Structure of the NADPH-Binding Motif of Glutathione Reductase: Efficiency Determined by Evolution

Abstract

The role of the second glycine residue (Gly-176) of the conserved GXGXXA "fingerprint" motif in the NADPH-binding domain of Escherichia coli glutathione reductase has been studied by means of site-directed mutagenesis. This glycine residue occurs at the N-terminus of the alpha-helix in the beta alpha beta fold that characterizes the dinucleotide-binding domain, in close proximity to the pyrophosphate bridge of the bound coenzyme. Introducing an alanine residue (G176A), the minimum possible change, at this position virtually inactivated the enzyme, as did the introduction of valine, leucine, isoleucine, glutamic acid, histidine, or arginine residues. Only the replacement by serine--a natural substitute for this glycine residue in some forms of mercuric reductase, a related flavoprotein disulfide oxidoreductase--produced a mutant enzyme (G176S) that retained significant catalytic activity. It is conceivable that this is due to a favorable hydrogen bond being formed between the serine hydroxyl and a pyrophosphate oxygen atom. In most of the mutant enzymes, the Km for NADPH was substantially greater than that found for wild-type glutathione reductase, as expected, but this was accompanied by an unexpected decrease in the Km for GSSG. The latter can be explained by the observation that the reduction of the enzyme by NADPH, the first half-reaction of the ping-pong mechanism, had become a rate-limiting step of the overall reaction catalyzed, albeit poorly, by the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of Redox Balance by the Stringent Response Regulatory Protein Promotes Antioxidant Defenses of Salmonella

“…Further supporting this idea, the dksA mutant exhibits lower rates in the reduction of GSSG than wild-type bacteria. The pool of ϳ200 M NADPH in dksA mutant Salmonella is still above the estimated K m of 20 M that glutathione reductase has for NADPH (45). According to Michaelis-Menten kinetics, the velocity of an enzymatic reaction is dependent on substrate level (V ϭ V max [S]/K m ϩ [S], where V is velocity, V max is maximal velocity, and S is substrate concentration).…”

Salicylic Acid–Independent ENHANCED DISEASE SUSCEPTIBILITY1 Signaling inArabidopsisImmunity and Cell Death Is Regulated by the MonooxygenaseFMO1and the Nudix HydrolaseNUDT7

“…A BLASTP database search identified an FMO-like protein in rice (Oryza sativa) of unknown function with 56% amino acid identity (Os FMO, Figure 5A). Other analyses have demonstrated the importance of the conserved Gly residues in the FAD and NADPH binding sites for cofactor binding and enzymatic activity (Rescigno and Perham, 1994;Kubo et al, 1997). We therefore generated by site-directed mutagenesis variants of FMO1 in which the conserved Gly residues of these motifs were exchanged to Ala residues ( Figure 5A).…”

Microbial synthesis of triacetic acid lactone

“…The FAS-B ketoreductase domain was shown to share signature features with other members of the shortchain dehydrogenase/reductase (SDR) superfamily of NAD (P)/NAD(P)H-dependent enzymes (Joernvall et al, 1995;Price et al, 2001). Shared features included the well-known Rossman fold motif (Chen et al, 1990;Rescigno and Perham, 1994), necessary for cofactor binding, and highly conserved Y 2226 XXXK catalytic residues (Oppermann et al, 1997), involved in hydrogen bonding and proton transfer. Replacement of catalytically active tyrosine with chemically inert phenylalanine in the Y2226F FAS-B mutant eliminated ketoreductase activity (Zha et al, 2004).…”