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.
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. [Biochemistry16, 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).
Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Japan.
Mamoru
M
Yamanishi
Koichiro
K
Kinoshita
Masaki
M
Fukuoka
Takuya
T
Saito
Aya
A
Tanokuchi
Yuuki
Y
Ikeda
Hirokazu
H
Obayashi
Koichi
K
Mori
Naoki
N
Shibata
Takamasa
T
Tobimatsu
Tetsuo
T
Toraya
D002384:Catalysis; D020134:Catalytic Domain; D003038:Cobamides / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004351:Drug Resistance; D005990:Glycerol / Q000378:metabolism*; D007700:Kinetics; D041121:Klebsiella oxytoca / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011408:Propanediol Dehydratase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019946:Propylene Glycol / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013237:Stereoisomerism; D014805:Vitamin B 12 / Q000378:metabolism
D003038:Cobamides; D019946:Propylene Glycol; D011408:Propanediol Dehydratase; C000913:cobamamide; D014805:Vitamin B 12; D005990:Glycerol