Functional and structural studies on chlorite dismutase from Nitrospira defluvii

Das Enzym Chloritdismutase (Cld), eine Oxidoreduktase, katalysiert den Abbau von Chlorit zu Chlorid und molekularem Sauerstoff.Es ist hiermit eines von drei bekannten Enzymen, welches die Bildung einer molekularen Sauerstoffbindung als primäre Funtion durchführt. Ursprünglich waren Clds nur aus Perchlorat-respirierenden Bakterien (PCRB) bekannt, wo sie die Selbstvergiftung der Bakterien durch Abbau von cytotoxischem Chlorit, welches als Produkt der (Per)chloratreduktion entsteht, verhindern. Belastung des Trinkwassers durch anthropogenes (Per)chlorat wurde in den letzten 50 Jahren zu einer zunehmenden Bedrohung der öffentlichen Gesundheit in den Vereinigten Staaten. Bioremediation wäre ein geeignetes Mittel zur Entgiftung. Im Jahr 2008 wurden Cld - Homologe nicht nur in PCRBs, sondern in einer Vielzahl von Bakterien- und Archaeenstämmen entdeckt. Sie bilden nun die Chloritdismutase-Überfamilie, deren Mitglieder CFPs (Chlorite dismutase family proteins) genannt werden. Nur ein kleiner Anteil dieser katalysieren den Abbau von Chlorit; die Funktion der meisten ist noch unbekannt, auch wenn z.B. das CFP aus L. monocytogenes essentiell für das Pathogen ist und damit einen Ansatzpunkt für Medikamentenentwicklung bieten könnte. Um Chloritdismutaseaktivität in sequenzbasiert in CFPs vorhersagen zu können, wurden konservierte und teilweise konservierte Aminosäurereste identifiziert und als mögliche Signaturreste für Cld-Aktivität vorgeschlagen. In dieser Arbeit wurden diese Aminosäurereste in der Cld von Candidatus Nitrospira defluvii, eines wichtigen nitrifizierenden Klärbakteriums durch ortsspezifische Mutagenese ausgetauscht. Die Mutanten wurden durch biochemische Enzymaktivitätsmessungen und Röntgenkristallografie charakterisiert. Die wichtige Rolle des zuvor identifizierten Aminosäurerests Arginin 173 wurde nochmals bestätigt und erweitert. Für elf Mutanten und eine Cobalt Protoporphyrin IX Variante wurden Fliessgleichgewichtskinetiken gemessen und die Struktur vierer Mutanten sowie der apo-Variante von NdCld wurde durch Röntgenkristallografie gelöst. Des weiteren wurde die Relevanz elektostatischer bzw. hydrophober Interaktionen, welche zur Stabilität der dimeren Chloritdismutase von Nitrobacter winogradskyi beitragen, untersucht.The enzyme chlorite dismutase (Cld), a heme b binding oxidoreductase, catalyzes the degradation of chlorite to chloride and molecular oxygen. It is therefore one of three known enzymes catalyzing the formation of a molecular O-O bond as its primary function. Clds were originally thought to be present in (per)chlorate respiring bacteria (PCRB), where they prevent self-toxification of the bacteria by degradation of cytotoxic chlorite, which is formed as a product of (per)chlorate reduction. Because freshwater pollution by anthropogenic perchlorate has become a serious public health threat in the United States, bioremediation would present an efficient method of detoxification. In 2008, many homologues to Cld were discovered not only in PCRBs, but in a large number of diverse bacterial and archaeal phylae. They comprise now a chlorite dismutase superfamily, whose members are called CFPs (Chlorite dismutase family proteins). Only a small subset of those were found to degrade chlorite, the function of the large moiety remains unknown, though e.g. the CFP from L. monocytogenes was shown to be essential for the organism, making the protein a suitable drug target. To predict chlorite dismutase activity in CFPs on a sequence basis, conserved and partially conserved residues were previously identified and suggested as possible signature residues for Cld activity. In this work, these residues in the active site of the Cld from Candidatus Nitrospira defluvii (NdCld), a key nitrifier in wastewater treatment, were exchanged by site-directed mutagenesis and the mutants characterized by biochemical enzyme activity assays (steady-state kinetics) and structurally by X-ray crystallography. The role of the previously identified catalytic residue arginine 173 was confirmed and steady-state kinetics measured for eleven mutants and a cobalt protoporphyrine IX variant of NdCld. The structure of four mutants and apo-NdCld was solved by X-ray crystallography. In addition, the respective contributions of electrostatic and hydrophobic interactions to the stability of the dimer interface in the chlorite dismutase from Nitrobacter winogradskyi were experimentally determined

A combination of chitooligosaccharide and lipochitooligosaccharide recognition promotes arbuscular mycorrhizal associations in Medicago truncatula.

Plants associate with beneficial arbuscular mycorrhizal fungi facilitating nutrient acquisition. Arbuscular mycorrhizal fungi produce chitooligosaccharides (COs) and lipo-chitooligosaccharides (LCOs), that promote symbiosis signalling with resultant oscillations in nuclear-associated calcium. The activation of symbiosis signalling must be balanced with activation of immunity signalling, which in fungal interactions is promoted by COs resulting from the chitinaceous fungal cell wall. Here we demonstrate that COs ranging from CO4-CO8 can induce symbiosis signalling in Medicago truncatula. CO perception is a function of the receptor-like kinases MtCERK1 and LYR4, that activate both immunity and symbiosis signalling. A combination of LCOs and COs act synergistically to enhance symbiosis signalling and suppress immunity signalling and receptors involved in both CO and LCO perception are necessary for mycorrhizal establishment. We conclude that LCOs, when present in a mix with COs, drive a symbiotic outcome and this mix of signals is essential for arbuscular mycorrhizal establishment

Manipulating Conserved Heme Cavity Residues of Chlorite Dismutase: Effect on Structure, Redox Chemistry and Reactivity

Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from \u201cCandidatus Nitrospira defluvii\u201d (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV\u2013vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an O\u2550O bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E\ub0\u2032 = 12113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation

An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

International audienc

A combination of chitooligosaccharide and lipochitooligosaccharide recognition promotes arbuscular mycorrhizal associations in Medicago truncatula

Abstract: Plants associate with beneficial arbuscular mycorrhizal fungi facilitating nutrient acquisition. Arbuscular mycorrhizal fungi produce chitooligosaccharides (COs) and lipo-chitooligosaccharides (LCOs), that promote symbiosis signalling with resultant oscillations in nuclear-associated calcium. The activation of symbiosis signalling must be balanced with activation of immunity signalling, which in fungal interactions is promoted by COs resulting from the chitinaceous fungal cell wall. Here we demonstrate that COs ranging from CO4-CO8 can induce symbiosis signalling in Medicago truncatula. CO perception is a function of the receptor-like kinases MtCERK1 and LYR4, that activate both immunity and symbiosis signalling. A combination of LCOs and COs act synergistically to enhance symbiosis signalling and suppress immunity signalling and receptors involved in both CO and LCO perception are necessary for mycorrhizal establishment. We conclude that LCOs, when present in a mix with COs, drive a symbiotic outcome and this mix of signals is essential for arbuscular mycorrhizal establishment

Receptor-mediated chitin perception in legume roots is functionally separable from Nod factor perception

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Structural signatures in EPR3 define a unique class of plant carbohydrate receptors

Receptor-mediated perception of surface-exposed carbohydrates like lipo- and exo-polysaccharides (EPS) is important for non-self recognition and responses to microbial associated molecular patterns in mammals and plants. In legumes, EPS are monitored and can either block or promote symbiosis with rhizobia depending on their molecular composition. To establish a deeper understanding of receptors involved in EPS recognition, we determined the structure of the Lotus japonicus (Lotus) exopolysaccharide receptor 3 (EPR3) ectodomain. EPR3 forms a compact structure built of three putative carbohydrate-binding modules (M1, M2 and LysM3). M1 and M2 have unique βαββ and βαβ folds that have not previously been observed in carbohydrate binding proteins, while LysM3 has a canonical βααβ fold. We demonstrate that this configuration is a structural signature for a ubiquitous class of receptors in the plant kingdom. We show that EPR3 is promiscuous, suggesting that plants can monitor complex microbial communities though this class of receptors

A combination of chitooligosaccharide and lipochitooligosaccharide recognition promotes arbuscular mycorrhizal associations in Medicago truncatula.

Plants associate with beneficial arbuscular mycorrhizal fungi facilitating nutrient acquisition. Arbuscular mycorrhizal fungi produce chitooligosaccharides (COs) and lipo-chitooligosaccharides (LCOs), that promote symbiosis signalling with resultant oscillations in nuclear-associated calcium. The activation of symbiosis signalling must be balanced with activation of immunity signalling, which in fungal interactions is promoted by COs resulting from the chitinaceous fungal cell wall. Here we demonstrate that COs ranging from CO4-CO8 can induce symbiosis signalling in Medicago truncatula. CO perception is a function of the receptor-like kinases MtCERK1 and LYR4, that activate both immunity and symbiosis signalling. A combination of LCOs and COs act synergistically to enhance symbiosis signalling and suppress immunity signalling and receptors involved in both CO and LCO perception are necessary for mycorrhizal establishment. We conclude that LCOs, when present in a mix with COs, drive a symbiotic outcome and this mix of signals is essential for arbuscular mycorrhizal establishment