caa a22 4500 445321903 CHVBK 20180317142706.0 cr unu---uuuuu 170323e20111001xx s 000 0 eng 10.1007/s10534-011-9442-5 doi (NATIONALLICENCE)springer-10.1007/s10534-011-9442-5 Nitrate reduction associated with respiration in Sinorhizobium meliloti 2011 is performed by a membrane-bound molybdoenzyme [Elektronische Daten] [Felix Ferroni, María Rivas, Alberto Rizzi, María Lucca, Nora Perotti, Carlos Brondino] The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K m=97±11μM, V=9.4±0.5μMmin−1, and k cat=12.1±0.6s−1) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI. Springer Science+Business Media, LLC., 2011 Sinorhizobium meliloti nationallicence Membrane-bound nitrate reductase nationallicence Nitrogen metabolism nationallicence Kinetic studies nationallicence Ferroni Felix Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, S3000ZAA, Santa Fe, Argentina aut Rivas María REQUIMTE/CQFB, Departamento de Quimica, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal aut Rizzi Alberto Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, S3000ZAA, Santa Fe, Argentina aut Lucca María Planta Piloto de Procesos Industriales (PROIMI)-CONICET, Av. Belgrano y Caseros, 4000, Tucumán, Argentina aut Perotti Nora Planta Piloto de Procesos Industriales (PROIMI)-CONICET, Av. Belgrano y Caseros, 4000, Tucumán, Argentina aut Brondino Carlos Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, S3000ZAA, Santa Fe, Argentina aut BioMetals Springer Netherlands 24/5(2011-10-01), 891-902 0966-0844 24:5<891 2011 24 10534 https://doi.org/10.1007/s10534-011-9442-5 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10534-011-9442-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ferroni Felix Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, S3000ZAA, Santa Fe, Argentina aut NATIONALLICENCE 700 1- Rivas María REQUIMTE/CQFB, Departamento de Quimica, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal aut NATIONALLICENCE 700 1- Rizzi Alberto Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, S3000ZAA, Santa Fe, Argentina aut NATIONALLICENCE 700 1- Lucca María Planta Piloto de Procesos Industriales (PROIMI)-CONICET, Av. Belgrano y Caseros, 4000, Tucumán, Argentina aut NATIONALLICENCE 700 1- Perotti Nora Planta Piloto de Procesos Industriales (PROIMI)-CONICET, Av. Belgrano y Caseros, 4000, Tucumán, Argentina aut NATIONALLICENCE 700 1- Brondino Carlos Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, S3000ZAA, Santa Fe, Argentina aut NATIONALLICENCE 773 0- BioMetals Springer Netherlands 24/5(2011-10-01), 891-902 0966-0844 24:5<891 2011 24 10534 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer