Evolution of enzymatic activities in the enolase superfamily: D-tartrate dehydratase from Bradyrhizobium japonicum

We focus on the assignment of function to and elucidation of structure-function relationships for a member of the mechanistically diverse enolase superfamily encoded by the Bradyrhizobium japonicum genome (b116730; GI:27381841). As suggested by sequence alignments, the active site contains the same functional groups found in the active site of mandelate racemase (MR) that catalyzes a 1,1-proton transfer reaction: two acid/base catalysts, Lys 184 at the end of the second β-strand, and a His 322-Asp 292 dyad at the ends of the seventh and sixth β-strands, respectively, as well as ligands for an essential Mg²⁺, Asp 213, Glu 239, and Glu 265 at the ends of the third, fourth, and fifth β-strands, respectively. We screened a library of 46 acid sugars and discovered that only D-tartrate is dehydrated, yielding oxaloacetate as product. The kinetic constants (k_cat = 7.3 s ^-1; k_cat/K_M = 8.5 × 10⁴ M ^-1 s^-1) are consistent with assignment of the D-tartrate dehydratase (TarD) function. The kinetic phenotypes of mutants as well as the structures of liganded complexes are consistent with a mechanism in which Lys 184 initiates the reaction by abstraction of the α-proton to generate a Mg²⁺-stabilized enediolate intermediate, and the vinylogous β-elimination of the 3-OH group is general acid-catalyzed by the His 322, accomplishing the anti-elimination of water. The replacement of the leaving group by solvent-derived hydrogen is stereorandom, suggesting that the enol tautomer of oxaloacetate is the product; this expectation was confirmed by its observation by ¹H NMR spectroscopy. Thus, the TarD-catalyzed reaction is a "simple" extension of the two-step reaction catalyzed by MR: base-catalyzed proton abstraction to generate a Mg²⁺-stabilized enediolate intermediate followed by acid-catalyzed decomposition of that intermediate to yield the product.