The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2′‐phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343 :38–43). In order to analyze the structural changes involved, we have determined 4 high‐resolution crystal structures, i.e., the structures of the wild‐type enzyme (1.86 Å resolution, R ‐factor of 16.8%), of the wild‐type enzyme ligated with NADP (2.0 Å, 20.8%), of the NAD‐dependent mutant (1.74 Å, 16.8%), and of the NAD‐dependent mutant ligated with NAD (2.2 Å, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD‐binding site assumes a less rigid structure than the NADP site of the wild‐type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 Å from the sites of the mutations, this observation reminds us that mutations may have important long‐range consequences that are difficult to anticipate.

The crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R ‐factor of 16.8% at 1.86 Å resolution. The molecular 2‐fold axis of the dimer is local but very close to a possible crystallographic 2‐fold axis; the slight asymmetry could be rationalized from the packing contacts. The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity. The structure was compared with the well‐known structure of the homologous enzyme from human erythrocytes with 52% sequence identity. Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E. coli enzyme has an antiparallel β‐sheet connecting the subunits. The differences at the glutathione binding site and in particular a deformation caused by a Leu‐Ile exchange indicate why the E. coli enzyme accepts trypanothione much better than the human enzyme. The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones.

NAD and NADP are ubiquitous coenzymes in biological redox reactions. They have distinct metabolic functions, yet they differ only by an additional phosphate group esterified at the 2'-hydroxyl group of the AMP moiety of NADP. The natural specificity of Escherichia coli glutathione reductase for NADP has previously been converted into a marked preference for NAD by introducing seven point mutations into the beta alpha beta-fold of the NADP-binding domain of the protein based on the known structure of the human enzyme. Among them was the replacement of Ala179 by glycine (A179G) in the alpha-helix of the fold, a change suggested by a difference in a sequence fingerprint previously found in the dinucleotide-binding domains of a number of dehydrogenases. Although this position is at a distance of 10 A from the bound 2'-phosphate group of NADP in glutathione reductase, the A179G mutation was found to be synergistic and beneficial. We have now carried out X-ray crystallographic analyses of the NAD-dependent mutant without and with bound NADH. A comparison of the structures of the mutant and wild-type enzymes reveals a flip of the peptide bond between Gly174 and Ala175 such that the side-chain of another introduced amino acid, Glu197, is fixed and can participate in binding the adenine ribose of NAD, thereby contributing to the ability of the mutated enzyme to exert its selectivity for the "wrong" coenzyme.