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Methionine is an essential amino acid involved in the formation of polyamines and a precursor metabolite for DNA and protein methylation. The dependence of cancer cells on methionine has triggered extensive investigations aimed at its targeting for cancer therapy, including the exploitation as a therapeutic tool of methionine γ-lyase (MGL), a bacterial enzyme that degrades methionine, capable of inhibiting cancer cells growth due to methionine starvation. We have exploited the high-resolution power of mass spectrometry to compare the effects of reduced availability of the methyl donor SAM, induced by MGL treatment, on the post-translational modifications of the histone tails in normal Hs27 and cancer HT-29 cells. In the absence of MGL, our analysis detected a three-fold higher relative abundance of trimethylated K25 of H1.4 in HT-29 than Hs27 cells, and a complex pattern of methylated, unmethylated and acetylated peptides in H2 and H3.3. In the presence of MGL, in HT-29, the peptide H2A1_4_11 is predominantly unmodified with mono-methylated K5 increasing upon treatment, whereas in Hs27 cells, H2A1_4_11 is monomethylated at K5 and K9 with these marks decreasing upon treatment. The time dependence of the effects of MGL-mediated methionine depletion on PTMs of histone variants in HT-29 cancer cells was also monitored. Overall, our present data on histone variants H1, H2A, H2B as well as H3.3 integrated with our previous studies on histones H3 and H4, shed light on the epigenetic modifications associated with methionine starvation and associated cancer cell death.

Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of the most menacing threats to public health worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals, therefore, its exploitation as a drug target is particularly promising. In this article, we report a series of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the virtual screening of an in-house library of compounds that led to the selection of seven structurally unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either from the original library or from medicinal chemistry efforts, were tested to determine a preliminary structure–activity relationship and, especially, to improve the inhibitory potency of the derivatives, that was indeed ameliorated by several folds compared to hit compound 5 Despite these progresses, at this stage, the most promising compound failed to interfere with bacterial growth when tested on a Gram-negative model organism, anticipating the need for further research efforts.

[Image: see text] In ϒ-proteobacteria and Actinomycetales, cysteine biosynthetic enzymes are indispensable during persistence and become dispensable during growth or acute infection. The biosynthetic machinery required to convert inorganic sulfur into cysteine is absent in mammals; therefore, it is a suitable drug target. We searched for inhibitors of Salmonella serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of l-cysteine biosynthesis. The virtual screening of three ChemDiv focused libraries containing 91 243 compounds was performed to identify potential SAT inhibitors. Scaffold similarity and the analysis of the overall physicochemical properties allowed the selection of 73 compounds that were purchased and evaluated on the recombinant enzyme. Six compounds displaying an IC(50)

Serine racemase is a pyridoxal 5′‑phosphate dependent enzyme responsible for the synthesis of d ‑serine, a neuromodulator of the NMDA receptors. Its activity is modulated by several ligands, including ATP, divalent cations and protein interactors. The murine orthologue is inhibited by S-nitrosylation at Cys113, a residue adjacent to the ATP binding site. We found that the time course of inhibition of human serine racemase by S-nitrosylation is markedly biphasic, with a fast phase associated with the reaction of Cys113. Unlike the murine enzyme, two additional cysteine residues, Cys269, unique to the human orthologue, and Cys128 were also recognized as S-nitrosylation sites through mass spectrometry and site-directed mutagenesis. The effect of S-nitrosylation on the fluorescence of tryptophan residues and on that of the pyridoxal phosphate cofactor indicated that S-nitrosylation produces a partial interruption of the cross-talk between the ATP binding site and the active site. Overall, it appears that the inhibition results from a conformational change rather than the direct displacement of ATP.

The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits kcat and kcat /Km for methionine γ-elimination reaction that are 2.4- and 1.36-fold higher than C. freundii enzyme, respectively, whereas absorption, fluorescence, and circular dichroism spectra are very similar, as expected on the basis of 87% sequence identity and high conservation of active site residues. The reactivity of cysteine residues with DTNB and iodoacetamide was investigated as well as the impact of their chemical modification on catalytic activity. This information is relevant because for increasing bioavailability and reducing immunogenity, MGL should be decorated with polyethylene glycol (PEG). It was found that Cys118 is a faster reacting residue, which results in a significant decrease in the γ-elimination activity. Thus, the protection of Cys118 before conjugation with cysteine-reacting PEG represents a valuable strategy to preserve MGL activity. The anticancer action of C. novyi MGL, evaluated in vitro against prostate (PC-3), chronic myelogenous leucemia (K562), and breast (MDA-MB-231 and MCF7) cancer cells, exhibits IC50 of 1.3 U mL-1 , 4.4 U mL-1 , 1.2 U mL-1 , and 3.4 U mL-1 , respectively. A higher cytotoxicity of C. novyi MGL was found against cancer cells with respect to C. freundii MGL, with the exception of PC-3, where a lower cytotoxicity was observed. © 2017 IUBMB Life, 69(9):668-676, 2017.

The impact of gender and diet on the proteome of Longissimus dorsi was addressed by 2D-PAGE analysis of male and female pigs, fed with a barley-based control diet and a diet enriched with extruded linseed and plant extracts. No statistically significant difference in protein number between female and male samples was found. Furthermore, PCA excluded gender-dependent protein clusters. For both the control and enriched diet, several spots exhibited at least a 1.5-fold intensity difference, but none showed a statistically relevant variation. Protein profiles PCA for both diets indicated that the first two principal components account up to 47% of total variance, with two diet-dependent separated clusters. Among 176 common spots, 29 exhibited >1.5 fold change, mostly more abundant in the control diet. PMF identified 14 distinct proteins, including myofibrillar proteins, glycolytic enzymes and myoglobin, thus suggesting a diet-dependent meat quality. A statistically significant increase in carbonylated proteins of enriched diet samples was detected using the 2,4-dinitrophenylhydrazine method but not using fluorescein-5-thiosemicarbazide-labeled bands. ROS induction and DNA oxidative damage, detected in a human cell line exposed to digested meat from both diets, further support the notion that the enriched diet does not protect against oxidative stress. SIGNIFICANCE: The comparison of the protein profile of female and male Longissimus dorsi from pigs fed by a control diet and a diet enriched with polyphenols, indicate no gender effect, whereas diet affects the abundance of several proteins, possibly linked to meat quality. Protein carbonylation was statistically higher in meat from the enriched diet, suggesting that polyphenols at the concentration present in the diet did not exert a protective effect against oxidation.

We investigated the pH dependence of the fluorescence spectra of ADIFAB (FFA Sciences), a probe used for the quantification of free fatty acids (FFA). Data reports the change in the emission peak of ADIFAB and in the affinity for FFA as a function of pH. An algorithm based on spectral deconvolution allowed to correct ADIFAB fluorescence spectra for the spectroscopic effect caused by pH. Kd values were calculated at each pH based on a calibration with oleic acid. This method allows estimating FFA concentration by ADIFAB in media at different pH. The current data are related to the research article "Phospholipid components of the synthetic pulmonary surfactant CHF5633 probed by fluorescence spectroscopy" (Faggiano et al., 2018) [1]. (C) 2018 The Authors. Published by Elsevier Inc.

CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic pulmonary surfactant currently under clinical development for the treatment of Respiratory Distress Syndrome in premature infants. The product is composed of phospholipids in liposomal organization, together with two peptide analogues of human surfactant proteins B and C. Phospholipids in liposomes can undergo oxidation of unsaturated lipids and hydrolysis, with formation of fatty acids and lysolipids, both affecting the physico-chemical properties of the formulation. We exploited two fluorescence probes, Prodan and ADIFAB, to evaluate the stability of the phospholipid components of CHF5633. While Prodan enters the phospholipid bilayer and probes the polarity of this environment, ADIFAB binds free fatty acids in the aqueous phase, allowing to determine their concentration. Changes of Prodan fluorescence emission indicated an increase in the polarity of the phospholipid bilayer as a function of time. This behavior is coupled with an increase in fatty acids concentration in the aqueous phase, as determined by ADIFAB, and an increase in lysolipids concentration, as determined by HPLC-MS. Prodan and ADIFAB resulted efficient probes to monitor phospholipids hydrolysis in liposomes, reporting an increased stability of CHF5633 at pH values higher than 6.5.

Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine ?-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in KM, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC50 lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis.

Methionine γ-lyase is a pyridoxal 5'-phosphate dependent tetramer that catalyzes the α,γ-elimination of methionine in ammonia, methanethiol and α-ketobutyrate. MGL catalytic power has been exploited as a therapeutic strategy to reduce the viability of cancer cells or bacteria. In order to obtain a stable enzyme to be delivered at the site of action, MGL can be encapsulated in a variety of matrices. As a reference encapsulation strategy we have prepared MGL nanoporous wet silica gels. Immobilized MGL gels were characterized with regards to activity, stability, absorption, circular dichroism and fluorescence properties and compared with soluble MGL. We found that MGL gels exhibit (i) spectroscopic properties very similar to MGL in solution, (ii) a higher stability with respect to the soluble enzyme and (iii) catalytic activity six-fold lower than in solution. These findings prove that MGL encapsulation is a suitable strategy for therapeutic applications.

The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits k

Saffron is a spice comprised of the dried stigmas and styles of Crocus sativus L. flowers and, since it is very expensive, it is frequently adulterated. So far, proteomic tools have never been applied to characterize the proteome of saffron or identify possible cases of fraud. In this study, 1D-Gel Electrophoresis was carried out to characterize the protein profile of (i) fresh stigmas and styles of the plant; (ii) dried stigmas and styles from different geographical origins (Spanish, Italian, Greek and Iranian) that had been stored for various periods of time after their processing; and (iii) two common plant adulterants, dried petals of Carthamus tinctorius L. and dried fruits of Gardenia jasminoides Ellis. A selective protein extraction protocol was applied to avoid interference from colored saffron metabolites, such as crocins, during electrophoretic analyses of saffron. We succeeded in separating and assigning the molecular weights to more than 20 proteins. In spite of the unavailability of the genome of saffron, we were able to identify five proteins by Peptide Mass Fingerprinting: phosphoenolpyruvate carboxylase 3, heat shock cognate 70 KDa protein, crocetin glucosyltransferase 2, α-1,4-glucan-protein synthase and glyceraldehydes-3-phosphate dehydrogenase-2. Our findings indicate that (i) few bands are present in all saffron samples independently of origin and storage time, with amounts that significantly vary among samples and (ii) aging during saffron storage is associated with a reduction in the number of detectable bands, suggesting that proteases are still active. The protein pattern of saffron was quite distinct from those of two common adulterants, such as the dried petals of Carthamus tinctorius and the dried fruits of Gardenia jasminoides indicating that proteomic analyses could be exploited for detecting possible frauds.

Interferon α (IFN α) subtypes are important protein drugs that have been used to treat infectious diseases and cancers. Here, we studied the reactivity of IFN α-2b to microbial transglutaminase (TGase) with the aim of obtaining a site-specific conjugation of this protein drug. Interestingly, TGase allowed the production of two monoderivatized isomers of IFN with high yields. Characterization by mass spectrometry of the two conjugates indicated that they are exclusively modified at the level of Gln101 if the protein is reacted in the presence of an amino-containing ligand (i.e., dansylcadaverine) or at the level of Lys164 if a glutamine-containing molecule is used (i.e., carbobenzoxy-l-glutaminyl-glycine, ZQG). We explained the extraordinary specificity of the TGase-mediated reaction on the basis of the conformational features of IFN. Indeed, among the 10 Lys and 12 Gln residues of the protein, only Gln101 and Lys164 are located in highly flexible protein regions. The TGase-mediated derivatization of IFN was then applied to the production of IFN derivatives conjugated to a 20 kDa polyethylene glycol (PEG), using PEG-NH

The transglutaminase (TGase) from Streptomyces mobaraensis catalyzes transamidation reactions in a protein substrate leading to the modification of the side chains of Gln and Lys residues according to the A-CONH(2) + H(2)N-B → A-CONH-B + NH(3) reaction, where both A and B can be a protein or a ligand. A noteworthy property of TGase is its susbstrate specificity, so that often only a few specific Gln or Lys residues can be modified in a globular protein. The molecular features of a globular protein dictating the site-specific reactions mediated by TGase are yet poorly understood. Here, we have analyzed the reactivity toward TGase of apomyoglobin (apoMb), α-lactalbumin (α-LA), and fragment 205-316 of thermolysin. These proteins are models of protein structure and folding that have been studied previously using the limited proteolysis technique to unravel regions of local unfolding in their amino acid sequences. The three proteins were modified by TGase at the level of Gln or Lys residues with dansylcadaverine or carbobenzoxy-l-glutaminylglycine, respectively. Despite these model proteins containing several Gln and Lys residues, the sites of TGase derivatization occur over restricted chain regions of the protein substrates. In particular, the TGase-mediated modifications occur in the "helix F" region in apoMb, in the β-domain in apo-α-LA in its molten globule state, and in the N-terminal region in fragment 205-316 of thermolysin. Interestingly, the sites of limited proteolysis are located in the same chain regions of these proteins, thus providing a clear-cut demonstration that chain flexibility or local unfolding overwhelmingly dictates the site-specific modification by both TGase and a protease.

The regulation of enzyme activity through the transient formation of multiprotein assemblies plays an important role in the control of biosynthetic pathways. One of the first regulatory complexes to be discovered was cysteine synthase (CS), formed by the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). These enzymes are at the branch point of the sulfur, carbon, and nitrogen assimilation pathways. Understanding the mechanism of complex formation helps to clarify the role played by CS in the regulation of sulfur assimilation in bacteria and plants. To this goal, stopped-flow fluorescence spectroscopy was used to characterize the interaction of SAT with OASS, at different temperatures and pH values, and in the presence of the physiological regulators cysteine and bisulfide. Results shed light on the mechanism of complex formation and regulation, so far poorly understood. Cysteine synthase assembly occurs via a two-step mechanism involving rapid formation of an encounter complex between the two enzymes, followed by a slow conformational change. The conformational change likely results from the closure of the active site of OASS upon binding of the SAT C-terminal peptide. Bisulfide, the second substrate and a feedback inhibitor of OASS, stabilizes the CS complex mainly by decreasing the back rate of the isomerization step. Cysteine, the product of the OASS reaction and a SAT inhibitor, slightly affects the kinetics of CS formation leading to destabilization of the complex.

Tryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) complex catalyzing the last two steps of tryptophan biosynthesis in bacteria, plants and fungi. Structural, dynamic and functional studies, carried out over more than 40 years, have unveiled that: (1) alpha- and beta-active sites are separated by about 20 A and communicate via the selective stabilization of distinct conformational states, triggered by the chemical nature of individual catalytic intermediates and by allosteric ligands; (2) indole, formed at alpha-active site, is intramolecularly channeled to the beta-active site; and (3) naturally occurring as well as genetically generated mutants have allowed to pinpoint functional and regulatory roles for several individual amino acids. These key features have made tryptophan synthase a text-book case for the understanding of the interplay between chemistry and conformational energy landscapes.

We have used a nanosecond pH-jump technique, coupled with simultaneous transient absorption and fluorescence emission detection, to characterize the dynamics of the acid-induced spectral changes in the GFPmut2 chromophore. Disappearance of the absorbance at 488 nm and the green fluorescence emission occurs with a thermally activated, double exponential relaxation. To understand the source of the two transients we have introduced mutations in amino acid residues that interact with the chromophore (H148G, T203V, and E222Q). Results indicate that the faster transient is associated with proton binding from the solution, while the second process, smaller in amplitude, is attributed to structural rearrangement of the amino acids surrounding the chromophore. The protonation rate shows a 3-fold increase for the H148G mutant, demonstrating that His148 plays a key role in protecting the chromophore from the solvent. The deprotonation rate for T203V is an order of magnitude smaller, showing that the hydrogen bond with the hydroxyl of Thr203 is important in stabilizing the deprotonated form of the chromophore. A kinetic model suggests that, in addition to protecting the chromophore from the solvent, His148 may act as the primary acceptor for the protons on the way to the chromophore.

O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5'-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions.

The pyridoxal 5'-phosphate (PLP)-dependent tryptophan synthase is a alpha(2)beta(2) complex. The alpha-beta subunit interaction plays a critical role both in the reciprocal activation of the individual subunits and in the allosteric regulation. We have investigated whether mutations of alpha loop6 Gly(181) and beta helix6 Ser(178) affect intersubunit communication. The loss of the hydrogen bond between these residues, achieved by proline substitution, does not significantly influence the intersubunit catalytic activation, but completely abolishes ligand-induced intersubunit signaling. The comparison of the crystal structure of the wild type and beta Ser(178)Pro mutant, in the absence and presence of alpha-subunit ligands, indicates that the removal of the interaction between beta Ser(178) and alpha Gly(181) strongly affects the equilibrium between active (closed) and inactive (open) conformations of the alpha-active site, the latter being stabilized in both mutants.

The blood is a complex body fluid containing a large variety of cells and molecules serving vital functions. In particular, hemoglobin, contained in the red cells, loads, carries, and delivers oxygen and carbon dioxide in the circulation. In the last 30 years intense efforts have been made (1) to develop allosteric effectors of hemoglobin that affect oxygen affinity to treat diseases such as sickle-cell anemia, ischemia, hypoxia, and to improve the efficiency of radio- and chemotherapy; (2) to obtain blood substitutes based either on organic molecules that solubilize oxygen or on genetically and/or chemically modified hemoglobins; and (3) to formulate solutions that maintain the blood volume and oncotic pressure in surgery and hemorrhagic events. In spite of the pharmacological relevance of these molecules, only plasma expanders are already available on the U.S. market. This is an indication of the difficulties in mimicking the multifaceted roles of hemoglobin. Keywords: oxygen; hemoglobin; blood substitutes; plasma expanders; allosteric effectors; perfluorochemicals

Pyridoxine-5'-phosphate oxidase catalyzes the oxidation of either the C4' alcohol group or amino group of the two substrates pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to an aldehyde, forming pyridoxal 5'-phosphate. A hydrogen atom is removed from C4' during the oxidation and a pair of electrons is transferred to tightly bound FMN. A new crystal form of the enzyme in complex with pyridoxal 5'-phosphate shows that the N-terminal segment of the protein folds over the active site to sequester the ligand from solvent during the catalytic cycle. Using (4'R)-[(3)H]PMP as substrate, nearly 100 % of the radiolabel appears in water after oxidation to pyridoxal 5'-phosphate. Thus, the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon atom of pyridoxamine 5'-phosphate. Site mutants were made of all residues at the active site that interact with the oxygen atom or amine group on C4' of the substrates. Other residues that make interactions with the phosphate moiety of the substrate were mutated. The mutants showed a decrease in affinity, but exhibited considerable catalytic activity, showing that these residues are important for binding, but play a lesser role in catalysis. The exception is Arg197, which is important for both binding and catalysis. The R197 M mutant enzyme catalyzed removal of the proS hydrogen atom from (4'R)-[(3)H]PMP, showing that the guanidinium side-chain plays an important role in determining stereospecificity. The crystal structure and the stereospecificity studies suggests that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion.

Parma dry-cured ham is a typical Italian food product, manufactured according to a technological process based on empirical observations and traditional recipes, consisting of steps of salting, resting, drying and ageing. The salting phase is crucial because salt acts as a bacteriostatic agent, reduces water content, and plays an active role in the development of the sensory and quality parameters of the final product. The salting consists of two phases: in the first phase, the pork legs are covered with sea salt and then left in a room for 5-7 days at controlled temperature (0-3 °C) and humidity (75-90 %). In the second phase, the hams, after the removal of the remained salt, are covered again with fresh sea salt and stored for 12-15 days in the above mentioned environmental conditions. Two kinds of salt are used to cover the pork legs. A wet salt is used in the ham rind while dry salt is used to cover the unskinned parts. During the salting phases an exudate rich in proteins is produced. We have determined by LC-MS/MS the protein pattern of exudates obtained under different technological conditions.

The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pKa values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein.

The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.

Cellular death is characterized by a complex pattern of molecular events that depend on cell type. Specifically, muscle cells first undergo rigor mortis due to ATP depletion, and later, on the time scale of days, muscle fiber degradation due to proteolytic enzyme activity. In the present review, we will refer to proteomic investigations on the post-mortem evolution of the protein patterns of animal muscle cells. These studies, carried out with the application of either bottom-up or top-down methods, are relevant for understanding the biochemical reactions that i) convert muscle to meat, ii) are associated with meat aging and iii) impact on meat tenderness, a feature of significant commercial value. We also report on the proteomic investigations that have been made to analyze the transformation of meat in industrial processes. These studies are primarily aimed at identifying protein patterns and/or individual proteins diagnostic of the quality of the final product.

Farm animal meat is a worldwide food with poultry, pork and bovine meat supplying a large portion of proteins to human diet. Because animal muscles cannot be consumed immediately after slaughtering, aging is required to achieve the transformation of muscles to meat. This process is associated with a complex pattern of molecular events that depends on many variables, including animal genetic profile, diet, slaughtering procedures and temperatures. Correlations between such variables and protein pattern have been and are still being thoroughly studied applying proteomic methods [1–4].

Proteomics is a powerful tool for the identification of proteins that are characteristic of the transformation of meat in industrial processes and might be diagnostic of the quality of the final product. Parma dry cured ham is a typical Italian food product, manufactured according to established technological steps mainly based on subjective empirical observations and traditional recipes. Meat salt intake, dehydration and water activity decrease are some of the key events that lead to a food product that can be stored at room temperature for several months. Specifically, the development of the sensory and quality traits of the cured ham is accomplished by a process that includes salting, resting, drying and maturation

Cysteine is the final product of the reductive sulfate assimilation pathway in bacteria and plants and serves as the precursor for all sulfur-containing biological compounds, such as methionine, S-adenosyl methionine, iron–sulfur clusters and glutathione. Moreover, in several microorganisms cysteine plays a role as a reducing agent, eventually counteracting host oxidative defense strategies. Cysteine is synthesized by the PLP-dependent O-acetylserine sulfhydrylase, a dimeric enzyme belonging to the fold type II, catalyzing a beta-replacement reaction. In this review, the spectroscopic properties, catalytic mechanism, three-dimensional structure, conformational changes accompanying catalysis, determinants of enzyme stability, role of selected amino acids in catalysis, and the regulation of enzyme activity by ligands and interaction with serine acetyltransferase, the preceding enzyme in the biosynthetic pathway, are described. Given the key biological role played by O-acetylserine sulfhydrylase in bacteria, inhibitors with potential antibiotic activity have been developed. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.

We have used fluorescence spectroscopy techniques such as fluorescence correlation spectroscopy and fluorescence anisotropy decay on a wide time range, from nanoseconds to seconds, to investigate the unfolding kinetics induced by guanidinium chloride of GFPMut2 and its point mutation H148G, which has proved to be relevant for GFP photochemistry and photophysics. The mutation affects the unfolding kinetics of GFP leading to a much faster process at alkaline pH values, where protonation dynamics is negligible, that can be ascribed to a twofold role of His148, either as a proton shutter towards the chromophore and as a conformation stabiliser. For both mutants a soft region located near beta-strand 3 is found that starts to gain flexibility in the ns range at denaturant concentrations far lower than those required to turn off the chromophore fluorescence, as derived from the anisotropy decay of an extrinsic probe covalently bound to the proteins. © 2011 Elsevier B.V. All rights reserved.

New probes for kinetic intracellular measurements in the millisecond range are desirable to monitor protein biochemical dynamics essential for catalysis, allosteric regulation and signalling. Good candidates to this aim are the photo-switchable mutants of the Green Fluorescent Protein, whose anionic fluorescence, primed by blue light, is markedly enhanced under an additional excitation at a shorter wavelength and relaxes within few milliseconds. The aim of this report is to study how the brightness enhancement kinetics depends on the physical-chemical and spectroscopic parameters and to provide proof-of-concepts experiments for the use of the fluorescence enhancement in conditions in which the protein diffusion is hindered and thereby photo-bleaching can be a limiting critical issue. Future, direct applications of photo-chromic mutants for modulated excitation imaging would in fact require such a detailed knowledge. We present here an extensive study of the photo-switching mechanism of the E222Q mutant of GFPMut2 (Mut2Q), pumped by visible 488 nm light and probed at 400-420 nm, as a function of pH, viscosity, temperature and light intensity. In solution, two characteristic photo-switching times are found by means of modulated double beam fluorescence correlation spectroscopy in the 1-30 ms range, depending on the solution pH. The photo-switching kinetics is solved in terms of the eigenvalues and the eigenvectors of a specific energy diagram and directly used to fit the data, suggesting that the observed photo-switching amplitudes and kinetics are related to a single three-levels transition loop. Finally we give in-vitro examples of the use of modulated excitation microscopy, based on fluorescence enhancement amplitude and kinetics detection, on Mut2Q protein samples immobilized in acrylamide gels.

GFP mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Because serine 65, threonine 203, glutamate 222, and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, and S72A GFP) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components are found in the fluorescence autocorrelation functions of GFPmut2: a 10-100 micros pH-dependent component and a 100-500 micros laser-power-dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a 3-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the histidine 148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, is due to an excited-state process that is probably related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.

The tryptophan synthase alpha2beta2 complex catalyzes the last two steps in the biosynthesis of l-tryptophan in bacteria, plants, and fungi, the conversion of indole-3-glycerol phosphate and l-serine to l-tryptophan, glyceraldehyde 3-phosphate, and water. The beta-subunit binds pyridoxal 5'-phosphate and catalyzes the beta-replacement reaction with serine and indole. Structural, spectral, and kinetic studies indicate that different monovalent cations stabilize the alternative enzyme conformations and equilibrium distribution of the internal, external, and alpha-aminoacrylate Schiff base. To improve our understanding of the role of monovalent cations, the pH dependence of steady-state and pre-steady-state kinetic parameters and primary kinetic deuterium isotope effects were measured in the presence of l-serine and [alpha-2H]-l-serine in the absence and presence of Na+, K+, and Cs+. For the interpretation of the data obtained in this study, it was necessary to re-interpret a number of results published previously. Overall, data suggest that the enzyme exists in two conformers that equilibrate slowly either in the absence of substrates and monovalent cations or in the presence of K+ or Cs+, whereas they equilibrate faster in the presence of Na+. The rate of interconversion of the conformers increases as a group on the enzyme with a pKa of approximately 8 becomes deprotonated. The pH dependence of deuterium isotope effects is suggestive of a mechanism in which a pH-dependent conformational change that closes the active site precedes the chemical steps, likely a result of formation of one or more salt bridges. As the pH increases, the reaction becomes more committed to proceed to products, which causes the deuterium isotope effect to decrease to a value of unity at high pH. The closure of the site is modulated by the different monovalent cations and is fastest in the presence of Na+, which exhibits the maximum isotope effect of 5.7 (likely the intrinsic effect) on V/Kserine, and slowest in the presence of Cs+, which exhibits the smallest isotope effect of approximately 1.5. The isotope effect on V, in all cases, indicates a contribution to rate limitation from steps in the second half of the reaction. Finally, in the presence of Na+, the steady-state isotope effect on V is greater than that on the pre-steady-state rate constant for decay of the external Schiff base, suggesting that the rate of conversion of the two conformers of the internal aldimine contributes to the pre-steady-state rate, but not the steady-state rate because the high serine concentration traps the enzyme in the active E-serine complex before it can decay to the less active form.

Ligand binding to the alpha-subunit of the alpha2beta2 complex of tryptophan synthase induces the alphaloop6 closure over the alpha-active site. This conformational change is associated with the formation of a hydrogen bond between alphaGly181 NH group and betaSer178 carbonyl oxygen, a key event for the triggering of intersubunit allosteric signals. Mutation of betaSer178 to Pro and alphaGly181 to Pro, Ala, Phe and Val abolishes the ligand-induced intersubunit communication. Molecular dynamics methods were applied to simulate the conformation of the highly flexible and crystallographically undetectable open state of alphaloop6 in the wild type and in the alpha181 mutants. The open conformation of alphaloop6 is favoured in the wild type enzyme in the absence of alpha-ligands, and in the alpha181 mutants both in the presence and absence of bound ligands. A very good correlation was found between the extent of limited tryptic proteolysis and both the hydrogen bond distance between alphaX181 and betaSer178, obtained from the molecular dynamics simulation, and the hydrogen bond strength, evaluated by HINT, an empirical force field that takes into account both enthalpic and entropic contributions. Comparison of the open and closed conformations of alphaloop6 suggests a pathway for substrate entrance into the alpha-active site and provides an explanation for the limited catalytic efficiency of the open state.

The pyridoxal 5'-phosphate-dependent tryptophan synthase alpha2beta2 complex is a paradigmatic protein for substrate channeling and allosteric regulation. The enzymatic activity is modulated by a ligand-mediated equilibrium between open (inactive) and closed (active) conformations of the alpha- and beta-subunit, predominantly involving the mobile alpha loop 6 and the beta-COMM domain that contains beta helix 6. The alpha ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the carbonyl oxygen of betaSer-178 of beta helix 6 and the NH group of alphaGly-181 of alpha loop 6. We investigated whether and to what extent mutations of alphaGly-181 and betaSer-178 affect allosteric regulation by the replacement of betaSer-178 with Pro or Ala and of alphaGly-181 with either Pro to remove the amidic proton that forms the hydrogen bond or Ala, Val, and Phe to analyze the dependence on steric hindrance of the open-closed conformational transition. The alpha and beta activity assays and the equilibrium distribution of beta-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced alpha-to beta-subunit signaling, demonstrating distinct pathways for alpha-beta-site communication. Limited proteolysis experiments indicate that the removal of the interaction between betaSer-178 and alphaGly-181 strongly favors the more trypsin-accessible open conformation of the alpha-active site. When the hydrogen bond cannot be formed, the alpha-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface.

Proteins belonging to the superfamily of pyridoxal 5'-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types. The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimensional structures of pyridoxal 5'-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the beta-family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo- and holoprotein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, (31)P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization. Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form. The data are discussed in light of the fact that the role of the coenzyme in structural stabilization varies among the pyridoxal 5'-phosphate-dependent enzymes and does not seem to be linked to the particular enzyme fold type.

Pyridoxal 5′-phosphate (PLP) is the biologically active form of B6 vitamers. PLP plays the role as coenzyme in more than 160 different enzymes acting on a variety of substrates and inhibitors predominantly possessing amine and carboxylate moieties. The key feature of PLP-dependent enzymes is the catalytic versatility, carrying out transamination, decarboxylation, and beta and gamma elimination and substitution via several catalytic intermediates. Catalysis frequently accompanies a conformational transition between an open, less active state and a closed, fully active state. Recent investigations on functional genomics of PLP-dependent enzymes and the search of novel targets for therapeutic activity indicate that this class of enzymes has not been fully explored in the past and calls for more intensive and focussed studies.

The rising emergence of antibiotic resistance urges the search for new strategies to defeat microorganisms that lead to persistent infections of the host. Tolerant to antibiotics, slowly replicating bacteria often cause latent and persistent infections that are the most challenging for pharmacological treatment. Persistence inside the host requires an extensive re-programming of the pathogen metabolic functions, due to the extremely hostile environment they face. Therefore, targeting key metabolic functions could result in better antibiotic treatments, shortened latency periods, and increased susceptibility to traditional antibiotics. Bacteria, differently from mammals, assimilate inorganic sulfur into cysteine, the precursor of a number of key metabolites including reducing agents, cofactors and membrane components. Inhibition of cysteine biosynthesis was proven to interfere heavily with the ability of pathogens to fight oxidative stress, to infect the host and to establish long-term infections. This review has the purpose of i) briefly summarizing the key structural and functional properties of transporters and enzymes involved in sulfur assimilation, ii) presenting biological evidence that supports the exploitation of this pathway for the identification of potential targets and, iii) highlighting intense efforts and advancements in the search of promising candidates for the development of novel compounds that enhance antibiotics therapy.