Extrinsic factors potassium chloride and glycerol induce thermostability in recombinant anthranilate synthase from Archaeoglobus fulgidus

Thermostable anthranilate synthase from the marine sulfate-reducing hyperthermophile Archaeoglobus fulgidus has been expressed in Escherichia coli, purified, and characterized. The functional enzyme is an alpha(2)beta(2) heterotetrameric complex of molecular mass 150 +/- 15 kDa. It is composed of two TrpE ( 50 kDa) and two TrpG ( 18 kDa) subunits. The extrinsic factors glycerol (25%) and potassium chloride ( 2 M) stabilized the recombinant enzyme against thermal inactivation. In the presence of these extrinsic factors, the enzyme was highly thermostable, exhibiting a half-life of thermal inactivation of about 1 h at 85 degreesC. The kinetic constants for the enzyme under these conditions were: K-m (chorismate) 84 muM, K-m (glutamine) 7.0 mM, k(cat) 0.25 s(-1), and pH optimum 8.0. The enzyme was competitively, though non-cooperatively, inhibited by tryptophan.