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 mechanism of assembly of the bis(molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase (DMSOR). R. sphaeroides DMSOR expressed in a mobA(-) Escherichia coli strain lacks molybdopterin and molybdenum but contains a full complement of guanine in the form of GMP and GDP. Escherichia coli MobA, molybdopterin-Mo, GTP, and MgCl(2) 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 activation. 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 wild-type 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 dinucleotide has been demonstrated directly without using the activation of a molybdoenzyme as an indicator for cofactor formation.
Temple
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CA
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Rajagopalan
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KV
D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006150:Guanine Nucleotides / Q000378:metabolism*; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D009942:Organometallic Compounds / Q000378:metabolism*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000378:metabolism*
D006150:Guanine Nucleotides; D007506:Iron-Sulfur Proteins; D009942:Organometallic Compounds; C106961:bis(molybdopterin guanine dinucleotide)molybdenum; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase