Structural and mutational characterization of the catalytic A-module of the mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii

Alginate is a family of linear copolymers of (1→4)-linked β-d-mannuronic acid and its C-5 epimer α-l-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-Å resolution. AlgE4A folds into a right-handed parallel β-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The β-helix is composed of four parallel β-sheets, comprising 12 complete turns, and has an amphipathic α-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp(152), an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr(149), His(154), and Asp(178) as being essential for activity. Tyr(149) probably acts as the proton acceptor, whereas His(154) is the proton donor in the epimerization reaction

Guluronic acid content as a factor affecting turbidity removal potential of alginate

Alginates are natural polymers composed of mannuronic and guluronic acid residues. They are currently extracted from brown algae; however, alginate can also be synthesized by some species of Azotobacter and Pseudomonas. Alginates with different proportion of mannuronic and guluronic acids are known to have different characteristics and form gels at different extents in the presence of calcium ions. The aim of this work was to investigate the usefulness of alginate as a non-toxic coagulant used in purification of drinking water. This study utilized alginates from Azotobacter vinelandii having different guluronic acid levels. These were obtained partly by changing the cultivation parameters, partly by epimerizing a purified alginate sample in vitro using the A. vinelandii mannuronan C-5 epimerase AlgE1. The different alginates were then used for coagulation together with calcium. The results showed that turbidity removal capability was dependent on the content of guluronic acid residues. For the best performing samples, the turbidity decreased from 10 NTU to 1 NTU by the use of only 2 mg/L of alginate and 1.5 mM of calcium chloride

Genome Sequence of Azotobacter vinelandii , an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes

Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.Fil: Setubal, João C.. Virginia Polytechnic Institute; Estados UnidosFil: Dos Santos, Patricia Carolina. Wake Forest University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Goldman, Barry S.. Monsanto Company; Estados UnidosFil: Ertesvag, Helga. Norwegian University of Science and Technology; NoruegaFil: Espin, Guadelupe. Universidad Nacional Autónoma de México; MéxicoFil: Rubio, Luis M.. Instituto Imdea Energia; EspañaFil: Valla, Svein. Norwegian University of Science and Technology; NoruegaFil: Almeida, Nalvo F.. Virginia Polytechnic Institute; Estados Unidos. Universidade Federal do Mato Grosso do Sul; BrasilFil: Balasubramanian, Divya. Hiram College; Estados UnidosFil: Cromes, Lindsey. Hiram College; Estados UnidosFil: Curatti, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Fundación para Investigaciones Biológicas Aplicadas. Centro de Estudios de Biodiversidad y Biotecnología; ArgentinaFil: Du, Zijin. Monsanto Company; Estados UnidosFil: Godsy, Eric. Monsanto Company; Estados UnidosFil: Goodner, Brad. Hiram College; Estados UnidosFil: Hellner Burris, Kaitlyn. Hiram College; Estados UnidosFil: Hernandez, José A.. Midwestern University; Estados UnidosFil: Houmiel, Katherine. Seattle Pacific University; Estados UnidosFil: Imperial, Juan. Centro de Biotecnologia y Genomica de Plantas; EspañaFil: Kennedy, Christina. University of Arizona; Estados UnidosFil: Larson, Timothy J.. Virginia Polytechnic Institute; Estados UnidosFil: Latreille, Phil. Monsanto Company; Estados UnidosFil: Ligon, Lauren S.. Virginia Polytechnic Institute; Estados UnidosFil: Lu, Jing. Monsanto Company; Estados UnidosFil: Mærk, Mali. Norwegian University of Science and Technology; NoruegaFil: Miller, Nancy M.. Monsanto Company; Estados UnidosFil: Norton, Stacie. Monsanto Company; Estados UnidosFil: O'Carroll, Ina P.. Virginia Polytechnic Institute; Estados UnidosFil: Paulsen, Ian. Macquarie University; AustraliaFil: Raulfs, Estella C.. Virginia Polytechnic Institute; Estados UnidosFil: Roemer, Rebecca. Hiram College; Estados Unido