Pedro Lamosa

Thermostabilization of Proteins by Diglycerol Phosphate, a New Compatible Solute from the Hyperthermophile Archaeoglobus fulgidus

ABSTRACT Diglycerol phosphate accumulates under salt stress in the archaeonArchaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896–902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, bakers yeast alcohol dehydrogenase, andThermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins fromDesulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianumrubredoxins. In contrast, the stability of D. desulfuricansrubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.

Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine.

Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the β-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of β-alanine and its subsequent conversion into 3HP using a novel β-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3gL(-1) with a 0.14±0.0C-molC-mol(-1) yield on glucose in 80h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.

Pseudovitamin B12 is the corrinoid produced by Lactobacillus reuteri CRL1098 under anaerobic conditions

We have reported previously on the ability of Lactobacillus reuteri to produce a compound with vitamin B 12 activity. Here we report on the chemical characterisation of this corrinoid‐like molecule. High performance liquid chromatography coupled to an ultraviolet diode array detector, mass spectrometry and nuclear magnetic resonance spectroscopy has enabled us to identify the compound as Co α ‐[ α ‐(7‐adenyl)]‐Co β ‐cyanocobamide or pseudovitamin B 12 . This molecule differs from cobalamin in the α ‐ligand, where it has adenine instead of 5,6‐dimethylbenzimidazole bound in a α ‐glycosidic linkage to C‐1 of ribose. L. reuteri is the first lactic acid bacterium in which the production of a cobalamin‐like molecule has been identified and the first microorganism reported to produce exclusively pseudo‐ B 12 .

Processing the interspecies quorum-sensing signal autoinducer-2 (AI-2): characterization of phospho-(S)-4,5-dihydroxy-2,3-pentanedione isomerization by LsrG protein.

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsible for processing the phosphorylated signal. Because P-DPD is the intracellular inducer of the lsr operon, enzymes involved in P-DPD processing impact the levels of Lsr expression. Here we show that LsrG catalyzes isomerization of P-DPD into 3,4,4-trihydroxy-2-pentanone-5-phosphate. We present the crystal structure of LsrG, identify potential catalytic residues, and determine which of these residues affects P-DPD processing in vivo and in vitro. We also show that an lsrG deletion mutant accumulates at least 10 times more P-DPD than wild type cells. Consistent with this result, we find that the lsrG mutant has increased expression of the lsr operon and an altered profile of AI-2 accumulation and removal. Understanding of the biochemical mechanisms employed by bacteria to quench signaling of other species can be of great utility in the development of therapies to control bacterial behavior.

Molecular identification of N-acetylaspartylglutamate synthase and beta-citrylglutamate synthase.

The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and β-citrylglutamate, a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and l-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of β-citrylglutamate. The nature of the reaction products was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress in the understanding of the function of NAAG and β-citrylglutamate.

Organic solutes in Rubrobacter xylanophilus: the first example of di-myo-inositol-phosphate in a thermophile.

The thermophilic and halotolerant nature of Rubrobacter xylanophilus led us to investigate the accumulation of compatible solutes in this member of the deepest lineage of the Phylum Actinobacteria. Trehalose and mannosylglycerate (MG) were the major compounds accumulated under all conditions examined, including those for optimal growth. The addition of NaCl to a complex medium and a defined medium had a slight or negligible effect on the accumulation of these compatible solutes. Glycine betaine, di-myo-inositol-phosphate (DIP), a new phosphodiester compound, identified as di-N-acetyl-glucosamine phosphate and glutamate were also detected but in low or trace levels. DIP was always present, except at the highest salinity examined (5% NaCl) and at the lowest temperature tested (43°C). Nevertheless, the levels of DIP increased with the growth temperature. This is the first report of MG and DIP in an actinobacterium and includes the identification of the new solute di-N-acetyl-glucosamine phosphate.

Suberin isolation from cork using ionic liquids: characterisation of ensuing products

Cholinium alkanoates, a class of benign ionic liquids, were demonstrated to efficiently extract suberin domains from cork. A detailed characterisation of the extracted material has yet to be attained. In the present study the significance of the alkylic chain length of the anion and the ionic liquids basicity was investigated. The results obtained emphasise cholinium hexanoates selection; it proved to be a straightforward process, also ensuring the recyclability and reusability of the ionic liquid. The extracted suberinic material has been thoroughly characterised for the first time by ATR-FTIR, NMR, GC-MS and thermal analyses. Data showed that it is mainly composed of oligomeric or polymeric aliphatic esterified structures, resulting from suberin partial cleavage. More than 40 wt% of the extracted suberinic material was found to be cross-linked. Even though, the composing monomeric units were similar to those usually identified in suberin samples obtained by the conventional extraction processes. These data pave the way for advanced studies of suberin monomers/oligomers as building-blocks for the development of novel biopolymers and biomaterials.

Butyrate-rich Colonic Microenvironment Is a Relevant Selection Factor for Metabolically Adapted Tumor Cells

The short chain fatty acid (SCFA) buyrate is a product of colonic fermentation of dietary fibers. It is the main source of energy for normal colonocytes, but cannot be metabolized by most tumor cells. Butyrate also functions as a histone deacetylase (HDAC) inhibitor to control cell proliferation and apoptosis. In consequence, butyrate and its derived drugs are used in cancer therapy. Here we show that aggressive tumor cells that retain the capacity of metabolizing butyrate are positively selected in their microenvironment. In the mouse xenograft model, butyrate-preselected human colon cancer cells gave rise to subcutaneous tumors that grew faster and were more angiogenic than those derived from untreated cells. Similarly, butyrate-preselected cells demonstrated a significant increase in rates of homing to the lung after intravenous injection. Our data showed that butyrate regulates the expression of VEGF and its receptor KDR at the transcriptional level potentially through FoxM1, resulting in the generation of a functional VEGF:KDR autocrine growth loop. Cells selected by chronic exposure to butyrate express higher levels of MMP2, MMP9, α2 and α3 integrins, and lower levels of E-cadherin, a marker for epithelial to mesenchymal transition. The orthotopic model of colon cancer showed that cells preselected by butyrate are able to colonize the animals locally and at distant organs, whereas control cells can only generate a local tumor in the cecum. Together our data shows that a butyrate-rich microenvironment may select for tumor cells that are able to metabolize butyrate, which are also phenotypically more aggressive.

Linear and branched poly(ω-hydroxyacid) esters in plant cutins.

Poly(omega-hydroxyacid) ester oligomers were obtained from the cutin of four plant cuticles by partial depolymerization methanolysis. The structure of these oligomers was determined by electrospray ionization coupled to tandem mass spectrometry (ESI-MS/MS) analysis of their lithium-adducts, showing which monomers were present and their relative position. Dimers up to heptamers were identified, in a total yield of oligomers of up to 50% of the total cutin content. Two main structural types of poly(omega-hydroxyacid) ester oligomers were found. Poly(10,16-dihydroxyhexadecanoic acid) esters were the main oligomers in the cutin of Lycopersicum esculentum fruit, with a predominantly branched structure. The 1D and 2D NMR analysis of these oligomers showed that inter-monomer linkages were mostly in secondary midchain hydroxyls, in a ratio of 4.5 to 1, compared with esterification in the primary omega-hydroxyls. In Hedera helix leaves cutin, poly(9-epoxy-18-hydroxyoctadecanoic acid) esters were the dominant oligomers, composed of monomers linearly linked head-to-tail. It is proposed that the cutin polyesters have different poly(omega-hydroxyacid)ester domains, either built of linear chains, where the C18-epoxy omega-hydroxyacids are dominant, or forming a highly branched network, where the C16-dihydroxy omega-hydroxyacids predominate. It is hypothesized that the C18 linear polyester can account for the ordered lamellae seen at ultrastructural level in H. helix cuticle, and that the C16 mostly branched polyester will be the basis of the reticulate structure seen in L. esculentum cuticle.

α‐d‐Mannopyranosyl‐(1→2)‐α‐d‐glucopyranosyl‐(1→2)‐glycerate in the thermophilic bacterium Petrotoga miotherma − structure, cellular content and function

The intracellular accumulation of low molecular mass organic compounds in response to stressful conditions was investigated in the thermophilic bacterium Petrotoga miotherma, a member of the order Thermotogales. This led to the discovery of a new solute, whose structure was established as α‐d‐mannopyranosyl‐(1→2)‐α‐d‐glucopyranosyl‐(1→2)‐glycerate (MGG) by MMR spectroscopy and MS. Under optimum growth conditions (3% NaCl; 55 °C), MGG was the major solute [up to 0.6 µmol·(mg protein)−1]; α‐glutamate and proline were also present but in minor amounts [below 0.08 µmol·(mg protein)−1]. The level of MGG increased notably with the salinity of the growth medium up to the optimum NaCl concentration. At higher NaCl concentrations, however, the level of MGG decreased, whereas the levels of proline and α‐glutamate increased about five‐fold and 10‐fold, respectively. MGG plays a role during low‐level osmotic adaptation of Petrotoga miotherma, whereas α‐glutamate and, to a lesser extent, proline are used for osmoprotection under salt stress. MGG is not part of the cell strategy for coping with heat or oxidative stress. Nevertheless, MGG was an efficient protector of pig heart malate dehydrogenase against heat inactivation and freeze‐drying, although mannosylglycerate was better. This is the first report on the occurrence of MGG in living systems.