Regulation of the Tryptophan Synthetic Enzymes in Clostridium butyricum

Abstract

Experiments concerned with the regulation of the tryptophan synthetic enzymes in anaerobes were carried out with a strain of Clostridium butyricum. Enzyme activities for four of the five synthetic reactions were readily detected in wild-type cells grown in minimal medium. The enzymes mediating reactions 3, 4, and 5 were derepressed 4to 20-fold, and the data suggest that these enzymes are coordinately controlled in this anaerobe. The first enzyme of the pathway, anthranilate synthetase, could be derepressed approximately 90-fold under these conditions, suggesting that this enzyme is semicoordinately controlled. Mutants resistant to 5-methyl tryptophan were isolated, and two of these were selected for further analysis. Both mutants retained high constitutive levels of the tryptophan synthetic enzymes even in the presence of repressing concentrations of tryptophan. The anthranilate synthetase from one mutant was more sensitive to feedback inhibition by tryptophan than the enzyme from wild-type cells. The enzyme from the second mutant was comparatively resistant to feedback inhibition by tryptophan. Neither strain excreted tryptophan into the culture fluid. Tryptophan inhibits anthranilate synthetase from wild-type cells noncompetitively with respect to chorismate and uncompetitively with respect to glutamine. The Michaelis constants calculated for chorismate and glutamine are 7.6 x 10-5 M and 6.7 x 10-4 M, respectively. The molecular weights of the enzymes estimated by zonal centrifugation in sucrose and by gel filtration ranged from 24,000 to 89,000. With the possible exception of a tryptophan synthetase complex, there was no evidence for the existence of other enzyme aggregates. The data indicate that tryptophan synthesis is regulated by repression control of the relevant enzymes and by feedback inhibition of anthranilate synthetase. That this enzyme system more closely resembles that found in Bacillus than that found in enteric bacteria is discussed.

Regulation of a Ligand-Mediated Association-Dissociation System of Anthranilate Synthesis in Clostridium butyricum

“…The strain of C. butyricum (American Type Culture Collection, Rockville, Md., no. 6014) and the two 5-methyltryptophan-resistant derivatives, mutant mtr-4 and mutant mtr-8, used in this study have been described previously (3). The media and methods of cultivation and isolation of the 5-methyltryptophan-resistant strains have also been described previously (3).…”

“…Crude extracts were prepared from late logarithmic phase cells grown in minimal medium. The procedure used in the prepara-tion of these extracts was the same as previously reported (3). Except when otherwise stated, the extract buffer contained 0.05 M potassium phosphate (pH 7.5), 0.1 mM ethylenediaminetetraacetic acid, 6 mM #-mercaptoethanol, 0.5 M KCl, and 30% glycerol (vol/vol), (PEMKG buffer).…”

“…One unit of enzyme activity is expressed as that amount which catalyzes the utilization of Anthranilate synthetase activity was measured by using either glutamine or ammonia as substrates. The details of this assay using glutamine as the amide donor have been described previously (3). The ammonia-dependent reaction was performed in exactly the same manner except that the incubation mixture contained 100 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 9.0), 20 mM MgCl2, 100 mM NH4Cl, and 2 mM chorismic acid.…”

“…The tryptophan enzymes in two of the mutants selected for further study, mutant mtr-4 and mutant mtr-8, remained at elevated levels when these mutants were grown in minimal medium containing 20 ,ug of L-tryptophan per ml, a concentration more than sufficient to completely repress synthesis of the tryptophan syn-thetic enzymes in the parental wild type (3). We also demonstrated that the anthranilate synthetase from mutant mtr-4 was still sensitive to feedback inhibition by L-tryptophan, whereas the enzyme from mutant mtr-8 was comparatively resistant to feedback inhibition (3).…”

Tryptophan Metabolism in Ochromonas malhamensis

“…The ability of microorganisms, unlike mammals and higher animals, to synthesize tryptophan from a simple salt media containing a utilizable source of carbon has been observed (8). End product repression, precursor induction, and constitutive levels of enzyme production have been all reported within the pathway (1, 19, 24). I n contrast to bacterial cells, data on individual enzymatic reactions of the metabolic pathway of tryptophan in protozoans have been somewhat meager.…”