Mechanism of assembly of the bis(molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase

A fully defined in vitro system has been developed for studying the mechani sm of assembly of the bis(molybdopterin guanine dinucleotide)molybdenum cof actor in Rhodobacter sphaeroides dimethyl sulfoxide reductase (DMSOR). R. s phaeroides DMSOR expressed in a mobA(-) Escherichia coil strain lacks molyb dopterin and molybdenum but contains a full complement of guanine in the fo rm of GMP and GDP. Escherichia coil MobA, molybdopterin-Mo, GTP, and MgCl2, are required and sufficient for the in vitro activation of purified DMSOR expressed in the absence of MobA High levels of MobA inhibit the in vitro a ctivation. A chaperone is not required for the in vitro activation process. The reconstituted DMSOR can exhibit up to 73% of the activity observed in recombinant DMSOR purified from a wildtype strain. The use of radiolabeled GTP has demonstrated incorporation of the guanine moiety from the GTP into the activated DMSOR. No role was observed for E. coli MobB in the in vitro activation of apo-DMSOR. This work also represents the first time that the MobA-mediated conversion of molybdopterin to molybdopterin guanine dinucleo tide has been demonstrated directly without using the activation of a molyb doenzyme as an indicator for cofactor formation.