Purification and Characterization of Coenzyme F 390 Synthetase from Methanobacterium thermoautotrophicum (strain H)

Coenzyme F 390 synthetase catalyzes the formation of 8‐hydroxyadenylylated‐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 when these organisms were exposed to oxygen. To get more insight into the defined function of coenzyme F 390 , the coenzyme F 390 synthetase from Methanobacterium thermoautrophicum was purified from a cell‐free extract and its catalytic properties were determined. The synthetase was purified 150‐fold to a specific activity of 0.45 μmol · mim −1 · mg protein −1 . The enzyme consisted of one polypeptide of approximately 51 kDa. The isolated enzyme showed a tendency to aggregate into dimers and tetramers upon concentration. Co‐elution during purification of GTP‐dependent coenzyme F 390 synthetase activity suggested that the synthetase is also capable of 8‐hydroxyguanylylated‐coenzyme F 420 , (coenzyme F 390 ‐G) formation. Initial‐velocity measurements of the two‐substrate reaction showed that the enzyme kinetics for the coenzyme F 390 synthetase reaction proceeded by a ternary‐complex mechanism. The coenzyme F 390 synthetase displayed a K m for coenzyme F 420 of 39 μM and 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 conditions. Treatment with air was not required to obtain the enzyme in an active form. However, 1,5‐dihydro‐coenzyme F 420 (coenzyme F 420 H 2 ) appeared to be a potent competitive inhibitor ( K i 3 μM) with respect to coenzyme F 420 . The latter findings may explain why the enzyme could 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 are discussed in view of the proposed role for coenzyme F 390 in methanogenic metabolism.