The three-dimensional structure of Corynebacterium 2,5-diketo-D-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-Å resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate. Thus the threedimensional structure has now been solved for a prokaryotic example of the aldo-keto reductase superfamily. The details of the binding of the NADPH cofactor help to explain why 2,5-DKGR exhibits lower binding affinity for cofactor than the related human aldose reductase does. Furthermore, changes in the local loop structure near the cofactor suggest that 2,5-DKGR will not exhibit the biphasic cofactor binding characteristics observed in aldose reductase. Although the crystal structure does not include substrate, the two ordered water molecules present within the substrate-binding pocket are postulated to provide positional landmarks for the substrate 5-keto and 4-hydroxyl groups. The structural basis for several previously described active-site mutants of 2,5-DKGR A is also proposed. Recent research efforts have described a novel approach to the synthesis of L-ascorbate (vitamin C) by using a genetically engineered microorganism that is capable of synthesizing 2,5-DKG from glucose and subsequently is transformed with the gene for 2,5-DKGR. These modifications create a microorganism capable of direct production of 2-keto-L-gulonate from D-glucose, and the gulonate can subsequently be converted into vitamin C. In economic terms, vitamin C is the single most important specialty chemical manufactured in the world. Understanding the structural determinants of specificity, catalysis, and stability for 2,5-DKGR A is of substantial commercial interest.

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The shared surface topology of two chemically dissimilar but functionally equivalent molecular structures has been analyzed. A carbohydrate moiety (␣-D-mannopyranoside) and a peptide molecule (DVFYPYP-YASGS) bind to concanavalin A at a common binding site. The cross-reactivity of the polyclonal antibodies (pAbs) was used for understanding the topological relationship between these two independent ligands. The anti-␣-D-mannopyranoside pAbs recognized various peptide ligands of concanavalin A, and the anti-DVFY-PYPYASGS pAbs recognized the carbohydrate ligands, providing direct evidence of molecular mimicry. On the basis of differential binding of various rationally designed peptide analogs to the anti-␣-D-mannopyranoside pAbs, it was possible to identify different peptide residues critical for the mimicry. The comparison of circular dichroism profiles of the designed analogs suggests that the carbohydrate mimicking conformation of the peptide ligand incorporates a polyproline type II structural fold. The concanavalin A binding activity of these analogs was found to have a direct correlation with the topological relationship between peptide and carbohydrate ligands.The functional mimicry involving unrelated molecules is often encountered. Many times it occurs by design and is used as an effective control during various regulatory mechanisms. However, sometimes the accidental structural resemblances lead to aberrations, which are expressed in terms of clinical manifestations. For example, it has been suggested that the mimicry between microbial or viral peptides and the self-peptides presented inappropriately on a target tissue could initiate an autoimmune attack (1). On the other hand, many enzymatically regulated events are controlled by intervention of proteinaceous inhibitors, which mimic substrate binding. The complementarity between serine proteases and the corresponding substrates is matched by aprotinin, the chymotrypsin inhibitor, and many other such inhibitors (2, 3). In addition, molecular mimicry has implications for rational drug design applications. Considerable efforts have been invested in the development of peptidomimetic drugs using nonpeptidyl surrogates (4 -7).Systematic approaches involving computational as well as experimental tools have been used to analyze and exploit topological similarity between dissimilar molecules (8 -12). It is apparent that the structural rules governing molecular mimicry are required to be defined for its successful exploitation. Concanavalin A (ConA), 1 a lectin known to be specific for binding to certain mannose-containing carbohydrates, provides an appropriate model system for this purpose. Peptidyl ligands have been characterized that bind to ConA with affinities comparable with those of the carbohydrate ligand methyl ␣-Dmannopyranoside (13,14). We have shown that the peptide and the carbohydrate ligands of ConA are true topological mimics. Cross-reactivity of polyclonal antibodies (pAbs) against an assortment of designed peptide analogs is used here to delineate...