Studies of the CobA-Type ATP:Co(I)rrinoid Adenosyltransferase Enzyme of Methanosarcina mazei Strain Gö1

Abstract

Although methanogenic archaea use B 12 extensively as a methyl carrier for methanogenesis, little is known about B 12 metabolism in these prokaryotes or any other archaea. To improve our understanding of how B 12 metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain Gö1 (open reading frame MM3138, referred to as cobA Mmhere) was cloned and used to restore coenzyme B 12 synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme.cobA Mmprotein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca 2+ions, but the activity was not enhanced by Mg 2+and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn 2+. The CobAMmenzyme had a K mATP of 3 μM and a K mHOCbl of 1 μM. Unlike the S. enterica enzyme, CobAMmused cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the β phosphate of ATP was required for binding to the enzyme. A striking difference between CobASeand CobAMmwas the use of ADP as a substrate by CobAMm, suggesting an important role for the γ phosphate of ATP in binding. The results from 31 P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPP i) is the reaction by-product; no cleavage of PPP i was observed, and the enzyme was only slightly inhibited by pyrophosphate (PP i). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobASeand CobAMmenzymes.