Carmina Montiel

Kinetic and structural features of betaine aldehyde dehydrogenases: mechanistic and regulatory implications.

The betaine aldehyde dehydrogenases (BADH; EC 1.2.1.8) are so-called because they catalyze the irreversible NAD(P)(+)-dependent oxidation of betaine aldehyde to glycine betaine, which may function as (i) a very efficient osmoprotectant accumulated by both prokaryotic and eukaryotic organisms to cope with osmotic stress, (ii) a metabolic intermediate in the catabolism of choline in some bacteria such as the pathogen Pseudomonas aeruginosa, or (iii) a methyl donor for methionine synthesis. BADH enzymes can also use as substrates aminoaldehydes and other quaternary ammonium and tertiary sulfonium compounds, thereby participating in polyamine catabolism and in the synthesis of gamma-aminobutyrate, carnitine, and 3-dimethylsulfoniopropionate. This review deals with what is known about the kinetics and structural properties of these enzymes, stressing those properties that have only been found in them and not in other aldehyde dehydrogenases, and discussing their mechanistic and regulatory implications.

Amino Acid Residues Critical for the Specificity for Betaine Aldehyde of the Plant ALDH10 Isoenzyme Involved in the Synthesis of Glycine Betaine

Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of ω-primary or ω-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-π interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant Km(BAL) increases and Vmax/Km(BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the Vmax/Km(BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other ω-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants.

Characterization of Blue Agave Bagasse (BAB) as Raw Material for Bioethanol Production Processes by Gravimetric, Thermal, Chromatographic, X-ray Diffraction, Microscopy, and Laser Light Scattering Techniques

A detailed characterization of the main types of blue agave bagasse (BAB) obtained from the four largest tequila factories in the State of Jalisco (Mexico) is presented here. After milling/sieving the agave bagasses, two particle size fractions were identified, one rich in fibers and the other consisting of dust/fine particles. Both fractions were analyzed to determine the content of cellulose, hemicellulose, lignin, organic-soluble compounds, absorbed remaining sugars, minerals, and organic matter. After detailed analyses of both fractions by wet, thermal (thermo-gravimetric analysis (TGA)/differential thermo-gravimetric analysis (DTA)), and other methods (high-performance liquid chromatography (HPLC), microscopy, particle size by laser diffraction light scattering, and crystallinity by X-ray diffraction), a moderate-to-intensive method was devised for further processing the fibrous fraction, which had a high crystalline cellulose content, as well as for its subsequent enzymatic saccharification under well-defined moderate conditions. Alternative processing options were also devised for the dust/fine particle fraction, which has a moderate crystalline cellulose that is rich in adsorbed sugars and that has a high mineral matter content.

Reversible, partial inactivation of plant betaine aldehyde dehydrogenase by betaine aldehyde: mechanism and possible physiological implications

In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL.

Enzyme-catalyzed synthesis of heptyl-β-glycosides: Effect of water coalescence at high temperature

Alkyl glycosides can be synthesized by glycosidases in organic media with limited amounts of water. These systems, however, limit the solubility of the sugar substrates and decrease reaction yields. Herein we report the enzymatic synthesis of heptyl-β-glycosides in heptanol catalyzed by a hyperthermophilic β-glycosidase at 90°C. Our results indicate that dispersion of water in heptanol changes with time producing coalescence of water at the bottom of the reactor, playing a key role in the reaction yield. Water-soluble substrate, enzyme and products are concentrated in the aqueous phase, according to their partition coefficients, promoting side reactions that inactivate the enzyme. Reaction yield of heptyl-β-glycosides was 35% relative to lactose, at 7% water. The increase in the water phase to 12% diminished the enzyme inactivation and increased the heptyl-β-glycosides yield to 52%. Surface-active compounds, SDS and octyl glucoside, increased water dispersion but were unable to prevent coalescence.

Decoration of virus-like particles with an enzymatic activity of biomedical interest

The natural properties of virus-like particles (VLPs), like their nanometric size, polyvalence, monodispersity and biocompatibility, had called the attention of scientists from different fields. VLPs constitute an excellent platform for the development nanomaterials with a broad spectrum of applications, ranging from physics of soft matter to the development of vaccines and biological nanocarriers. To expand the repertoire of functions of VLPs, they can be decorated with different molecules. In this research, the α-glucosidase Ima1p of Saccharomyces cerevisiae was attached to the surface of in vitro assembled VLPs of parvovirus B19, by using the SpyTag/SpyCatcher system. The resulting particles were structurally characterized displaying a noticeable increase in size compared to the non-decorated VLPs. The study of the biochemical properties of the coupled enzyme indicate that it increased its Vmax by three-fold toward p-nitrophenyl-α-D-glucopyranoside (p-NPG) as substrate. In addition, the linked enzyme displayed a notorious 10 °C shift in its optimal temperature, from 35 °C for the non-attached enzyme, to 45 °C for the enzyme attached to VLPs. The decorated VLPs were also able to act on glycogen; therefore, these particles may be further developed as part of the therapy for treatment of lysosomal storage diseases derived from defects in the human acid α-glucosidase.

Chemoenzymatic synthesis of polypeptides in neat 1,1,1,2-tetrafluoroethane solvent

Chemoenzymatic polypeptide synthesis offers several advantages over chemical or other biological routes, however, the use of aqueous-based media suffers from reverse hydrolysis reactions that challenge peptide chain propagation. Herein, the protease from subtilisin Carlsberg biocatalyzed the synthesis of poly-L-PheOEt, poly-L-LeuOEt, and the copolymers poly-L-PheOEt-co-L-LeuOEt from their amino acid ethyl ester substrates in a neat liquid 1,1,1,2-tetrafluoroethane solvent. The products, achieved in acceptable yields (ca. 50%), were fully characterized showing relatively high molar mass (ca. 20 000 Da for poly-L-PheOEt). This non-toxic low-boiling hydrofluorocarbon enhances enzymatic peptide propagation by limiting hydrolysis owing to its hydrophobic and relatively polar characteristics that sustain the protease activity and solubilize substrates and products. Computational molecular dynamic calculations were used to assess the L-PheOEt/L-LeuOEt-solvent and polypeptide-solvent interactions in this system. Additionally, the homopolypeptides displayed higher crystallinity than the copolypeptides with random incorporation of amino acid ethyl esters, notwithstanding the significantly highest specificity for Phe in this system. Interestingly, secondary structure characterization of the products by FTIR and circular dichroism suggests a non-common peptide folding.

Growth of epithelial cells on films of enzymatically synthesized poly(gallic acid) crosslinked to carboxymethylcellulose

Poly(gallic acid), a novel polymer obtained by enzymatic polymerization of gallic acid, was successfully cross-linked to carboxymethylcellulose using citric acid as a cross-linker. Non-woven sheets were prepared with the resulting copolymer and their in vitro biocompatibility was assessed. The results on the characterizations of the produced films by mechanical tests, water vapour permeability, contact angle, and antioxidant activity by electronic paramagnetic resonance were adequate for skin tissue regeneration. Films were also decomposed under physiological conditions using universal buffers at pH 3, 7 and 10. In vitro experiments with fibroblast-like and epithelial-like cells showed good adhesion and proliferation onto the PGAL-co-CMC sheets. These non-woven sheets can consequently be considered as novel biocompatible and biodegradable films with high-responsiveness for biomedical or tissue engineering applications.

Cytoprotective effect of the enzyme-mediated polygallic acid on fibroblast cells under exposure of UV-irradiation

The poly(gallic acid), produced by laccase-mediated oxidation of gallic acid in aqueous media (pH5.5) to attain a novel material with well-defined molecular structure and high water solubility (500mg/mL at 25°C), has been investigated to understand its potential biological activities. In this regard, a biomedical approach based on cytoprotective effect on human fibroblast cells exposed to UV-irradiation in the presence of the polymer has been demonstrated. The results also shows that 200μg/mL of poly(gallic acid) inhibits the growth and migration of dermal fibroblasts and cancer cell lines without affecting cell viability. Poly(gallic acid) pretreatment with 10μg/mL protects dermal fibroblasts from UV induced cell death and additionally, the cytoprotective effect reduce ROS presence in the cells. This property can be correlated with the antioxidant power (IC50 of 23.5μg/mL) of this novel material, which was ascertained by electronic paramagnetic resonance spectroscopy and spectrophotometrically. Additionally, the antimicrobial activity of this material was corroborated with the inhibition of Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC 29212) strains (MIC=400mg/mL) common bacteria found in hospitals.