Purification and properties of coenzyme F390 hydrolase from Methanobacterium thermoautotrophicum (strain Marburg)

Abstract

8-Hydroxyadenylylated coenzyme F420 (coenzyme F390-A) is formed in methanogenic bacteria upon oxidative stress. After reinstatement of anaerobic conditions, coenzyme F390 is degraded into coenzyme F420 and AMP. The enzyme catalyzing the latter reaction, coenzyme F390 hydrolase, was purified to homogeneity from Methanobacterium thermoautotrophicum strain Marburg 355-fold to a specific activity of 12.1 mumol.min-1.mg protein-1. The enzyme consisted of one polypeptide of approximately 27 kDa. Coenzyme F390 hydrolase displayed an apparent Km for coenzyme F390 of 40 microM. The enzyme required the presence of a reducing agent like dithiothreitol to become active. Activity could be manipulated by applying various ratios of reduced and oxidized dithiothreitol. Activation proceeded by a two-electron reduction, which indicates that one S-S bridge is involved the activation/inactivation of the enzyme. Dithiothreitol could be replaced by the methanogenic C1-carrier 2-mercaptoethanesulfonate (H-S-CoM), but not by N7-mercaptoheptanoyl-L-threonine phosphate (H-S-HTP) or other naturally occurring thiol-containing compounds. The addition of the heterodisulfide of H-S-CoM and H-S-HTP (CoM-S-S-HTP) diminished the stimulatory effect of H-S-CoM.