title: Characterization of glycerol dehydratase expressed by fusing its alpha- and beta-subunits.
abstract: The gdh and gdhr genes, encoding B(12)-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the beta-subunit was lost during GDH purification when co-expressing alpha, beta and gamma subunit. This was overcome by fusing the beta-subunit to alpha- or gamma-subunit with/without the insertion of a linker peptide between the fusion moieties. The kinetic properties of the fusion enzymes were characterized and compared with wild type enzyme. The results demonstrated that the fusion protein GDHALB/C, constructed by linking the N-terminal of beta-subunit to the C-terminal of alpha subunit through a (Gly(4)Ser)(4) linker peptide, had the greatest catalytic activity. Similar to the wild-type enzyme, GDHALB/C underwent mechanism-based inactivation by glycerol during catalysis and could be reactivated by GDHR.
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
authors.forename: Xu-De
authors.initials: XD
authors.lastname: Wang
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
authors.forename: Yong-Chao
authors.initials: YC
authors.lastname: Guo
authors.forename: Zhi-Ping
authors.initials: ZP
authors.lastname: Zhang
authors.forename: Zhu-An
authors.initials: ZA
authors.lastname: Cao
authors.forename: Ya-Feng
authors.initials: YF
authors.lastname: Zhou
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006836:Hydro-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D006836:Hydro-Lyases; C020671:glycerol dehydratase