Evolution of enzymatic activities in the enolase superfamily: Identification of the general acid catalyst in the active site of D-glucarate dehydratase from Escherichia coli

D-Glucarate dehydratase from Escherichia coli (GlucD), a member of the enol ase superfamily, catalyzes the dehydration of both D-glucarate and L-idarat e to form 5-keto-4-deoxy-D-glucarate (KDG). Previous mutagenesis and struct ural studies identified Lys 207 and the His 339-Asp 313 dyad as the general basic catalysts that abstract the C5 proton from L-idarate and D-glucarate , respectively, thereby initiating the reaction by formation of a stabilize d enediolate anion intermediate [Gulick, A. M., Hubbard, B. K., Gerlt, J. A ., and Rayment, I. (2000) Biochemistry 39, 4590-4602]. The vinylogous elimi nation of the 4-OH group from this intermediate presumably requires a gener al acid catalyst. The structure of GlucD with KDG and 4-deoxy-D-glucarate b ound in the active site revealed that only His 339 and Asn 341 are proximal to the presumed position of the 4-OH leaving group. The N341D and N341L mu tants of GlucD were constructed and subjected to both mechanistic and struc tural analyses. The N341L but not N341D mutant catalyzed the dehydrofluorin ation of 4-deoxy-4-fluoro-D-glucarate, demonstrating that in this mutant th e initial proton abstraction from C5 can be decoupled from elimination of t he leaving group from C4. The kinetic properties and structures of these mu tants suggest that either Asn 341 participates in catalysis as the general acid that facilitates the departure of the 4-leaving group or is essential for proper positioning of His 339. In the latter scenario, His 339 would fu nction not only as the general base that abstracts the C5 proton from D-glu carate but also as the general acid that catalyzes both the departure of th e 4-OH group and the stereospecific incorporation of solvent hydrogen with retention of configuration to form the KDG product. The involvement of a si ngle functional group in this reaction highlights the plasticity of the act ive site design in members of the enolase superfamily.