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.

Fig. 1

The Anthranilate Synthase-Catalyzed Reaction

Fig. 2. Tryptophan Inhibition of SvAS Activity

Chorismate concentration was varied in the presence of the following fixed concentrations of tryptophan: none (closed circles), 2.5 μM (open circles), 5 μM (closed triangles), 10 μM (open triangles) and 20 μM (closed squares). See Materials and Methods for further details

Fig. 3. Determination of the Size of SvAS by Gel Filtration Chromatography

The position of the SvAS protein is indicated with a solid square. Albumin was run twice for this experiment. Inset (bottom panel), the results were obtained by removing an aliquot of the active fraction corresponding to a molecular mass of 67 kDa from the gel filtration column, adding it to the assay cuvette, and immediately measuring the change in fluorescence intensity (in counts per second) over time (in sec). The fact that no lag is observed at the beginning of the assay suggests that assembly of the monomers into higher-order structures (dimers) is not required for activity. See Materials and Methods for details

Fig. 3. Determination of the Size of SvAS by Gel Filtration Chromatography

The position of the SvAS protein is indicated with a solid square. Albumin was run twice for this experiment. Inset (bottom panel), the results were obtained by removing an aliquot of the active fraction corresponding to a molecular mass of 67 kDa from the gel filtration column, adding it to the assay cuvette, and immediately measuring the change in fluorescence intensity (in counts per second) over time (in sec). The fact that no lag is observed at the beginning of the assay suggests that assembly of the monomers into higher-order structures (dimers) is not required for activity. See Materials and Methods for details

Fig. 4. Variable Association Modes for Tetrameric Anthranilate Synthases

Stereo ribbon diagrams of: A, the S. enterica (typhimurium) heterotetramer showing that the TrpE-TrpG and TrpE’-TrpG’ dimers associate via TrpE subunits; B, the S. solfataricus heterotetramer showing that the TrpE-TrpG and TrpE’-TrpG’ dimers associate via TrpG subunits. Courtesy of Michael J. Eck. Adapted by permission from Macmillan Publishers Ltd.: Nature Structural Biology [18], copyright 2001