Thiourea-Based Receptors for Anion Recognition and Signaling.

This work reports on two thiourea-based receptors with pyridine and amine units including 1-naphthyl (MT1N) and 4-nytrophenyl (MT4N) as signaling units. For both compounds, their affinity and signaling ability toward various anions of different geometry and basicity in DMSO were studied using UV-vis, fluorescence, and 1H NMR techniques. Anion recognition studies revealed that both MT1N and MT4N have, in general, high affinities toward basic anions. In this regard, a higher acidity of the MT4N receptor was demonstrated. Furthermore, MT4N has a higher affinity for fluoride (log K1 = 5.98) than for the other anions and can effectively detect it through colorimetric changes that can be monitored by the UV-vis technique. The interaction between receptors and anions mainly involves the hydrogens of the amino and thiourea groups of the former. Complementary single-crystal X-ray diffraction studies and molecular modeling at the DFT level were also performed.

Review of the Greening Reaction by Thermal Treatment: New Insights Exploring the Structural Implications of Myoglobin.

Myoglobin is the main factor responsible for muscle pigmentation in tuna; muscle color depends upon changes in the oxidative state of myoglobin. The tuna industry has reported muscle greening after thermal treatment involving metmyoglobin (MetMb), trimethylamine oxide (TMAO), and free cysteine (Cys). It has been proposed that this pigmentation change is due to a disulfide bond between a unique cysteine residue (Cys10) found in tuna MetMb and free Cys. However, no evidence has been given to confirm that this reaction occurs. In this review, new findings about the mechanism of this greening reaction are discussed, showing evidence of how free radicals produced from Cys oxidation under thermal treatment participate in the greening of tuna and horse muscle during thermal treatment. In addition, the reaction conditions are compared to other green myoglobins, such as sulfmyoglobin, verdomyoglobin, and cholemyoglobin.

Sulfmyoglobin production by free cysteine during thermal treatment: Involvement of heme iron in the production of free radicals.

The meat greening is an abnormal pigmentation related to microbiological contamination and lipid oxidation during storage. This color change results from sulfmyoglobin (SulfMb) production promoted by the reaction between metmyoglobin (MetMb), H2O2, and thiol compounds. Spectral studies on cooked meat suggested the production of SulfMb, probably due to the increment of free radicals during thermal treatment. Thus, we evaluated the involvement of free radicals and heme iron in the SulfMb production from horse MetMb and free cysteine (Cys) during thermal treatment. The results confirm that the reaction of SulfMb production at meat muscle pH (5.7-7.2) during heat treatment is a product of free radicals formed from Cys oxidation (SH) and reactive oxygen species (O2-, H2O2). This is catalyzed by the release of heme iron, thus promoting a consecutive reaction having MbFe(IV)O as a reaction intermediate.

The greening reaction of skipjack tuna (Katsuwonus pelamis) metmyoglobin promoted by free cysteine during thermal treatment.

Background: Tuna muscle greening is a problem that occurs after heating. A hypothesis has been postulated to address this problem, involving a conserved Cys residue at position 10 (Cys-10) present on tuna myoglobin (Mb) that is exposed during the thermic treatment, forming a disulfide bond with free cysteine (Cys) in the presence of trimethylamine oxide (TMAO), resulting in the greening of the tuna Mb. Methods: We present a study using skipjack tuna (Katsuwonus pelamis) metmyoglobin (MbFe(III)-H2O) where the effect of free Cys (1-6 mM), TMAO (1.33 mM), and catalase on the greening reaction (GR) was monitored by UV-vis spectrometry during thermal treatment at 60 °C for 30 min. Moreover, the participation of Cys-10 on the GR was evaluated after its blocking with N-ethymaleimide. Results: The GR occurred in tuna MbFe(III)-H2O after heat treatment with free Cys, forming sulfmyoglobin (MbFe(II)-S) as the responsible pigment for the tuna greening. However, the rate constants of MbFe(II)-S production depended on Cys concentration (up to 4 mM) and occurred regardless of the TMAO presence. We postulate that two consecutive reactions involve an intermediate ferrylmyoglobin (promoted by H2O2) species with a subsequent MbFe(II)-S formation since the presence of catalase fosters the reduction of the rate reaction. Moreover, GR occurred even with blocked Cys-10 residues in tuna Mb and horse Mb (without Cys in its sequence). Discussion: We found that GR is not exclusive to tuna Mb´s, and it can be promoted in other muscle systems. Moreover, Cys and thermal treatment are indispensable for promoting this pigmentation anomaly.

A Novel Glutathione S-Transferase Gtt2 Class (VpGSTT2) Is Found in the Genome of the AHPND/EMS Vibrio parahaemolyticus Shrimp Pathogen.

Glutathione S-transferases are a family of detoxifying enzymes that catalyze the conjugation of reduced glutathione (GSH) with different xenobiotic compounds using either Ser, Tyr, or Cys as a primary catalytic residue. We identified a novel GST in the genome of the shrimp pathogen V. parahaemolyticus FIM- S1708+, a bacterial strain associated with Acute Hepatopancreatic Necrosis Disease (AHPND)/Early Mortality Syndrome (EMS) in cultured shrimp. This new GST class was named Gtt2. It has an atypical catalytic mechanism in which a water molecule instead of Ser, Tyr, or Cys activates the sulfhydryl group of GSH. The biochemical properties of Gtt2 from Vibrio parahaemolyticus (VpGSTT2) were characterized using kinetic and crystallographic methods. Recombinant VpGSTT2 was enzymatically active using GSH and CDNB as substrates, with a specific activity of 5.7 units/mg. Low affinity for substrates was demonstrated using both Michaelis-Menten kinetics and isothermal titration calorimetry. The crystal structure showed a canonical two-domain structure comprising a glutathione binding G-domain and a hydrophobic ligand H domain. A water molecule was hydrogen-bonded to residues Thr9 and Ser 11, as reported for the yeast Gtt2, suggesting a primary role in the reaction. Molecular docking showed that GSH could bind at the G-site in the vicinity of Ser11. G-site mutationsT9A and S11A were analyzed. S11A retained 30% activity, while T9A/S11A showed no detectable activity. VpGSTT2 was the first bacterial Gtt2 characterized, in which residues Ser11 and Thr9 coordinated a water molecule as part of a catalytic mechanism that was characteristic of yeast GTT2. The GTT2 family has been shown to provide protection against metal toxicity; in some cases, excess heavy metals appear in shrimp ponds presenting AHPND/EMS. Further studies may address whether GTT2 in V. parahaemolyticus pathogenic strains may provide a competitive advantage as a novel detoxification mechanism.

Biochemical Characterization of a Novel α/ß-Hydrolase/FSH from the White Shrimp Litopenaeus vannamei.

(1) Background: Lipases and esterases are important enzymes that share the α/ß hydrolase fold. The activity and cellular localization are important characteristics to understand the role of such enzymes in an organism. (2) Methods: Bioinformatic and biochemical tools were used to describe a new α/ß hydrolase from a Litopenaeus vannamei transcriptome (LvFHS for Family Serine Hydrolase). (3) Results: The enzyme was obtained by heterologous overexpression in Escherichia coli and showed hydrolytic activity towards short-chain lipid substrates and high affinity to long-chain lipid substrates. Anti-LvFHS antibodies were produced in rabbit that immunodetected the LvFSH enzyme in several shrimp tissues. (4) Conclusions: The protein obtained and analyzed was an α/ß hydrolase with esterase and lipase-type activity towards long-chain substrates up to 12 carbons; its immunodetection in shrimp tissues suggests that it has an intracellular localization, and predicted roles in energy mobilization and signal transduction.

Mechanism of general acid-base catalysis in transesterification of an RNA model phosphodiester studied with strongly basic catalysts.

Using 80% vol aqueous DMSO as the reaction medium for transesterification of an RNA model substrate 2-hydroxypropyl 4-nitrophenyl phosphate allows one to observe catalysis in buffer mixtures composed of highly basic components such as guanidines, amidines or alkylamines, which provide up to 10(3)-fold accelerations over the background reaction in the 0.01-0.1 M concentration range. The rate law k(obs) = k(1)[B] + k(2)[B][BH(+)] was established indicating contributions from both simple general base catalysis and the reaction involving concerted action of neutral (B) and protonated (BH(+)) forms of the buffer. The catalytic efficiency of guanidinium and amidinium cations is 10 times larger than that of more acidic ammonium cations. Rate constants k(1) and k(2) obey the Brønsted equations with the slopes 0.77 and 0.69 respectively. Proton inventory for k(2) (B = guanidine) in D(2)O/H(2)O mixtures gives two fractionation factors phi(1) = 0.48 and phi(2) = 1.26 for normal and inverse isotope effects respectively. The former results from the proton transfer to B and the latter from the binding of guanidinium cation to the phosphate group as follows from observation of an inverse solvent isotope effect for the binding of guanidinium and amidinium cations to a phosphodiester anion. The results of kinetic studies together with analysis of transition state stabilization free energies for guanidinium and amidinium cations show that the protonated buffer component acts via electrostatic transition state stabilization rather than proton transfer, which may be possible for a guanidinium assisted hydroxide catalyzed reaction.