caa a22 4500 606236368 CHVBK 20210128101247.0 cr unu---uuuuu 210128e20150201xx s 000 0 eng 10.1007/s11010-014-2256-3 doi (NATIONALLICENCE)springer-10.1007/s11010-014-2256-3 The fused anthranilate synthase from Streptomyces venezuelae functions as a monomer [Elektronische Daten] [Meseret Ashenafi, Prasad Reddy, James Parsons, W. Byrnes] Recently, we showed that the fused chorismate-utilizing enzyme from the antibiotic-producing soil bacterium Streptomyces venezuelae is an anthranilate synthase (designated SvAS), not a 2-amino-2-deoxyisochorismate (ADIC) synthase, as was predicted based on its amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE (an ADIC synthase). Here, we report the characterization of SvAS using steady-state kinetics, gel filtration chromatography, and laser light scattering. The recombinant His-tagged enzyme has Michaelis constants Km with respect to substrates chorismate and glutamine of 8.2±0.2μM and 0.84±0.05mM, respectively, and a catalytic rate constant k cat of 0.57±0.02s−1 at 30°C. Unlike most other anthranilate synthases, SvAS does not utilize ammonia as a substrate. The enzyme is competitively but non-cooperatively inhibited by tryptophan (K i=11.1±0.1μM) and is active as a monomer. The finding that SvAS is a monomer jibes with the variety of association modes that have been observed for anthranilate synthases from different microorganisms, and it identifies the enzyme's minimal functional unit as a single TrpE-TrpG pair. Springer Science+Business Media New York, 2014 Anthranilate synthase nationallicence Chorismate-utilizing enzyme nationallicence Fused enzyme nationallicence Streptomyces venezuelae nationallicence Ashenafi Meseret Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520W Street, NW, 20059, Washington, DC, USA aut Reddy Prasad Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Stop 1070, 20899, Gaithersburg, MD, USA aut Parsons James University of Maryland, Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, 20850, Rockville, MD, USA aut Byrnes W. Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520W Street, NW, 20059, Washington, DC, USA aut Molecular and Cellular Biochemistry Springer US; http://www.springer-ny.com 400/1-2(2015-02-01), 9-15 0300-8177 400:1-2<9 2015 400 11010 https://doi.org/10.1007/s11010-014-2256-3 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s11010-014-2256-3 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ashenafi Meseret Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520W Street, NW, 20059, Washington, DC, USA aut NATIONALLICENCE 700 1- Reddy Prasad Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Stop 1070, 20899, Gaithersburg, MD, USA aut NATIONALLICENCE 700 1- Parsons James University of Maryland, Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, 20850, Rockville, MD, USA aut NATIONALLICENCE 700 1- Byrnes W. Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520W Street, NW, 20059, Washington, DC, USA aut NATIONALLICENCE 773 0- Molecular and Cellular Biochemistry Springer US; http://www.springer-ny.com 400/1-2(2015-02-01), 9-15 0300-8177 400:1-2<9 2015 400 11010