Cloning, expression, and characterization of coenzyme-B12-dependent diol dehydratase from Lactobacillus diolivorans.
The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α2β2γ2) structure with a native molecular mass of 210 kDa. It requires coenzyme B12 for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K m values for coenzyme B12, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B12-dependent diol dehydratase and not a glycerol dehydratase.
Xuqin
X
Wei
Xiaolei
X
Meng
Yunlai
Y
Chen
Yutuo
Y
Wei
Liqin
L
Du
Ribo
R
Huang
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003038:Cobamides; D005990:Glycerol / Q000378:metabolism; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D010100:Oxygen / Q000378:metabolism; D011408:Propanediol Dehydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D003038:Cobamides; D011994:Recombinant Proteins; D011408:Propanediol Dehydratase; C000913:cobamamide; D005990:Glycerol; D010100:Oxygen