期刊
MOLECULAR AND CELLULAR BIOCHEMISTRY
卷 400, 期 1-2, 页码 9-15出版社
SPRINGER
DOI: 10.1007/s11010-014-2256-3
关键词
Anthranilate synthase; Chorismate-utilizing enzyme; Fused enzyme; Streptomyces venezuelae
类别
资金
- U.S. National Institutes of Health (NIH) RCMI [2G12RR003048-18]
- MBRS-SCORE [3S06GM0816-33S1, 1SC3GM083752]
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 K-m with respect to substrates chorismate and glutamine of 8.2 +/- A 0.2 mu M and 0.84 +/- A 0.05 mM, respectively, and a catalytic rate constant k (cat) of 0.57 +/- A 0.02 s(-1) at 30 A degrees 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 +/- A 0.1 mu M) 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.
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