7-mercaptoheptanoylthreonine-phosphate and cobamamide

Adenosylcobalamin (AdoCbl), or coenzyme B

Topics: Animals; Carbon; Catalysis; Cobalt; Cobamides; Models, Molecular

Control over transition metal redox state is essential for metalloprotein function and can be achieved via coordination chemistry and/or sequestration from bulk solvent. Human methylmalonyl-Coenzyme A (CoA) mutase (MCM) catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA using 5'-deoxyadenosylcobalamin (AdoCbl) as a metallocofactor. During catalysis, the occasional escape of the 5'-deoxyadenosine (dAdo) moiety leaves the cob(II)alamin intermediate stranded and prone to hyperoxidation to hydroxocobalamin, which is recalcitrant to repair. In this study, we have identified the use of bivalent molecular mimicry by ADP, coopting the 5'-deoxyadenosine and diphosphate moieties in the cofactor and substrate, respectively, to protect against cob(II)alamin overoxidation on MCM. Crystallographic and electron paramagnetic resonance (EPR) data reveal that ADP exerts control over the metal oxidation state by inducing a conformational change that seals off solvent access, rather than by switching five-coordinate cob(II)alamin to the more air stable four-coordinate state. Subsequent binding of methylmalonyl-CoA (or CoA) promotes cob(II)alamin off-loading from MCM to adenosyltransferase for repair. This study identifies an unconventional strategy for controlling metal redox state by an abundant metabolite to plug active site access, which is key to preserving and recycling a rare, but essential, metal cofactor.

Topics: Adenosine Diphosphate; Humans; Methylmalonyl-CoA Mutase; Molecular Mimicry; Oxidation-Reduction; Vitamin B 12

Cobalamin (or vitamin B

Topics: Adenosine Triphosphate; Adrenodoxin; Humans; Vitamin B 12

Adenosylcobalamin (AdoCbl) or coenzyme B

Topics: Cobamides; Coenzymes; Ethanolamine Ammonia-Lyase; Glycerol; Hydro-Lyases; Molecular Chaperones; Phosphothreonine; Propanediol Dehydratase

Adenosylcobalamin (AdoCbl) or coenzyme B

Topics: Animals; Cobamides; Ethanolamine Ammonia-Lyase; Glycerol; Hydro-Lyases; Phosphothreonine

Catalysis by radical enzymes dependent on coenzyme B

Topics: Adenosine; Catalysis; Cobamides; Intramolecular Transferases; Methylmalonyl-CoA Mutase; Phosphothreonine

The X-ray structures of coenzyme B

Topics: Cobamides; Ethanolamine Ammonia-Lyase; Kinetics; Propanediol Dehydratase

D-Ornithine aminomutase from Clostridium sticklandii comprises two strongly associating subunits, OraS and OraE, with molecular masses of 12,800 and 82,900 Da. Previous studies have shown that in Escherichia coli the recombinant OraS protein is synthesized in the soluble form and OraE as inclusion bodies. Refolding experiments also indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate (PLP) or adenosylcobalamin (AdoCbl) play important roles in the refolding process. In this study, the DNA fragment containing both genes was cloned into the same expression vector and coexpression of the oraE and oraS genes was carried out in E. coli. The solubility of the coexpressed OraS and OraE increases with decreasing isopropyl thio-beta-D-galactoside induction temperature. Among substrate analogues tested, only 2,4-diamino-n-butyric acid displays competitive inhibition of the enzyme with a K(i) of 96 +/- 14 microm. Lys629 is responsible for the binding of PLP. The apparent K(d) for coenzyme B(6) binding to d-ornithine aminomutase is 224 +/- 41 nm as measured by equilibrium dialysis. The mutant protein, OraSE-K629M, is successfully expressed. It is catalytically inactive and unable to bind PLP. Because no coenzyme is involved in protein folding during in vivo translation of OraSE-K629M in E. coli, in vitro refolding of the enzyme employs a different folding mechanism. In both cases, the association of the S and E subunit is important for D-ornithine aminomutase to maintain an active conformation.

Topics: Binding Sites; Catalysis; Cloning, Molecular; Clostridium sticklandii; Cobamides; DNA; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Genetic Vectors; Intramolecular Transferases; Kinetics; Mutation; Oligonucleotides; Phosphothreonine; Plasmids; Protein Binding; Protein Conformation; Protein Folding; Protein Structure, Tertiary; Pyridoxal Phosphate; Recombinant Proteins; Ribosomes; Temperature; Thiogalactosides; Ultraviolet Rays