Jesús Fernández-Lucas

Lactobacillus reuteri 2′-Deoxyribosyltransferase, a Novel Biocatalyst for Tailoring of Nucleosides

ABSTRACT A novel type II nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% α-helix, 10% β-strand, 16% β-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (Tm) of 64°C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40°C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2′-fluorodeoxyribonucleosides was carried out.

Psycho-physiological response in an automatic parachute jump

ABSTRACT Parachute jump is an extreme activity that elicits an intense stress response that affects jumpers’ body systems being able to put them at risk. The present research analysed modifications in blood oxygen saturation (BOS), heart rate (HR), cortisol, glucose, lactate, creatine kinase (CK), muscles strength, cortical arousal, autonomic modulation, pistol magazine reload time (PMRT) and state anxiety before and after an automatic open parachute jump in 38 male Spanish soldiers (25.6 ± 5.9 years). A MANOVA with samples as a fixed factor and Effect Size (ES) were conducted. MANOVA showed (Wilks lambda = .225; F = 5.980; P = .000) a significantly increase in cortisol (6.2 ± 3.2 vs. 8.2 ± 4.3 nmol/l; P = .025; ES = .47), HR (75.0 ± 14.6 vs. 87.4 ± 17.3 bpm; P = .004; ES = .72), lactate (1.8 ± 1.2 vs. 4.4 ± 2.2 mmol · l−1; P = .002; ES = 1.18), sympathetic nervous system and leg strength manifestation after the parachute jump. By contrary BOS, PMRT (55.6 ± 27.6 vs. 48.0 ± 16.7 s; P = .021; ES = .46) and somatic anxiety (SA), evaluated by CSAI2R questionnaire, decreased. An automatic parachute jump increased physiological and cortical response and decreased SA of participants. This stress response can affect the jumpers’ abilities and allow us to have a better understanding of the organism stress response and to improve training for both military and sport parachute jumps.

Multienzymatic synthesis of nucleic acid derivatives: a general perspective

Living cells are most perfect synthetic factory. The surprising synthetic efficiency of biological systems is allowed by the combination of multiple processes catalyzed by enzymes working sequentially. In this sense, biocatalysis tries to reproduce nature’s synthetic strategies to perform the synthesis of different organic compounds using natural catalysts such as cells or enzymes. Nowadays, the use of multienzymatic systems in biocatalysis is becoming a habitual strategy for the synthesis of organic compounds that leads to the realization of complex synthetic schemes. By combining several steps in one pot, a significant step economy can be realized and the potential for environmentally benign synthesis is improved. Using this sustainable synthetic system, several work-up steps can be avoided and pure products are ideally isolated after a series of reactions in one single vessel after just one straightforward purification step. In recent years, enzymatic methodology for the preparation of nucleic acid derivatives (NADs) has become a standard technique for the synthesis of a wide variety of natural NADs. Enzymatic methods have been shown to be an efficient alternative for the synthesis of nucleoside and nucleotide analogs to the traditional multistep chemical methods, since chemical glycosylation reactions include several protection–deprotection steps and the use of chemical reagents and organic solvents that are expensive and environmentally harmful. In this minireview, we want to illustrate what we consider the most current relevant examples of in vivo and in vitro multienzymatic systems used for the synthesis of nucleic acid derivatives showing advantages and disadvantages of each methodology. Finally, a detailed perspective about the impact of -omics in multienzymatic systems has been described.

Experience modulates the psychophysiological response of airborne warfighters during a tactical combat parachute jump

We aimed to analyse the effect of experience level in the psychophysiological response and specific fine motor skills of novel and expert parachute warfighters during a tactical combat parachute jump. We analysed blood oxygen saturation, heart rate, salivary cortisol, blood glucose, lactate and creatinkinase, leg strength, isometric hand-grip strength, cortical arousal, specific fine motor skills and cognitive anxiety, somatic anxiety and self-confident before and after a tactical combat parachute jump in 40 warfighters divided in two group, novel (n=17) and expert group (n=23). Novels presented a higher heart rate, lactate, cognitive anxiety, somatic anxiety and a lower self-confident than experts during the jump. We concluded that experience level has a direct effect on the psychophysiological response since novel paratroopers presented a higher psychophysiological response than compared to the expert ones, however this result neither affected the specific fine motor skills nor the muscle structure after a tactical combat parachute jump.

Enzymatic production of non‐natural nucleoside‐5'-monophosphates by a novel thermostable uracil phosphoribosyltransferase

The use of enzymes as biocatalysts applied to synthesis of modified nucleoside‐5′‐monophosphates (NMPs) is an interesting alternative to traditional multistep chemical methods which offers several advantages, such as stereo‐, regio‐, and enantioselectivity, simple downstream processing, and mild reaction conditions. Herein we report the recombinant expression, production, and purification of uracil phosphoribosyltransferase from Thermus themophilus HB8 (TtUPRT). The structure of TtUPRT has been determined by protein crystallography, and its substrate specificity and biochemical characteristics have been analyzed, providing new structural insights into the substrate‐binding mode. Biochemical characterization of the recombinant protein indicates that the enzyme is a homotetramer, with activity and stability across a broad range of temperatures (50–80 °C), pH (5.5–9) and ionic strength (0–500 mm NaCl). Surprisingly, TtUPRT is able to recognize several 5 and 6‐substituted pyrimidines as substrates. These experimental results suggest TtUPRT could be a valuable biocatalyst for the synthesis of modified NMPs.

Enzymatic Synthesis of Therapeutic Nucleosides using a Highly Versatile Purine Nucleoside 2’-DeoxyribosylTransferase from Trypanosoma brucei

The use of enzymes for the synthesis of nucleoside analogues offers several advantages over multistep chemical methods, including chemo‐, regio‐ and stereoselectivity as well as milder reaction conditions. Herein, the production, characterization and utilization of a purine nucleoside 2’‐deoxyribosyltransferase (PDT) from Trypanosoma brucei are reported. TbPDT is a dimer which displays not only excellent activity and stability over a broad range of temperatures (50–70 °C), pH (4–7) and ionic strength (0–500 mM NaCl) but also an unusual high stability under alkaline conditions (pH 8–10). TbPDT is shown to be proficient in the biosynthesis of numerous therapeutic nucleosides, including didanosine, vidarabine, cladribine, fludarabine and nelarabine. The structure‐guided replacement of Val11 with either Ala or Ser resulted in variants with 2.8‐fold greater activity. TbPDT was also covalently immobilized on glutaraldehyde‐activated magnetic microspheres. MTbPDT3 was selected as the best derivative (4200 IU/g, activity recovery of 22 %), and could be easily recaptured and recycled for >25 reactions with negligible loss of activity. Finally, MTbPDT3 was successfully employed in the expedient synthesis of several nucleoside analogues. Taken together, our results support the notion that TbPDT has good potential as an industrial biocatalyst for the synthesis of a wide range of therapeutic nucleosides through an efficient and environmentally friendly methodology.

Purine and pyrimidine salvage pathway in thermophiles: a valuable source of biocatalysts for the industrial production of nucleic acid derivatives

Due to their similarity to natural counterparts, nucleic acid derivatives (nucleobases, nucleosides, and nucleotides, among others) are interesting molecules for pharmaceutical, biomedical, or food industries. For this reason, there is increasing worldwide demand for the development of efficient synthetic processes for these compounds. Chemical synthetic methodologies require numerous protection-deprotection steps and often lead to the presence of undesirable by-products or enantiomeric mixtures. These methods also require harsh operating conditions, such as the use of organic solvents and hazard reagents. Conversely, enzymatic production by whole cells or enzymes improves regio-, stereo-, and enantioselectivity and provides an eco-friendly alternative. Because of their essential role in purine and pyrimidine scavenging, enzymes from purine and pyrimidine salvage pathways are valuable candidates for the synthesis of many different nucleic acid components. In recent years, many different enzymes from these routes, such as nucleoside phosphorylases, nucleoside kinases, 2′-deoxyribosyltransferases, phosphoribosyl transferases, or deaminases, have been successfully employed as biocatalysts in the production of nucleobase, nucleoside, or nucleotide analogs. Due to their great activity and stability at extremely high temperatures, the use of enzymes from thermophiles in industrial biocatalysis is gaining momentum. Thermophilic enzymes not only display unique characteristics such as temperature, chemical, and pH stability but also provide many different advantages from an industrial perspective. This mini-review aims to cover the most representative enzymatic approaches for the synthesis of nucleic acid derivatives. In this regard, we provide detailed comments about enzymes involved in crucial steps of purine and pyrimidine salvage pathways in thermophiles, as well as their biological role, biochemical characterization, active site mechanism, and substrate specificity. In addition, the most interesting synthetic examples reported in the literature are also included.

Marine chitinolytic enzymes, a biotechnological treasure hidden in the ocean?

Chitinolytic enzymes are capable to catalyze the chitin hydrolysis. Due to their biomedical and biotechnological applications, nowadays chitinolytic enzymes have attracted worldwide attention. Chitinolytic enzymes have provided numerous useful materials in many different industries, such as food, pharmaceutical, cosmetic, or biomedical industry. Marine enzymes are commonly employed in industry because they display better operational properties than animal, plant, or bacterial homologs. In this mini-review, we want to describe marine chitinolytic enzymes as versatile enzymes in different biotechnological fields. In this regard, interesting comments about their biological role, reaction mechanism, production, functional characterization, immobilization, and biotechnological application are shown in this work.