PURIFICATION AND CHARACTERIZATION OF COENZYME F-390 SYNTHETASE FROM METHANOBACTERIUM-THERMOAUTOTROPHICUM (STRAIN DELTA-H)

Coenzyme F-390 synthetase catalyzes the formation of 8-hydroxyadenylyl ated-coenzyme F-420 (coenzyme F-390-A) from coenzyme F-420 and ATP in some methanogenic Archaea. The presence of coenzyme F-390 was found wh en these organisms were exposed to oxygen. To get more insight into th e defined function of coenzyme F-390 the coenzyme F-390 synthetase fro m Methanobacterium thermoautrophicum was purified from a cell-free ext ract and its catalytic properties were determined. The synthetase was purified 150-fold to a specific activity of 0.45 mu mol . min(-1) . mg protein(-1). The enzyme consisted of one polypeptide of approximately 51 kDa. The isolated enzyme showed a tendency to aggregate into dimer s and tetramers upon concentration. Co-elution during purification of GTP-dependent coenzyme F-390 synthetase activity suggested that the sy nthetase is also capable of 8-hydroxyguanylylated-coenzyme F-420 (coen zyme F-390-G) formation. Initial-velocity measurements of the two-subs trate reaction showed that the enzyme kinetics for the coenzyme F-390 synthetase reaction proceeded by a ternary-complex mechanism. The coen zyme F-390 synthetase displayed a K-m for coenzyme F-420 of 39 mu M an d a K-m for ATP of 1.7 mM. In contrast to the enzyme in the cell-free extract, the isolated enzyme was active under aerobic and anaerobic co nditions. Treatment with air was not required to obtain the enzyme in an active form. However, 1,5-dihydro-coenzyme F-420 (coenzyme F420H2) appeared to be a potent competitive inhibitor (K-i 3 mu M) with respec t to coenzyme F-420. The latter findings may explain why the enzyme co uld only be detected in crude extracts that had been exposed to air, i .e. treatment with air causes the oxidation of reduced coenzyme F-420 present in anaerobic extracts. The results of this study sue discussed in view of the proposed role for coenzyme F-390 in methanogenic metab olism.