Purification and Properties of an NAD(P)+-linked Formaldehyde Dehydrogenase from Methylococcus capsulatus (Bath)

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Abstract
Crude soluble extracts of M. capsulatus strain Bath, grown on methane, contained NAD(P)+-linked formaldehyde dehydrogenase activity. Activity in the extract was lost on dialysis against phosphate buffer, but could be restored by supplementing with inactive, heat-treated extract (70.degree. C for 12 min). The non-dialysate, heat-sensitive component was isolated and purified, and has a MW of about 115,000. Sodium dodecyl sulfate gel electrophoresis of the protein suggested there were 2 equal subunits with MW of 57,000. The heat-stable fraction, which was necessary for activity of the heat-sensitive protein, was trypsin-sensitive and presumed to be a low MW protein or peptide. A number of thiol compounds and other common cofactors could not replace the component present in the heat-treated soluble extract. The purified formaldehyde dehydrogenase oxidized 3 other aldehydes with the following Km values: 0.68 mM (formaldehyde); 0.075 mM (glyoxal); 7.0 mM (glycolaldehyde); and 2.0 mM (DL-glyceraldehyde). NAD+ or NADP+ was required for activity, with Km values of 0.063 and 0.155 mM, respectively, and could not be replaced by any of the artificial electron acceptors tested. The enzyme was heat-stable at 45.degree. C for at least 10 min and had temperature and pH optima of 45.degree. C and pH 7.2, respectively. A number of metal-binding agents and substrate analogs were not inhibitory. Thiol reagents gave varying degrees of inhibition, the most potent being p-hydroxymercuribenzoate which at 1 mM gave 100% inhibition. The importance of possessing an NAD(P)+-linked formaldehyde dehydrogenase, with respect to M. capsulatus, is discussed.