Biochemical characterization of a variant of Escherichia coli glutamate decarboxylase with improved GABA production at alkaline pH.

The Federation of European Microbiological Societies (FEMS) and the Spanish Society for Microbiology (SEM) have joined​ forces to give you the best of microbiology. Come to the modern and friendly Mediterranean city Valencia, meet fellow microbiologists and update your knowledge with the state of the art on antimicrobial resistance and infections, food microbiology, sustainability, climate change and many more topics. Key disciplines including molecular approaches, biodiversity, bioremediation, eukaryotic microbes, virology and others will be examined in order to advance our understanding of current and future challenges.​​

Glutamate decarboxylase in bacteria

gamma -Aminobutyrate (GABA) is a non-proteinaceous amino acid which plays different roles in the living systems. GABA is biochemically produced by the irreversible a-decarboxylation of l-glutamate catalysed by glutamate decarboxylase (Gad), a widespread enzyme distributed among eukaryotes and prokaryotes. Structural features of the plant, mammalian and bacterial Gads are reported in the literature. In pathogenic bacteria, Gad activity has been linked to protection from acid stress, but the possibility cannot be excluded that it might perform other physiological roles. Indeed, the screening of lactic acid bacteria (LAB) based on their capacity to synthesize GABA opens a new perspective for the production of GABA containing dairy foods. The aim of this chapter is to provide an overview of the best characterized Gad enzymes in pathogenic and non-pathogenic bacteria, along with a description of the potential use of Gad as a source of GABA in functional foods and in biotechnological products

Acid survival mechanisms in neutralophilic bacteria

Numerous commensal and pathogenic Gram-negative and Gram-positive bacteria are referred to as neutralophiles because they grow best at pH levels close to neutrality. Thus, exposure to harsh-to-mild acidic environments, such as those encountered in the digestive tract of animal hosts, in the phagosome of macrophages, in fermented foods, but also in the soil or in acid mine drainage, is a rather common encounter for neutralophiles during their life cycle. As a result, it is not surprising that most of them have evolved sophisticated molecular mechanisms to cope with low pH. These protective mechanisms provide neutralophiles with the ability to sense acid pH and keep under control the intracellular acidification of the cytoplasm, thus avoiding protons from reaching such harmful levels as to compromise cellular vitality, which relies on the proper functioning of many biological macromolecules at pH levels near neutrality. The aim of this chapter is to provide an overview of the most commonly employed, and best characterized, molecular systems in a number of Gram-positive and Gram-negative bacteria. How they work inside the cell and how their activity can be linked to virulence are highlighted. The biochemistry and distribution of the glutamate-dependent acid resistance system among orally acquired bacteria are described in some detail

Biochemical Insights into Glutamate decarboxylase from Mycobacterium tuberculosis

Background: Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen which establishes a life-long infection by residing within macrophages and halting the phagosome-lysosome fusion. In response to IFN-γ, activated macrophages deliver the pathogen to the acidic compartment of the phagolysosome where pH fluctuates between 4.5-5.0. M. tuberculosis possesses a gene coding for a putative glutamate decarboxylase (MtGadB), the role of which could be to overcome the acidity of the phagolysosome or, as part of the GABA-shunt, to compensate for the lack of the α-ketoglutarate dehydrogenase activity in M. tuberculosis. Objectives: With the current study we investigated the biochemical properties of MtGadB to unveil its physiological role in M. tuberculosis. Methods: MtGadB (carrying an N-terminal His6-tag) was expressed and purified in Escherichia coli via affinity chromatography and its pH-dependent spectroscopic and enzymatic properties were investigated and compared with those of the extensively studied E. coli homologue (EcGadB). Results: We developed a successful protocol for the expression and purification of His6-MtGadB. We found that the His6-tag negatively influences the oligomeric structure and the enzymatic activity of MtGadB, in agreement with the structural role of the N-terminus in EcGadB. UV-Visible and fluorescence spectroscopic studies showed that MtGadB shares with the EcGadB counterpart many biochemical similarities. This is in line with the full conservation of GAD-signature residues observed in MtGadB. Given the possible involvement of MtGadB in the GABA-shunt, this work sets the basis for a better understanding of the M. tuberculosis GABA-shunt enzymes

Microbial stress meeting: from systems to molecules and back

International audienceThe 4th Microbial Stress Meeting: from Systems to Moleculesand Back was held in April 2018 in Kinsale, Ireland. The meeting covered five main topics: 1. Stress at the systems and structural level; 2. Responses to osmotic and acid stress; 3. Stress responses in single cells; 4. Stress in host-pathogen interactions; and 5. Biotechnological optimisation of microorganisms through engineering and evolution, over three days. Almost 130 delegates, from 24 countries and both the industrial and academic sectors, attended the meeting, presenting 9 lectures, 28 short talks and 52 posters. The meeting showcased the diverse and rapid advancements in microbial stress research, from the single cell level to mixed populations. In this report, a summary of the highlights from the meeting is presented

Analoghi e derivati di amminoacidi dicarbossilici come antibatterici

Questa invenzione si colloca nell’ambito scientifico della lotta alle resistenze multiple sviluppate dai batteri verso gli antibiotici attualmente in commercio. L’invenzione stravolge il concetto che gli antibiotici possano avere solo una certa natura chimica, intorno alla quale si è sviluppato un mercato che non riesce più ad essere innovativo, e che ha prodotto molecole chimicamente imparentate tra loro. Con questa invenzione inoltre, si vuole portare all’attenzione di soggetti interessati l’efficacia di molecole che agiscono sul metabolismo centrale nei batteri, e per le quali allo stato attuale e non ci sono indicazioni di tossicità sugli animali che ne precluderebbero l’impiego sistemico/topico

Biochemical characterization of Glutamate decarboxylase from Mycobacterium tuberculosis

During the infectious process Mycobacterium tuberculosis typically encounters an acid stress in the human macrophage phago(lyso)some (pH 4.5-6.0) [1]. Nonetheless, how M. tuberculosis survives this challenge is little understood. We have characterized M. tuberculosis glutamate decarboxylase (MtGadB). GadB catalyzes the decarboxylation of L-glutamate to yield GABA, while consuming one H+/catalytic cycle. It is a key enzyme in the potent glutamate-dependent acid resistance system found in many neutralophilic bacteria [2]. So far the only role proposed for MtGadB is to fill the interrupted TCA cycle in M. tuberculosis [3]. However, the perfect conservation of all key residues known as “GAD signature” [2] suggests that MtGadB may also play a role in protecting M. tuberculosis from acid stress. MtgadB was cloned into two different vectors, for expression with/without a His-tag. The best conditions for expression were screened by a rapid colorimetric assay. MtGadB was purified using either an ion exchange or an affinity chromatography. The oligomeric state was assessed by gel filtration chromatography. The pH-dependent activity and titration curves were compared with those of the E. coli and Brucella microti GadBs that we have characterized [4]. This study sets the basis to uncover the role of GadB in M. tuberculosis biology, and its potential as a “druggable” target. [1] Vandal et al. (2009) J Bacteriol. 191:4714-4721. [2] De Biase and Pennacchietti (2012) Mol. Microbiol 86:770-786. [3] Tian et al. (2005) PNAS 102:10670-10675. [4] Grassini et al. (2015) FEBS Open Bio 5:209-18

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

Enzymatic kinetic resolution of desmethylphosphinothricin indicates that phosphinic group is a bioisostere of carboxyl group

Escherichia coli glutamate decarboxylase (EcGadB), a pyridoxal 5’-phosphate (PLP)-dependent enzyme, is highly specific for L-glutamate and was demonstrated to be effectively immobilised for the production of γ-aminobutyric acid (GABA), its decarboxylation product. Herein we show that EcGadB quantitatively decarboxylates the L-isomer of D,L-2-amino-4-(hydroxyphosphinyl)butyric acid (D,L-Glu-γ-PH), a phosphinic analogue of glutamate containing C-P-H bonds. This yields 3-aminopropylphosphinic acid (GABA-PH), a known GABAB receptor agonist and provides previously unknown D-Glu-γ-PH, allowing us to demonstrate that L-Glu-γ-PH, but not D-Glu-γ-PH, is responsible for D,L-Glu-γ-PH antibacterial activity. Furthermore, using GABase, a preparation of GABA-transaminase and succinic semialdehyde dehydrogenase, we show that GABA-PH is converted to 3-(hydroxyphosphinyl)propionic acid (Succinate-PH). Hence, PLP-dependent and NADP+-dependent enzymes are herein shown to recognise and metabolise phosphinic compounds, leaving unaffected the P-H bond. We therefore suggest that the phosphinic group is a bioisostere of the carboxyl group and the metabolic transformations of phosphinic compounds may offer a ground for prodrug design