Diol dehydratase‐reactivating factor is a reactivase – evidence for multiple turnovers and subunit swapping with diol dehydratase

Abstract

Adenosylcobalamin-dependent diol dehydratase (DD) undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co-C bond. The damaged cofactor remains tightly bound to the active site. The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg 2+ by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme. In this study, we demonstrated that this reactivating factor mediates the cobalamin exchange not stoichiometrically but catalytically in the presence of ATP and Mg 2+ . Therefore, we concluded that the reactivating factor is a sort of enzyme. It can be designated DD reactivase. The reactivase showed broad specificity for nucleoside triphosphates in the activation of the enzymeAEcyanocobalamin complex. This result is consistent with the lack of specific interaction with the adenine ring of ADP in the crystal structure of the reactivase. The specificities of the reactivase for divalent metal ions were also not strict. DD formed 1 : 1 and 1 : 2 complexes with the reactivase in the presence of ADP and Mg 2+ . Upon complex formation, one b subunit was released from the (ab) 2 tetramer of the reactivase. This result, together with the similarity in amino acid sequences and folds between the DD b subunit and the reactivase b subunit, suggests that subunit displacement or swapping takes place upon formation of the enzymeAEreactivase complex. This would result in the dissociation of the damaged cofactor from the inactivated holoenzyme, as suggested by the crystal structures of the reactivase and DD.

The Biocatalytic Production of 3-Hydroxypropionaldehyde and Evaluation of Its Stability

“…There was no significant difference in 3-HPA as the bioconversion time increased further. The 3-HPA bioconversion was very rapid, in that about 90% of this product was produced within 0.5 h. The dhaB enzyme, which converts glycerol to 3-HPA and uses vitamin B 12 as a cofactor, is eventually inactivated because the cells cannot regenerate coenzyme B 12 ; instead the catalytic cycle is interrupted by the formation of 5'-deoxyadenosine and the catalytically inactive cobalamin [22], and the altered cofactor remains tightly bound to the active site [23]. A dhaB-reactivating factor in Klebsiella reactivates the inactivated enzyme-bound cyanocobalamin in the presence of free vitamin B 12 , adenosine triphosphate (ATP), and Mg 2+ by mediating the exchange of the tightly bound altered cofactor for a free intact cofactor [24].…”

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

“…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 diol‐dehydratase‐reactivating factor which demonstrated that the reactivating factor is an enzyme ‘reactivase’ [19].…”

Exploring Bacterial Organelle Interactomes: A Model of the Protein-Protein Interaction Network in the Pdu Microcompartment

“…These cases were largely consistent with existing experimental data, where they were available. An example of a positive result is the agreement between our predictions and structural data relating to the reactivation mechanism of the diol dehydratase [ 80 ].…”

“…Although no structural information about this complex in Salmonella is available, crystal structures of highly similar homologs from Klebsiella oxytoca have been solved [ 22 , 78 , 79 ]. Studies with these same homologs demonstrated that the binding mechanism involved a subunit exchange between the dehydratase and the reactivase, where one PduH subunit is released from the reactivase and replaced by one PduD subunit [ 80 ].…”