title: Ni2+-activated glyoxalase I from Escherichia coli: substrate specificity, kinetic isotope effects and evolution within the βαβββ superfamily.
abstract: The Escherichia coli glyoxalase system consists of the metalloenzymes glyoxalase I and glyoxalase II. Little is known regarding Ni(2+)-activated E. coli glyoxalase I substrate specificity, its thiol cofactor preference, the presence or absence of any substrate kinetic isotope effects on the enzyme mechanism, or whether glyoxalase I might catalyze additional reactions similar to those exhibited by related βαβββ structural superfamily members. The current investigation has shown that this two-enzyme system is capable of utilizing the thiol cofactors glutathionylspermidine and trypanothione, in addition to the known tripeptide glutathione, to convert substrate methylglyoxal to non-toxic D-lactate in the presence of Ni(2+) ion. E. coli glyoxalase I, reconstituted with either Ni(2+) or Cd(2+), was observed to efficiently process deuterated and non-deuterated phenylglyoxal utilizing glutathione as cofactor. Interestingly, a substrate kinetic isotope effect for the Ni(2+)-substituted enzyme was not detected; however, the proton transfer step was observed to be partially rate limiting for the Cd(2+)-substituted enzyme. This is the first non-Zn(2+)-activated GlxI where a metal ion-dependent kinetic isotope effect using deuterium-labelled substrate has been observed. Attempts to detect a glutathione conjugation reaction with the antibiotic fosfomycin, similar to the reaction catalyzed by the related superfamily member FosA, were unsuccessful when utilizing the E. coli glyoxalase I E56A mutein.
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada. kymullings@gmail.com
authors.forename: Kadia Y
authors.initials: KY
authors.lastname: Mullings
authors.forename: Nicole
authors.initials: N
authors.lastname: Sukdeo
authors.forename: Uthaiwan
authors.initials: U
authors.lastname: Suttisansanee
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Ran
authors.forename: John F
authors.initials: JF
authors.lastname: Honek
mesh_terms: D002104:Cadmium / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D019143:Evolution, Molecular; D007700:Kinetics; D007791:Lactoylglutathione Lyase / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D009532:Nickel / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D002104:Cadmium; D009532:Nickel; D007791:Lactoylglutathione Lyase