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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.

The role of histidine 148 and glutamate 222 is analyzed by studying the fluorescence dynamics of green fluorescence protein mutant (GFPmut) and its H148G and E222Q mutants. The power dependent but pH-independent relaxation mode is due to an excited-state process that is related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.

Background Metallo-β-lactamases (MBLs) have emerged as one of the most important bacterial resistance mechanisms because of their ability to hydrolyse virtually all β-lactam agents. MBL-producing Pseudomonas aeruginosa (MBL-PA) are an important cause of nosocomial infections, particularly in intensive care units (ICUs), where they are associated with serious infections and present a significant clinical risk. Aim To assess the molecular epidemiology, risk factors and outcomes of nosocomial infections caused by MBL-PA in a teaching hospital in Southern Brazil. Methods From January 2001 to December 2008, 142 carbapenem-resistant P. aeruginosa strains were isolated from distinct clinical samples from hospitalized patients. These isolates were screened for MBLs, and underwent polymerase chain reaction, sequencing and pulsed-field gel electrophoresis (PFGE). Patients infected with carbapenem-resistant MBL-PA were considered as cases, and patients infected with non-MBL-PA were considered as controls. Findings Eighty-four of 142 patients with positive carbapenem-resistant P. aeruginosa cultures met the criteria of the Centers for Disease Control and Prevention for infection. Fifty-eight patients were infected with MBL-PA (69%) and 26 patients were infected with non-MBL-PA (31%). Multi-variate analysis revealed that ICU stay [P = 0.003, odds ratio (OR) 4.01, 95% confidence interval (CI) 1.15-14.01] and urinary tract infection (P = 0.001, OR 9.67, 95% CI 1.72-54.48) were important risk factors for MBL-PA infection. Patients infected with MBL-PA showed faster onset of infection (P = 0.002) and faster progression to death (P = 0.04). Conclusions These results showed the severity of MBL-PA infections, and demonstrated the urgent need for strategies to improve infection control measures to prevent an increase in these nosocomial infections. [ABSTRACT FROM AUTHOR]

Methionine gamma lyase (MGL) is a bacterial and plant enzyme that catalyzes the conversion of methionine in methanthiol, 2-oxobutanoate and ammonia. The enzyme belongs to fold type I of the pyridoxal 5'-dependent family. The catalytic mechanism and the structure of wild type MGL and variants were determined in the presence of the natural substrate as well as of many sulfur-containing derivatives. Structure-function relationship studies were pivotal for MGL exploitation in the treatment of cancer, bacterial infections, and other diseases. MGL administration to cancer cells leads to methionine starvation, thus decreasing cells viability and increasing their vulnerability towards other drugs. In antibiotic therapy, MGL acts by transforming prodrugs in powerful drugs. Numerous strategies have been pursued for the delivering of MGL in vivo to prolong its bioavailability and decrease its immunogenicity. These include conjugation with polyethylene glycol and encapsulation in synthetic or natural vesicles, eventually decorated with tumor targeting molecules, such as the natural phytoestrogens daidzein and genistein. The scientific achievements in studying MGL structure, function and perspective therapeutic applications came from the efforts of many talented scientists, among which late Tatyana Demidkina to whom we dedicate this review., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Editorial Board member of Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics A.M. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The pyridoxal 5'-dependent enzyme methionine γ-lyase (MGL) catalyzes the degradation of methionine. This activity has been profitable to develop an antitumor agent exploiting the strict dependence of most malignant cells on the availability of methionine. Indeed, methionine depletion blocks tumor proliferation and leads to an increased susceptibility to anticancer drugs. Here, we explore the conjugation of MGL to gold nanoparticles capped with citrate (AuNPs) as a novel strategy to deliver MGL to cancer cells. Measurements of Transmission Electron Microscopy, Dynamic Light Scattering, Asymmetrical Flow Field-Flow Fractionation, X-ray Photoelectron Spectroscopy, and Circular Dichroism allowed to achieve an extensive biophysical and biochemical characterization of the MGL-AuNP complex including particle size, size distribution, MGL loading yield, enzymatic activity, and impact of gold surface on protein structure. Noticeably, we found that activity retention was improved over time for the enzyme adsorbed to AuNPs with respect to the enzyme free in solution. The acquired body of knowledge on the nanocomplex properties and this encouraging stabilizing effect upon conjugation are the necessary basis for further studies aimed at the evaluation of the therapeutic potential of MGL-AuNP complex in a biological milieu., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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.

The high numbers of COVID-19 cases and deaths in Brazil have made Latin America an epicentre of the pandemic. SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, but important gaps remain in our understanding of virus transmission dynamics at a national scale. We use 17,135 near-complete genomes sampled from 27 Brazilian states and bordering country Paraguay. From March to November 2020, we detected co-circulation of multiple viral lineages that were linked to multiple importations (predominantly from Europe). After November 2020, we detected large, local transmission clusters within the country. In the absence of effective restriction measures, the epidemic progressed, and in January 2021 there was emergence and onward spread, both within and abroad, of variants of concern and variants under monitoring, including Gamma (P.1) and Zeta (P.2). We also characterized a genomic overview of the epidemic in Paraguay and detected evidence of importation of SARS-CoV-2 ancestor lineages and variants of concern from Brazil. Our findings show that genomic surveillance in Brazil enabled assessment of the real-time spread of emerging SARS-CoV-2 variants., (© 2022. The Author(s).)

Background: Healthcare-associated infections (HAIs) impact morbidity, mortality, and hospitalization costs. The contribution of viruses to the overall burden of HAIs is not well described., Aim: To evaluate the prevalence and clinical findings in patients with HAIs caused by respiratory viruses., Methods: An observational, analytical, cross-sectional study was conducted to evaluate patients with a viral nosocomial respiratory infection, occurring between January 2013 and December 2019. Outcomes, comorbidities, cause of hospitalization, seasonality, and presence of bacterial co-infection were assessed., Findings: In all, 161 cases of HAIs with community respiratory viruses (CRVs) were identified through six years; 76.4% of patients had a median age of 2.8 years (interquartile range: 0.28-15.4 years). The main comorbidities in immunosuppressed patients were haematologic neoplasia (46.5%), myelodysplastic syndrome (33.8%), and haematopoietic stem cell transplantation (18.3%). In non-immunosuppressed patients, the most prevalent comorbidities were prematurity (49.1%), respiratory tract diseases (21.0%), and congenital malformations (19.3%). The viruses detected were human rhinovirus (36.6%), respiratory syncytial virus (21.7%), and the parainfluenza group (18.6%). The fatality rate was low (4.6%), and a higher incidence of HAIs occurred in the CRV seasonality period in southern Brazil., Conclusion: CRV circulation in the hospital environment is frequent, and likely involves healthcare workers and visitors as well as patients. More guidance on preventive measures in healthcare settings is required. In addition, care teams should consider these aetiologic agents in the differential diagnosis of patients with nosocomial pneumonia, giving opportunities to limit antibiotic use., (Copyright © 2022. Published by Elsevier Ltd.)

Brazil has experienced some of the highest numbers of COVID-19 cases and deaths globally and from May 2021 made Latin America a pandemic epicenter. Although SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, important gaps remain in our understanding of virus transmission dynamics at the national scale. Here, we describe the genomic epidemiology of SARS-CoV-2 using near-full genomes sampled from 27 Brazilian states and a bordering country - Paraguay. We show that the early stage of the pandemic in Brazil was characterised by the co-circulation of multiple viral lineages, linked to multiple importations predominantly from Europe, and subsequently characterized by large local transmission clusters. As the epidemic progressed under an absence of effective restriction measures, there was a local emergence and onward international spread of Variants of Concern (VOC) and Variants Under Monitoring (VUM), including Gamma (P.1) and Zeta (P.2). In addition, we provide a preliminary genomic overview of the epidemic in Paraguay, showing evidence of importation from Brazil. These data reinforce the usefulness and need for the implementation of widespread genomic surveillance in South America as a toolkit for pandemic monitoring that provides a means to follow the real-time spread of emerging SARS-CoV-2 variants with possible implications for public health and immunization strategies.

Influenza is an acute viral infectious respiratory disease worldwide, presenting in different clinical forms, from influenza-like illness (ILI) to severe acute respiratory infection (SARI). Although real-time quantitative polymerase chain reaction (qPCR) is already an important tool for both diagnosis and treatment monitoring of several viral infections, the correlation between the clinical aspects and the viral load of influenza is still unclear. This lack of clarity is primarily due to the low accuracy and reproducibility of the methodologies developed to quantify the influenza virus. Thus, this study aimed to develop and standardize a universal absolute quantification for influenza A by reverse transcription-quantitative PCR (RT-qPCR), using a plasmid DNA. The assay showed efficiency (Eff%) 98.6, determination coefficient (R 2 ) 0.998, linear range 10^ 1 to 10^ 10 , limit of detection (LOD) 6.77, limit of quantification (LOQ) 20.52 copies/reaction. No inter and intra assay variability was shown, and neither was the matrix effect observed. Serial measurements of clinical samples collected at a 72h interval showed no change in viral load. By contrast, immunocompetent patients have a significantly lower viral load than immunosuppressed ones. Absolute quantification in clinical samples showed some predictors associated with increased viral load: (H1N1)pdm09 (0.045); women (p = 0.049) and asthmatics (p = 0.035). The high efficiency, precision, and previous performance in clinical samples suggest the assay can be used as an accurate universal viral load quantification of influenza A. Its applicability in predicting severity and response to antivirals needs to be evaluated., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The development of safe and efficacious enzyme-based human therapies has increased greatly in the last decades, thanks to remarkable advances in the understanding of the molecular mechanisms responsible for different diseases, and the characterization of the catalytic activity of relevant exogenous enzymes that may play a remedial effect in the treatment of such pathologies. Several enzyme-based biotherapeutics have been approved by FDA (the U.S. Food and Drug Administration) and EMA (the European Medicines Agency) and many are undergoing clinical trials. Apart from enzyme replacement therapy in human genetic diseases, which is not discussed in this review, approved enzymes for human therapy find applications in several fields, from cancer therapy to thrombolysis and the treatment, e.g., of clotting disorders, cystic fibrosis, lactose intolerance and collagen-based disorders. The majority of therapeutic enzymes are of microbial origin, the most convenient source due to fast, simple and cost-effective production and manipulation. The use of microbial recombinant enzymes has broadened prospects for human therapy but some hurdles such as high immunogenicity, protein instability, short half-life and low substrate affinity, still need to be tackled. Alternative sources of enzymes, with reduced side effects and improved activity, as well as genetic modification of the enzymes and novel delivery systems are constantly searched. Chemical modification strategies, targeted-and/or nanocarrier-mediated delivery, directed evolution and site-specific mutagenesis, fusion proteins generated by genetic manipulation are the most explored tools to reduce toxicity and improve bioavailability and cellular targeting. This review provides a description of exogenous enzymes that are presently employed for the therapeutic management of human diseases with their current FDA/EMA-approved status, along with those already experimented at the clinical level and potential promising candidates., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Methionine is an essential amino acid used, beyond protein synthesis, for polyamine formation and DNA/RNA/protein methylation. Cancer cells require particularly high methionine supply for their homeostasis. A successful approach for decreasing methionine concentration is based on the systemic delivery of methionine γ-lyase (MGL), with in vitro and in vivo studies demonstrating its efficacy in cancer therapy. However, the mechanisms explaining how cancer cells suffer from the absence of methionine more significantly than non-malignant cells are still unclear. We analyzed the outcome of the human colorectal adenocarcinoma cancer cell line HT29 to the exposure of MGL for up to 72 h by monitoring cell viability, proteome expression, histone post-translational modifications, and presence of spurious transcription. The rationale of this study was to verify whether reduced methionine supply would affect chromatin decondensation by changing the levels of histone methylation and therefore increasing genomic instability. MGL treatment showed a time-dependent cytotoxic effect on HT29 cancer cells, with an IC 50 of 30 µg/ml, while Hs27 normal cells were less affected, with an IC 50 of >460 µg/ml. Although the levels of total histone methylation were not altered, a loss of the silencing histone mark H3K9me2 was observed, as well as a decrease in H4K20me3. Since H3K9me2/3 decorate repetitive DNA elements, we proved by qRT-PCR that MGL treatment leads to an increased expression of major satellite units. Our data indicate that selected histone methylation marks may play major roles in the mechanism of methionine starvation in cancer cells, proving that MGL treatment directly impacts chromatin homeostasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Raboni, Montalbano, Stransky, Garcia, Buschini, Bettati, Sidoli and Mozzarelli.)

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.

Enterovirus D68 (EV-D68) strain was confirmed in 36/69-52.2% of enterovirus-positive samples collected through surveillance networks for severe acute respiratory infections (SARI) and influenza-like illness (ILI) in southern Brazil in 2018. This finding settles the sustained circulation of EV-D68 in southern Brazil., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 <100 μM were identified via an indirect assay using Ellman's reagent and then tested on a Gram-negative model organism, with one of them being able to interfere with bacterial growth via SAT inhibition., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human serine racemase is a pyridoxal 5'-phosphate (PLP)-dependent dimeric enzyme that catalyzes the reversible racemization of L-serine and D-serine and their dehydration to pyruvate and ammonia. As D-serine is the co-agonist of the N-methyl-D-aspartate receptors for glutamate, the most abundant excitatory neurotransmitter in the brain, the structure, dynamics, function, regulation and cellular localization of serine racemase have been investigated in detail. Serine racemase belongs to the fold-type II of the PLP-dependent enzyme family and structural models from several orthologs are available. The comparison of structures of serine racemase co-crystallized with or without ligands indicates the presence of at least one open and one closed conformation, suggesting that conformational flexibility plays a relevant role in enzyme regulation. ATP, Mg 2+ , Ca 2+ , anions, NADH and protein interactors, as well as the post-translational modifications nitrosylation and phosphorylation, finely tune the racemase and dehydratase activities and their relative reaction rates. Further information on serine racemase structure and dynamics resulted from the search for inhibitors with potential therapeutic applications. The cumulative knowledge on human serine racemase allowed obtaining insights into its conformational landscape and into the mechanisms of cross-talk between the effector binding sites and the active site.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The fluorescence of Green Fluorescent Protein (wtGFP) and variants has been exploited in distinct applications in cellular and analytical biology. GFPs emission depends on the population of the protonated (A-state) and deprotonated (B-state) forms of the chromophore. Whereas wtGFP is pH-independent, mutants in which Ser65 is replaced by either threonine or alanine (as in GFPmut2) are pH-dependent, with a p K a around 6. Given the wtGFP pH-independence, only the structure of the protonated form was determined. The deprotonated form was deduced on the basis of the crystal structure of the Ser65Thr mutant at basic pH, assuming that it corresponds to the conformation populated in solution. Here, we present an investigation where structures of the protonated and deprotonated forms of GFPmut2 were determined from crystals grown in either MPD at pH 6 or PEG at pH 8.5, and moved to either higher or lower pH. Both crystal forms of GFPmut2 were titrated monitoring the process via polarized absorption microspectrophotometry in order to precisely correlate the protonation process with the structures. We found that (i) in solution, chromophore titration is not thermodynamically coupled with any residue and Glu222 is always protonated independent of the protonation state of the chromophore; (ii) the lack of coupling is reflected in the structural behavior of the chromophore and Glu222 environments, with only the former showing variations with pH; (iii) titrations of low-pH and high-pH grown crystals exhibit a Hill coefficient of about 0.75, indicating an anticooperative behavior not observed in solution; (iv) structures where pH was changed in the crystal point to Glu222 as the ionizable group responsible for the outset of the anticooperative behavior; and (v) in GFPmut2 the canonical GFP proton wire involving the chromophore is not interrupted at the level of Ser205 and Glu222 at basic pH as in the Ser65Thr mutant. This allows proposing the structure of the deprotonated state of GFPmut2 as an alternative model for the analogous state of wtGFP.

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. K d 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].

Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-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 K M , 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 IC 50 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., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)