Characterization, Sequencing, and Expression of the Genes Encoding a Reactivating Factor for Glycerol-inactivated Adenosylcobalamin-dependent Diol Dehydratase

Abstract

. These open reading frames, designated as ddrA and ddrB genes, were identified as the genes of a putative reactivating factor for inactivated diol dehydratase. The genes encoded polypeptides consisting of 610 and 125 amino acid residues with predicted molecular weights of 64,266 and 13,620, respectively. Co-expression of the open reading frame in the 5-flanking region was stimulatory but not obligatory for conferring the reactivating activity upon E. coli. Thus, the product of this gene was considered not an essential component of the reactivating factor.

A Reactivating Factor for Coenzyme B12-dependent Diol Dehydratase

“…The NH 2 -terminal 10-amino acid sequences of the A and B polypeptides determined by Edman sequencing were MRYIAGIDIG and MNGNHSAPAI, respectively. These sequences agreed com- pletely with those deduced from the nucleotide sequences of the ddrA and ddrB genes (20). Thus, the A and B polypeptides were undoubtedly identified as the ddrA and ddrB gene products, respectively.…”

“…Construction of Expression Plasmids-We have constructed expression plasmid pCXV(6/5b) for the ddrAB genes using vector pCXV (20). Because copy numbers of plasmids containing replication origin of p15A, like pCXV are less than those of plasmids containing replication origin of pBR322, like pUSI2E (23), (10 -12 and 15-20 copies/cell, respectively (24)), we transferred the genes from pCXV(6/5b) to another expression vector, pUSI2ENd.…”

“…Therefore, it was clear that the two polypeptides were co-purified as a tight complex. Because the predicted molecular weights of the DdrA and DdrB proteins are 64,266 and 13,620, respectively (20), it was highly suggested that the A and B polypeptides are the products of these genes. The NH 2 -terminal 10-amino acid sequences of the A and B polypeptides determined by Edman sequencing were MRYIAGIDIG and MNGNHSAPAI, respectively.…”

“…Because the reactivation was detectable only in situ but not in vitro, it remained unclear whether the reactivation is caused by a specific proteinous factor. Recently, we have identified the two open reading frames located in the 3Ј-flanking of the K. oxytoca diol dehydratase genes as the genes encoding a reactivating factor for glycerol-inactivated diol dehydratase and designated them as ddrA and ddrB genes (19,20).…”

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

“…The radical mechanism of action of these enzymes, involving coenzyme B 12 as essential cofactor, was initially proposed by Abeles and coworkers (52,53). These enzymatic reactions are subject to suicide inactivation by the substrate glycerol (54). More recently, the crystal structure of K. oxytoca diol dehydratase-cyanocobalamin complex was solved (55).…”