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  • GlxI is a member of the

    2022-05-26

    GlxI is a member of the βαβββ superfamily of proteins, consisting of fosfomycin resistance protein (FosA), methylmalonyl-CoA epimerase (MMCE), extradiol dioxygenase (DIOX), mitomycin C resistance protein (MRP) and bleomycin resistance protein (BRP) [29], [30], [31]. These proteins are believed to have evolved from a common one-module ancestor, which, upon gene duplication and fusion as well as through accumulated mutagenesis, resulted in high structural similarity but with different biological functionality within this superfamily [29], [30], [32]. Among these structural members, FosA is mechanistically close to GlxI, as both are metalloenzymes that utilize GSH as a cofactor/cosubstrate, and both enzymes form an active site octahedral geometry around the metal center (Fig. 2) [24], [25], [26], [33], [34]. However, FosA employs three water molecules and three metal binding ligands that coordinate to the metal center via vicinal chelation [33], [35], while GlxI utilizes two water molecules and four metal binding residues around the active metal [26]. FosA employs two metal binding subsites, one for the catalytically critical metal Mn and another for a monovalent K+ (Fig. 3) [36], [37], [38]. GlxI, on the other hand, utilizes only one metal binding site (Fig. 3) [24], [25], [26]. Fosfomycin, the substrate of FosA, is an antibiotic useful in the treatment of lower urinary tract and gastrointestinal infections due to its stability and broad spectrum of activity [39], [40], [41], [42]. This antibiotic affects both Gram-positive and Gram-negative bacteria by interfering with MurA, the first enzyme in the bacterial cell wall biosynthetic pathway [42], a target that is lacking in humans. FosA likely evolved from “housing keeping” gene(s) within bacterial Coenzyme Q10 to resist the cytotoxicity of the natural product fosfomycin. FosA activates the epoxide moiety in fosfomycin for nucleophilic attack by glutathione, resulting in the formation of an inactive GS-fosfomycin adduct (Fig. 2). We hypothesize that FosA activity might be engineered from some proteins in the same superfamily, more specifically GlxI, due to similarities between these enzymes as stated above. Herein, we report an investigation of the cofactor specificity of the E. coli Glx system using a set of thiol cofactors (GSH, GspdSH and T(SH)2) to probe the extent of cofactor promiscuity by these E. coli enzymes and to obtain additional insight into the molecular interactions that occur between substrate and metalloprotein. As well, we report our studies on evaluating the deuterium kinetic isotope effect (KIE) on E. coli GlxI catalysis by utilizing α-deuteriophenylglyoxal (α-deuterioPG) or non-deuterated phenylglyoxal (PG) as substrates and GSH as the cofactor. To determine if the extent of any KIE could be metal ion dependent, both Ni- as well as Cd-activated GlxI were utilized in these studies. Lastly, intrigued by the mechanistic relatedness between the enzymes GlxI and FosA, we prepared the E56A mutant of E. coli GlxI in order to produce a GlxI mimicking the metal coordination geometry of FosA. Fosfomycin conjugation competency was evaluated for this GlxI mutein in the presence of various M metal ions.
    Experimental
    Results and discussion
    Conclusions The current investigation has extended our understanding of the Ni-activated GlxI class of metalloenzyme. The E. coli Glx system, composed of GlxI and GlxII, has been shown to utilize a variety of thiol cofactors. GSH, GspdSH and T(SH)2 cofactors can be utilized to stereospecifically convert MG to d-lactate by the E. coli Glx system. As well, the metal ion-dependent presence of a deuterium isotope effect in E. coli GlxI is the first non Zn-dependent GlxI enzyme characterized by such experimental approaches. An observable deuterium kinetic isotope effect has been detected for the Cd-substituted E. coli GlxI indicating that α-proton transfer is partially rate-limiting in the enzymatic reaction.