold, with enzyme titers in the 76U/mL range. The crude extracellular enzyme was thereafter partially characterized. The optimal temperature and pH values were found to be 80°C and 9.0, respectively. 100% of the initial enzyme activity could be recovered after incubation for 24h at 80°C, therefore, proving the very high thermostability of the enzyme preparation.

Characterization and application of a novel nicotinamide mononucleotide adenylyltransferase from Thermus thermophilus HB8

Herein, we describe a novel enzymatic cycling method to measure nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NaMN), which are precursors of NAD biosynthesis. A gene encoding an NMN adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified in Thermus thermophilus HB8. The gene from T.thermophilus (TtNMNAT) was engineered for expression in Escherichia coli and the recombinant enzyme found to be stable, retaining full activity after incubation for 45min at 70°C. The Km values for NMN and ATP were calculated to be 0.263 and 1.27mM, respectively, with a Vmax value of 60.3μmoL/min/mg. TtNMNAT was successfully applied to the colorimetric NMN or NaMN assays, which employed (i) adenylation of NMN to NAD by TtNMNAT or adenylation of NaMN to deamido-NAD (NaAD) by TtNMNAT followed by amidation of NaAD to NAD by NAD synthetase (NADS, EC 6.3.1.5) and (ii) an NAD cycling reaction using 12α-hydroxysteroid dehydrogenase (12α-HSD, EC 1.1.1.176) and diaphorase (DI, EC 1.6.99.3) to accumulate reduced WST-8. This enzymatic cycling method enabled detection of 0.5μM (12.2nM in the reaction mixture) NMN or NaMN in an automatic clinical analyzer.

Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I) cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene.

Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes’ expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified ‘codon randomization’ strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.

13C metabolic flux analysis of three divergent extremely thermophilic bacteria: Geobacillus sp. LC300, Thermus thermophilus HB8, and Rhodothermus marinus DSM 4252

Thermophilic organisms are being increasingly investigated and applied in metabolic engineering and biotechnology. The distinct metabolic and physiological characteristics of thermophiles, including broad substrate range and high uptake rates, coupled with recent advances in genetic tool development, present unique opportunities for strain engineering. However, poor understanding of the cellular physiology and metabolism of thermophiles has limited the application of systems biology and metabolic engineering tools to these organisms. To address this concern, we applied high resolution 13C metabolic flux analysis to quantify fluxes for three divergent extremely thermophilic bacteria from separate phyla: Geobacillus sp. LC300, Thermus thermophilus HB8, and Rhodothermus marinus DSM 4252. We performed 18 parallel labeling experiments, using all singly labeled glucose tracers for each strain, reconstructed and validated metabolic network models, measured biomass composition, and quantified precise metabolic fluxes for each organism. In the process, we resolved many uncertainties regarding gaps in pathway reconstructions and elucidated how these organisms maintain redox balance and generate energy. Overall, we found that the metabolisms of the three thermophiles were highly distinct, suggesting that adaptation to growth at high temperatures did not favor any particular set of metabolic pathways. All three strains relied heavily on glycolysis and TCA cycle to generate key cellular precursors and cofactors. None of the investigated organisms utilized the Entner-Doudoroff pathway and only one strain had an active oxidative pentose phosphate pathway. Taken together, the results from this study provide a solid foundation for future model building and engineering efforts with these and related thermophiles.

Identification and characterization of preferred DNA-binding sites for the Thermus thermophilus transcriptional regulator FadR.

One of the primary transcriptional regulators of fatty acid homeostasis in many prokaryotes is the protein FadR. To better understand its biological function in the extreme thermophile Thermus thermophilus HB8, we sought to first determine its preferred DNA-binding sequences in vitro using the combinatorial selection method Restriction Endonuclease Protection, Selection, and Amplification (REPSA) and then use this information to bioinformatically identify potential regulated genes. REPSA determined a consensus FadR-binding sequence 5´-TTRNACYNRGTNYAA-3´, which was further characterized using quantitative electrophoretic mobility shift assays. With this information, a search of the T. thermophilus HB8 genome found multiple operons potentially regulated by FadR. Several of these were identified as encoding proteins involved in fatty acid biosynthesis and degradation; however, others were novel and not previously identified as targets of FadR. The role of FadR in regulating these genes was validated by physical and functional methods, as well as comparative genomic approaches to further characterize regulons in related organisms. Taken together, our study demonstrates that a systematic approach involving REPSA, biophysical characterization of protein-DNA binding, and bioinformatics can be used to postulate biological roles for potential transcriptional regulators.

Development of a new host-vector system for colour selection of cloned DNA inserts using a newly designed β-galactosidase gene containing multiple cloning sites in Thermus thermophilus HB27.

We constructed a new Thermus thermophilus cloning vector which enables the colour selection of cloned DNA inserts in the T. thermophilus HB27 host strain (β-gal-) on growth plates containing 3,4-cyclohexenoesculetin β-D-galactopyranoside (S-gal) in the medium. This vector harbors a modified β-galactosidase gene (TTP0042 of T. thermophilus HB27) with 12 unique restriction enzyme sites (Acc65I, AvrII, BlpI, BssHII, EcoRI, EcoRV, HindIII, NruI, SalI, SpeI, SphI and XbaI) as multiple cloning sites under the control of the T. thermophilus slpA promoter. This host-vector system facilitates cloning procedures in T. thermophilus HB27.

Cre/lox-based multiple markerless gene disruption in the genome of the extreme thermophile Thermus thermophilus.

Markerless gene-disruption technology is particularly useful for effective genetic analyses of Thermus thermophilus (T. thermophilus), which have a limited number of selectable markers. In an attempt to develop a novel system for the markerless disruption of genes in T. thermophilus, we applied a Cre/lox system to construct a triple gene disruptant. To achieve this, we constructed two genetic tools, a loxP-htk-loxP cassette and cre-expressing plasmid, pSH-Cre, for gene disruption and removal of the selectable marker by Cre-mediated recombination. We found that the Cre/lox system was compatible with the proliferation of the T. thermophilus HB27 strain at the lowest growth temperature (50°C), and thus succeeded in establishing a triple gene disruptant, the (∆TTC1454::loxP, ∆TTC1535KpnI::loxP, ∆TTC1576::loxP) strain, without leaving behind a selectable marker. During the process of the sequential disruption of multiple genes, we observed the undesired deletion and inversion of the chromosomal region between multiple loxP sites that were induced by Cre-mediated recombination. Therefore, we examined the effects of a lox66-htk-lox71 cassette by exploiting the mutant lox sites, lox66 and lox71, instead of native loxP sites. We successfully constructed a (∆TTC1535::lox72, ∆TTC1537::lox72) double gene disruptant without inducing the undesired deletion of the 0.7-kbp region between the two directly oriented lox72 sites created by the Cre-mediated recombination of the lox66-htk-lox71 cassette. This is the first demonstration of a Cre/lox system being applicable to extreme thermophiles in a genetic manipulation. Our results indicate that this system is a powerful tool for multiple markerless gene disruption in T. thermophilus.

An ArsR/SmtB family member regulates arsenic resistance genes unusually arranged in Thermus thermophilus HB27.

SummaryArsenic resistance is commonly clustered in ars operons in bacteria; main ars operon components encode an arsenate reductase, a membrane extrusion protein, and an As‐sensitive transcription factor. In the As‐resistant thermophile Thermus thermophilus HB27, genes encoding homologues of these proteins are interspersed in the chromosome. In this article, we show that two adjacent genes, TtsmtB, encoding an ArsR/SmtB transcriptional repressor and, TTC0354, encoding a Zn2+/Cd2+‐dependent membrane ATPase are involved in As resistance; differently from characterized ars operons, the two genes are transcribed from dedicated promoters upstream of their respective genes, whose expression is differentially regulated at transcriptional level. Mutants defective in TtsmtB or TTC0354 are more sensitive to As than the wild type, proving their role in arsenic resistance. Recombinant dimeric TtSmtB binds in vitro to both promoters, but its binding capability decreases upon interaction with arsenate and, less efficiently, with arsenite. In vivo and in vitro experiments also demonstrate that the arsenate reductase (TtArsC) is subjected to regulation by TtSmtB. We propose a model for the regulation of As resistance in T. thermophilus in which TtSmtB is the arsenate sensor responsible for the induction of TtArsC which generates arsenite exported by TTC0354 efflux protein to detoxify cells.

Indispensable residue for uridine binding in the uridine-cytidine kinase family

Uridine-cytidine kinase (UCK), including human UCK2, are a family of enzymes that generally phosphorylate both uridine and cytidine. However, UCK of Thermus thermophilus HB8 (ttCK) phosphorylates only cytidine. This cytidine-restricted activity is thought to depend on Tyr93, although the precise mechanism remains unresolved. Exhaustive mutagenesis of Tyr93 in ttCK revealed that the uridine phosphorylation activity was restored only by replacement of Tyr93 with His or Gln. Replacement of His117 in human UCK2, corresponding to residue Tyr93 in ttCK, by Tyr resulted in a loss of uridine phosphorylation activity. These findings indicated that uridine phosphorylation activity commonly depends on a single residue in the UCK family.

RNomics of Thermus themophilus HB8 by DNA microarray and next-generation sequencing.

By using the data obtained by the DNA microarray analysis for the intergenic regions applied to RNA samples extracted from Thermus thermophilus HB8, seven small non-coding RNAs, TtR-1 to TtR-7, were found to be expressed in the cells growing in rich and/or minimal media. By analysing the time course of the expression for the cell growth in combination with the sequence comparison to the known RNAs, two RNAs, TtR-1 and TtR-2, are suggested to be riboswitches. The existence of the seven RNAs and the exact sequence and length, ranging 77-284 nt, were confirmed by the next-generation sequencing. By the combination of these two high-throughput techniques, our understanding of RNAs in the cell will be increased significantly.

Role of Archaeal HerA Protein in the Biology of the Bacterium Thermus thermophilus.

Intense gene flux between prokaryotes result in high percentage of archaeal genes in the genome of the thermophilic bacteria Thermus spp. Among these archaeal genes a homolog to the Sulfolobus spp. HerA protein appears in all of the Thermus spp. strains so far sequenced (HepA). The role of HepA in Thermus thermophilus HB27 has been analyzed using deletion mutants, and its structure resolved at low resolution by electron microscopy. Recombinant HepA shows DNA-dependent ATPase activity and its structure revealed a double ring, conically-shaped hexamer with an upper diameter of 150 Å and a bottom module of 95 Å. A central pore was detected in the structure that ranges from 13 Å at one extreme, to 30 Å at the other. Mutants lacking HepA show defective natural competence and DNA donation capability in a conjugation-like process termed “transjugation”, and also high sensitivity to UV and dramatic sensitivity to high temperatures. These data support that acquisition of an ancestral archaeal HerA has been fundamental for the adaptation of Thermus spp. to high temperatures.

Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases from Thermus thermophilus HB8

Purine phosphoribosyltransferases, purine PRTs, are essential enzymes in the purine salvage pathway of living organisms. They are involved in the formation of C-N glycosidic bonds in purine nucleosides-5′-monophosphate (NMPs) through the transfer of the 5-phosphoribosyl group from 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP) to purine nucleobases in the presence of Mg2+. Herein, we report a simple and thermostable process for the one-pot, one-step synthesis of some purine NMPs using xanthine phosphoribosyltransferase, XPRT or adenine phosphoribosyltransferase, APRT2, from Thermus thermophilus HB8. In this sense, the cloning, expression and purification of TtXPRT and TtAPRT2 is described for the first time. Both genes, xprt and aprt2 were expressed as his-tagged enzymes in E. coli BL21(DE3) and purified by a heat-shock treatment, followed by Ni-affinity chromatography and a final, polishing gel-filtration chromatography. Biochemical characterization revealed TtXPRT as a tetramer and TtAPRT2 as a dimer. In addition, both enzymes displayed a strong temperature dependence (relative activity >75% in a temperature range from 70 to 90 °C), but they also showed very different behaviour under the influence of pH. While TtXPRT is active in a pH range from 5 to 7, TtAPRT2 has a high dependence of alkaline conditions, showing highest activity values in a pH range from 8 to 10. Finally, substrate specificity studies were performed in order to explore their potential as industrial biocatalyst for NMPs synthesis.

The transjugation machinery of Thermus thermophilus: Identification of TdtA, an ATPase involved in DNA donation.

In addition to natural competence, some Thermus thermophilus strains show a high rate of DNA transfer via direct cell-to-cell contact. The process is bidirectional and follows a two-step model where the donor cell actively pushes out DNA and the recipient cell employs the natural competence system to take up the DNA, in a hybrid transformation-dependent conjugation process (transjugation). While the DNA uptake machinery is well known as in other bacterial species that undergo transformation, the pushing step of transjugation remains to be characterized. Here we have searched for hypothetical DNA translocases putatively involved in the pushing step of transjugation. Among candidates encoded by T. thermophilus HB27, the TdtA protein was found to be required for DNA pushing but not for DNA pulling during transjugation, without affecting other cellular processes. Purified TdtA shows ATPase activity and oligomerizes as hexamers with a central opening that can accommodate double-stranded DNA. The tdtA gene was found to belong to a mobile 14 kbp-long DNA element inserted within the 3′ end of a tRNA gene, flanked by 47 bp direct repeats. The insertion also encoded a homolog of bacteriophage site-specific recombinases and actively self-excised from the chromosome at high frequency to form an apparently non-replicative circular form. The insertion also encoded a type II restriction endonuclease and a NurA-like nuclease, whose activities were required for efficient transjugation. All these data support that TdtA belongs to a new type of Integrative and Conjugative Element which promotes the generalized and efficient transfer of genetic traits that could facilitate its co-selection among bacterial populations.

A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides

We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D-pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D-pentoses into 5-phosphates that were further converted into 5-phospho-α-D-pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.