Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

In orally acquired bacteria, the ability to counteract extreme acid stress (pH < 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known "classical" Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved

Unusual structural and spectroscopic properties of an alanine transaminase from Escherichia coli

Determining the functions of proteins encoded by genome sequences represents an important challenge of contemporary biology. Recently we became interested in the characterization of the protein YfdZ from Escherichia coli, an ‘orphan enzyme’, for which the catalyzed reaction has not been assigned with certainty. Two recent reports suggest that this enzyme is an alanine aminotransferase (1, 2). Biochemical characterization of YfdZ was performed in our laboratory by overexpressing and purifying it in E. coli as recombinant protein with a C-terminal hexa-histidine tag. Preliminary information on YfdZ catalytic properties, obtained by thin layer chromatography (TLC), confirmed its alanine transaminase activity. Hence, we developed a quantitative enzymatic assay for the alanine-forming reaction and collected kinetic data with the aim of determining the kinetic constants. Furthermore, the kinetic analysis allowed us to detect pyruvate formation (unfavourable direction), not detected by preliminary TLC tests. Interestingly, the spectroscopic analysis showed anomalies in the binding of pyridoxal-5’-phosphate (PLP). Although YfdZ performs a reaction dependent on PLP and meets the necessary requirements for the binding of the cofactor, the spectrum of the purified enzyme is devoid of PLP. Reconstitution of YfdZ with PLP affects its spectroscopic and catalytic properties. Indeed the cofactor needs to be added to the reaction mixture, otherwise no catalytic activity is detectable. By means of SDS-PAGE, we noticed a strong tendency of the enzyme to dimerize in the absence of an excess of reducing agents. This is probably due to the formation of intermolecular-disulfide bond(s) involving one or more of the 4 cysteine residues in the protein. Notably, in the presence of dithiothreitol (DTT), a reducing agent, YfdZ activity is positively affected. Therefore DTT acts sinergically with PLP. By gel filtration chromatography we found that YfdZ exists as a dimer in solution and its quaternary structure does not change in the presence of DTT. Hence, the disulfide bridges among monomers do not alter the dimeric assembly of the enzyme, rather they seem to restrict the conformational changes associated to the enzymatic activity

The role of an active site aspartate residue in the catalytic activity of Glutamate decarboxylase from Escherichia coli.

The EMBO Conference focuses on fundamental and applied aspects of biocatalysis, with an emphasis on the impact that enzyme research has at the interface of biology and chemistry. The sessions will cover an array of topics including computational, chemistry and structural approaches, as well as directed evolution, bioinformatics and spectroscopic methods, aimed towards better understanding of enzyme mechanisms and mechanisms of complex multifunctional enzyme systems, in vitro and in vivo. The importance of the electrostatic and dynamical properties of enzymes will be addressed. The impact of this knowledge for drug discovery research and research on non-natural biocatalytic systems will be highlighted. Established and emerging scientists from academic and industrial settings will be available to stimulate discussion and provide perspective on the topics of this conference. We strongly encourage participation of students and postdoctoral associates, providing opportunities for discussion and networking. Oral presentations chosen from submitted poster abstracts will provide additional opportunities for discussing new and innovative ideas. The speakers are encouraged to give a brief introduction of the field in which they work and allow for sufficient time for discussion.Escherichia coli glutamate decarboxylase is a homohexameric PLP-dependent enzyme and a major structural component of the glutamate-based acid resistance system in this microorganism as well as in many orally-acquired, neutralophilic bacteria [1,2]. In fact the decarboxylation of L-glutamate, besides yielding γ-aminobutyrate (GABA) and CO2, consumes 1 H+/catalytic cycle, an activity shown to be beneficial for protecting the cell under extreme acid stress [1]. We have extensively characterized one of the two E. coli isoforms, the B isoform (EcGadB) and shown that it displays pH-dependency in activity in the acid range, being maximally active at pH 4-5 while showing negligible or no activity at or above pH 6.5. Based on the crystal structures of EcGadB at neutral and acidic pH, as well as in the presence of halides, and of a mutant form deleted at the N-terminal, we hypothesize that together with His465 (the penultimate residues in the amino acid sequence), Asp86 is a likely candidate for controlling the acidic range of activity of EcGad [3,4]. Notably, both residues are highly conserved in bacterial Gad [1]. The contribution to EcGadB spectroscopic and catalytic properties by His465, a critical residue for controlling active site access, was previously investigated [5]. In the present work, we carried out detailed biochemical characterization of the EcGadB-Asp86 mutant. However, in order to appreciate the contribution of Asp86 to the catalytic properties of EcGadB, it was necessary to incorporate the mutation Asp86→Asn in the mutant GadB_H465A, thereby avoiding the masking effect of His465 at pH>5.5. Our data show that, unlike wild-type EcGadB and GadB_H465A, the double mutant GadB_D86N¬-H465A, while retaining substrate specificity, is a more robust catalyst in the pH range 7-8 and displays an altered solvent kinetic isotope effect. Hence, GadB_D86N¬-H465A is less sensitive to pH increase during the decarboxylation reaction. We proposed that immobilization of EcGadB can be exploited for GABA synthesis at the industrial level [6]. GABA in turn can be used as precursor of 2-pyrrolidone, an industrial solvent, and of nylon 4. Thus mutant forms of EcGadB less sensitive to pH increase (i.e. > 5.5) are highly desirable. Based on our data, pH is no longer a limiting reaction parameter for GadB_D86N¬-H465A. References [1] De Biase D, Pennacchietti E. (2012) Mol. Microbiol 86: 770-86. [2] Lund P, Tramonti A, De Biase D. (2014) FEMS Microbiol Rev 38: 1091–125. [3] Capitani G, De Biase D, et al. (2003) EMBO J. 22: 4027-4037. [4] Gut H, Pennacchietti E, et al. (2006) EMBO J. 25: 2643-2651. [5] Pennacchietti E, Lammens TM, et al. (2009) J Biol Chem. 284: 31587-96. [6] Lammens TM, De Biase D, et al. (2009) Green Chemistry 11: 1562-67

Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system.

International audienceIn orally acquired bacteria, the ability to counteract extreme acid stress (pH ⩽ 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known "classical" Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved

Periplasmic oxidized-protein repair during copper stress in E. coli: A focus on the metallochaperone CusF

International audienceMethionine residues are particularly sensitive to oxidation by reactive oxygen or chlorine species (ROS/RCS), leading to the appearance of methionine sulfoxide in proteins. This post-translational oxidation can be reversed by omnipresent protein repair pathways involving methionine sulfoxide reductases (Msr). In the periplasm of Escherichia coli , the enzymatic system MsrPQ, whose expression is triggered by the RCS, controls the redox status of methionine residues. Here we report that MsrPQ synthesis is also induced by copper stress via the CusSR two-component system, and that MsrPQ plays a role in copper homeostasis by maintaining the activity of the copper efflux pump, CusCFBA. Genetic and biochemical evidence suggest the metallochaperone CusF is the substrate of MsrPQ and our study reveals that CusF methionines are redox sensitive and can be restored by MsrPQ. Thus, the evolution of a CusSR-dependent synthesis of MsrPQ allows conservation of copper homeostasis under aerobic conditions by maintenance of the reduced state of Met residues in copper-trafficking proteins

Costo sociale annuo della dispepsia funzionale dopo l&apos;eradicazione dell&apos;Helicobacter pylori : risultati di un&apos;indagine in centri di endoscopia digestiva

Objective: The aim of this study was to evaluate socio-demographic and clinical characteristics of dyspeptic patients in Italy after the eradication of Helicobacter pylori infection and to evaluate the impact of this syndrome on daily activities, on loss of production, and, finally, to estimate its cost for both National Health Service and society. Design: An observational, prospective, one-year, multicenter study was conducted from April 1999 to April 2002 under the aegis of the Italian study group of digestive tract motility (GISMAD). The study was based on 577 consecutive functional dyspeptic patients after eradication of H. pylori. Data were collected by a Case Report Form (CRF). Setting: 91 Centres of Digestive Endoscopy (CED), all located in Italy. Results: The mean age of the 577 dyspeptic patients enrolled in the study was 52 yrs.\ub114 (13.3% was younger than 40 yrs old; mostly females: 56.7%). Sixty point seven percent of patients met expenses related with the treatment of dyspepsia. Patients were stratified into four groups according to their prevalent symptoms: ulcer-like (UL), dismotility-like (DL), reflux-like (RL) and 'other'. Annual mean cost estimated for each patient was \u20ac 145.54, with differences between females and males ( \u20ac 168.95 vs \u20ac 114.91 respectively). "RL" patients resulted at the highest cost (\u20ac 204.32). Total costs were divided into health related costs (\u20ac 60.87) and loss of productivity (\u20ac 84.67), the latter being mostly related absences (21%). Conclusions: During a follow-up period of 12 months, in each group there was a clear evidence of both a reduction of negative influence of dyspeptic symptoms on the patients' activities and a decrease of the mean cost for the treatment of dyspepsia. "RL" group presents cost 30% higher when compared with "UL" and "DL" groups, and 90% higher when compared with "other"