Evolution of enzymatic activities in the enolase superfamily: D-mannonate dehydratase from Novosphingobium aromaticivorans

The D-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of the wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg²⁺ and also in the presence of Mg²⁺ and the 2-keto-3-keto-D-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the D-mannonate substrate. As previously observed in the structures of other members of the enolase superfamily, ManD contains two domains, an N-terminal α+β capping domain and a (β/α)₇β-barrel domain. The barrel domain contains the ligands for the essential Mg ²⁺, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth β-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second β-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth β-strands, respectively, that are found in many members of the superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second β-strand and Arg 147 at the end of the second β-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third β-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-D-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg²⁺ are the only conserved residues in the enolase superfamily, establishing the primary functional importance of the Mg²⁺-assisted strategy for stabilizing the enolate anion intermediate.