Year-end Sale % OFF 
Abstract
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.
Highlights
It was demonstrated that the MobA-dependent activation of nitrate reductase (NR) from a mobϪ strain was enhanced by the addition of GTP and MgCl2 to the soluble cell extract utilized for activation (16)
Recombinant enzyme purified from the wild-type parent strain, MC4100, is referred to as rDMSOR to differentiate it from enzyme purified from R. sphaeroides
We present here a fully defined, in vitro system for studying the mechanism of assembly of the bis(MGD)Mo cofactor
Summary
Construction of Expression Plasmids—The DMSOR expression plasmid used in this work was created by digesting the pJH720 construct (19) (Table I) with NcoI and HindIII to release the complete mature R. sphaeroides DMSOR coding sequence including an N-terminal His tag. TP1000 cells (Table I) containing pJH820 and grown as described above were used to purify mobABϪ DMSOR. TP1000 cells containing pJH820 and either pCT300A or pCT300B were grown as described above with the addition of 34 g/ml chloramphenicol and used to purify mobBϪ and mobAϪ DMSOR, respectively. All forms of recombinant DMSOR were purified as described previously (19) except that cell lysis was achieved using a Microfluidics M110L Microfluidizer Processor (6), and no heat step was performed. Sufficient GTP and MgCl2 were added to produce a final concentration of 1 mM each, and 50 mM Tris/HCl, pH 7.5, was added to bring the total volume to 150 l This mixture was placed in the Coy chamber for a minimum of 15 min before 50 l of heat-denatured SO was added. After discarding the first 60 ml of the 10 mM HCl wash, 1.5-ml fractions were collected for the remainder of the column
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
