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 X-ray structure of the diol dehydratase–adeninylpentylcobalamin com-plex revealed that the adenine moiety of adenosylcobalamin is anchored in
the adenine-binding pocket of the enzyme by hydrogen bonding of N3
with the side chain OH group of Sera224, and of 6-NH2, N1 and N7 with
main chain amide groups of other residues.
Adenosylcobalamin-dependent diol and glycerol dehydratases are isofunc-tional enzymes and undergo mechanism-based inactivation by a physiologi-cal substrate glycerol during catalysis. Inactivated holoenzymes are
reactivated by their own reactivating factors that mediate the ATP-depen-dent exchange of an enzyme-bound, damaged cofactor for free adenosylco-balamin through intermediary formation of apoenzyme.
[x-(Adenosyl)alkyl]cobalamins (homoadenosylcobalamins) are useful ana-logues of adenosylcobalamin to get information about the distance between
Co and C5¢, which is critical for Co-C bond activation. In order to use
them as probes for exploring the active sites of enzymes, the coenzymic
properties of homoadenosylcobalamins for diol dehydratase and ethanol-amine ammonia-lyase were investigated. The kcat and kcat
adenosylmethylcobalamin were about 0.27% and 0.15% that for the regu-lar coenzyme with diol dehydratase, respectively. ...
The three genespduCDEencoding the diol dehydratase of
Lactobacillus collinoides, have been cloned for overexpres-sion in the pQE30 vector. Although the three subunits of the
protein were highly induced, no activity was detected in cell
extracts. The enzyme was therefore purified to near homo-geneity by ammoniumsulfate precipitation and gel filtration
chromatography. In fractions showing diol dehydratase
activity, three main bands were present after SDS/PAGE
with molecular masses of 63, 28 and 22 kDa, respectively....
Recombinant glycerol dehydratase ofKlebsiella pneumoniae
was purified to homogeneity. The subunit composition of
the enzyme was most probablya2b2c2
.When(R)- and (S)-propane-1,2-diolswere used independently as substrates, the
rate with the (R)-enantiomer was 2.5 times faster than that
with the (S)-isomer. In contrast to diol dehydratase,an iso-functional enzyme,the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than
that for the (R)-isomer (Km(R)
/Km(S) ¼1.5). ...