Molecular basis for specificities of reactivating factors for adenosylcobalamin‐dependent diol and glycerol dehydratases

Abstract

Diol dehydratase (1,2-propanediol hydro-lyase, EC 4.2.1.28) and glycerol dehydratase (glycerol hydro-lyase, EC 4.2.1.30) are isofunctional enzymes that catalyze adenosylcobalamin (AdoCbl) (coenzyme B 12 )-dependent conversion of 1,2-propanediol, 1,2-ethanediol, and glycerol to the corresponding aldehydes [1][2][3][4][5]. These enzymes encoded in the pdu (propanediol utilization) operon [6][7][8] and the dha (dihydroxyacetone) regulon [9][10][11][12], respectively, are involved in producing the electron acceptors propionaldehyde and Adenosylcobalamin-dependent diol and glycerol dehydratases are isofunctional enzymes and undergo mechanism-based inactivation by a physiological substrate glycerol during catalysis. Inactivated holoenzymes are reactivated by their own reactivating factors that mediate the ATP-dependent exchange of an enzyme-bound, damaged cofactor for free adenosylcobalamin through intermediary formation of apoenzyme. The reactivation takes place in two steps: (a) ADP-dependent cobalamin release and (b) ATP-dependent dissociation of the resulting apoenzyme-reactivating factor complexes. The in vitro experiments with purified proteins indicated that diol dehydratase-reactivating factor (DDR) cross-reactivates the inactivated glycerol dehydratase, whereas glycerol dehydratase-reactivating factor (GDR) did not cross-reactivate the inactivated diol dehydratase. We investigated the molecular basis of their specificities in vitro by using purified preparations of cognate and noncognate enzymes and reactivating factors. DDR mediated the exchange of glycerol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin, whereas GDR cannot mediate the exchange of diol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydratase-DDR complex was cross-formed, although the diol dehydratase-GDR complex was not formed. There were no specificities of reactivating factors in the ATP-dependent dissociation of enzyme-reactivating factor complexes. Thus, it is very likely that the specificities of reactivating factors are determined by the capability of reactivating factors to form complexes with apoenzymes. A modeling study based on the crystal structures of enzymes and reactivating factors also suggested why DDR cross-forms a complex with glycerol dehydratase, and why GDR does not cross-form a complex with diol dehydratase.

Novel Coenzyme B12-dependent Interconversion of Isovaleryl-CoA and Pivalyl-CoA

“…We note that although the MeaI domain of Gk IcmF exhibits ATPase activity it is functionally distinct from the ATP-dependent chaperones for AdoCbl-dependent eliminases, e.g. diol dehydratases (36,37).…”

Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair In Vitro and In Vivo

“…Given the availability of the reduced metals and redox equivalents, many metallocofactors can “self-assemble” in vitro into their active form with varying degrees of success. However, recent studies from many groups have established the importance or essentiality of biosynthetic pathways for cluster insertion (1, 2) and have suggested the importance of repair or maintenance pathways (3–5) for regenerating active cofactors from damaged metal clusters. Pioneering studies of the Dean group (2), followed by work from many labs, have contributed to our understanding of iron-sulfur (FeS) cluster biosynthesis and the complex machinery required (reviewed in Reference 6).…”

Redesign of coenzyme B12 dependent diol dehydratase to be resistant to the mechanism‐based inactivation by glycerol and act on longer chain 1,2‐diols

“…Specific protein factors that are responsible for the reactivation of diol dehydratase [12][13][14] and glycerol dehydratase [15][16][17] were designated diol-dehydratase-reactivating factor and glycerol-dehydratasereactivating factor, respectively. They reactivate the inactivated holoenzymes by mediating the exchange of a damaged cofactor for intact AdoCbl through a molecular-chaperone-like mechanism [13,14,16,18]. Recently, we reported evidence for multiple turnovers with dioldehydratase-reactivating factor which demonstrated that the reactivating factor is an enzyme 'reactivase' [19].…”