Purification and some properties of riboflavin synthetase from Bacillus stearothermophilus ATCC 8005

Abstract

A riboflavin synthetase was purified 51-fold from a thermophilic organism, Bacillus stearothermophilus ATCC 8005, that grew at 40 to 72 degrees C. Some of the properties of the enzyme are: (i) its temperature optimum was 95 degrees C, and the activity was negligible below 40 degrees C; (ii) the Arrhenius plot of the initial reaction rates was concave upward, with a break at 65 degrees C, and the apparent activation energies below and above 65 degrees C were 4.2 X 10(4) and 6.7 X 10(4) J/mol, respectively; (iii) the enzyme was fairly stable up to 60 degrees C without 6,7-dimethyl-8-ribityllumazine; this substance protected the enzyme from inactivation above 60 to 97 degrees C; (iv) the pH range for stability was 6.0 to 10.0 at 26 degrees C and 6.3 to 7.6 at 55 degrees C; (v) the enzyme was highly resistant at 26 degrees C to denaturation in 8 M urea, but the tolerance was extremely low at 55 degrees C; (vi) its molecular weight was estimated at 45,000; (vii) the Km for 6,7-dimethyl-8-ribityllumazine was 23 micrometer at 55 degrees C and 29 micrometer at 75 degrees C; (viii) its pH optimum was 6.7 to 7.2; (ix) 6-methyl-7-hydroxy-8-ribityllumazine was a competitive inhibitor (Ki = 0.18 micrometer); (x) the activity was sensitive to heavy-metal ions and thiol reagents; (xi) the enzyme did not require cofactor or a carbon donor; and (xii) the molar ratio of 6,7-dimethyl-8-ribityllumazine consumption to riboflavin formation was 2 throughout the entire reaction. Properties i through vi distinguish this enzyme from riboflavin synthetases purified by other investigators from mesophilic organisms, Ashbya gossypii, Eremothecium ashbyii, Escherichia coli, yeast, and spinach.