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Redesign of coenzyme B 12 dependent diol dehydratase to be resistant to the mechanism‐based inactivation by glycerol and act on longer chain 1,2‐diols
Author(s) -
Yamanishi Mamoru,
Kinoshita Koichiro,
Fukuoka Masaki,
Saito Takuya,
Tanokuchi Aya,
Ikeda Yuuki,
Obayashi Hirokazu,
Mori Koichi,
Shibata Naoki,
Tobimatsu Takamasa,
Toraya Tetsuo
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2012.08470.x
Subject(s) - glycerol , stereochemistry , chemistry , cofactor , enzyme , active site , dehydratase , biochemistry
Coenzyme B 12 dependent diol dehydratase undergoes mechanism‐based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co–C bond. Bachovchin et al. [ Biochemistry 16 , 1082–1092 (1977)] reported that glycerol bound in the G S conformation, in which the pro‐S ‐CH 2 OH group is oriented to the hydrogen‐abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X‐ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of ( S )‐1,2‐propanediol, i.e. in the G S conformation, with its 3‐OH group hydrogen bonded to Serα301, but not to nearby Glnα336. k inact of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild‐type enzyme. k cat / k inact showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild‐type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for ( S )‐1,2‐propanediol decreased on these mutations. The substrate activities towards longer chain 1,2‐diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2‐diols. Database Structural data are available in the Protein Data Bank under the accession number 3AUJ . Structured digital abstract• Diol dehydrase gamma subunit , Diol dehydrase beta subunit and Diol dehydrase alpha subunit physically interact by X‐ray crystallography ( View interaction )