Purification and characterization of coenzyme F390 synthetase from Methanobacterium thermoautotrophicum (strain delta H).
Coenzyme F390 synthetase catalyzes the formation of 8-hydroxyadenylylated-coenzyme F420 (coenzyme F390-A) from coenzyme F420 and ATP in some methanogenic Archaea. The presence of coenzyme F390 was found when these organisms were exposed to oxygen. To get more insight into the defined function of coenzyme F390, the coenzyme F390 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 mumol.min-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 F390 synthetase activity suggested that the synthetase is also capable of 8-hydroxyguanylylated-coenzyme F420 (coenzyme F390-G) formation. Initial-velocity measurements of the two-substrate reaction showed that the enzyme kinetics for the coenzyme F390 synthetase reaction proceeded by a ternary-complex mechanism. The coenzyme F390 synthetase displayed a Km for coenzyme F420 of 39 microM and a Km 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 F420 (coenzyme F420H2) appeared to be a potent competitive inhibitor (Ki 3 microM) with respect to coenzyme F420. 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 F420 present in anaerobic extracts. The results of this study are discussed in view of the proposed role for coenzyme F390 in methanogenic metabolism.
Department of Microbiology, Faculty of Science, University of Nijmegen, The Netherlands.
P
P
Vermeij
F J
FJ
Detmers
F J
FJ
Broers
J T
JT
Keltjens
C
C
Van der Drift
D002846:Chromatography, Affinity; D003066:Coenzyme A Ligases / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D017014:Methanobacterium / Q000201:enzymology*; D009713:Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
C069619:F390 synthetase; D009713:Nucleotidyltransferases; D003066:Coenzyme A Ligases