title: The fused anthranilate synthase from Streptomyces venezuelae functions as a monomer.
abstract: 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 ± 0.2 μM and 0.84 ± 0.05 mM, respectively, and a catalytic rate constant k cat of 0.57 ± 0.02 s(-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 ± 0.1 μM) 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520 W Street, NW, Washington, DC, 20059, USA.
authors.forename: Meseret
authors.initials: M
authors.lastname: Ashenafi
authors.forename: Prasad T
authors.initials: PT
authors.lastname: Reddy
authors.forename: James F
authors.initials: JF
authors.lastname: Parsons
authors.forename: W Malcolm
authors.initials: WM
authors.lastname: Byrnes
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000878:Anthranilate Synthase / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D007700:Kinetics; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D014364:Tryptophan
chemical_list: D014364:Tryptophan; D000878:Anthranilate Synthase