Radical catalysis of B 12 enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases

Abstract

Enzymatic radical catalysis is defined as a mechanism of catalysis by which enzymes catalyze chemically difficult reactions by utilizing the high reactivity of free radicals. Adenosylcobalamin (coenzyme B12) serves as a cofactor for enzymatic radical reactions. The recent structural analysis of adenosylcobalamin-dependent diol dehydratase revealed that the substrate 1,2-propanediol and an essential potassium ion are located inside a (beta/alpha)8 barrel. Two hydroxyl groups of the substrate coordinate directly to the potassium ion which binds to the negatively charged inner part of the cavity. Cobalamin bound in the base-on mode covers the cavity to isolate the active site from solvent. Based on the three-dimensional structure and theoretical calculations, a new mechanism for diol dehydratase is proposed in which the potassium ion plays a direct role in the catalysis. The mechanisms for generation of a catalytic radical by homolysis of the coenzyme Co-C bond and for protection of radical intermediates from undesired side reactions during catalysis are discussed based on the structure. The reactivating factors for diol and glycerol dehydratases have been identified. These factors are a new type of molecular chaperone which participate in reactivation of the inactivated holoenzymes by mediating ATP-dependent exchange of the modified coenzyme for free intact coenzyme.

Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

“…All of the glycerol and diol dehydratases characterized so far belong to class II of coenzyme B12-containing enzymes (50,51). The radical mechanism of action of these enzymes, involving coenzyme B 12 as essential cofactor, was initially proposed by Abeles and coworkers (52,53).…”

Protein Content of Polyhedral Organelles Involved in Coenzyme B 12 -Dependent Degradation of 1,2-Propanediol in Salmonella enterica Serovar Typhimurium LT2

“…These enzymes have a broad but uneven distribution among living forms and are vital to human health, are essential to the carbon cycle, and have important industrial applications (5, 27, 30). Historically, bacteria have provided excellent model systems for the study of vitamins, and recent investigations with several bacterial systems have found the molecular biology of B 12 -dependent processes to be unexpectedly complex (9,27,29,34,35). One of the most surprising findings in this area has been the identification of a polyhedral organelle involved in coenzyme B 12 -dependent 1,2-propanediol (1,2-PD) degradation by Salmonella enterica (9).…”

MeaB Is a Component of the Methylmalonyl-CoA Mutase Complex Required for Protection of the Enzyme from Inactivation

“…Other B 12 -dependent enzymes (glycerol and diol dehydratase) undergo suicide inactivation by the substrate during catalysis (6,26,27). The inactivation involves irreversible cleavage of the Co-C bond of AdoCbl, forming 5Ј-deoxyadenosine and an alkylcobalamin-like species (27).…”