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 alpha+beta capping domain and a (beta/alpha)7beta-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth beta-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second beta-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth beta-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 beta-strand and Arg 147 at the end of the second beta-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 beta-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.
Rakus
John F
JF
Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, Illinois 61801, USA.
Fedorov
Alexander A
AA
Fedorov
Elena V
EV
Glasner
Margaret E
ME
Vick
Jacob E
JE
Babbitt
Patricia C
PC
Almo
Steven C
SC
Gerlt
John A
JA
D000595:Amino Acid Sequence; D001665:Binding Sites; D005075:Biological Evolution; D003460:Crystallization; D018360:Crystallography, X-Ray; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D042301:Sphingomonadaceae / Q000201:enzymology / Q000235:genetics*
D006836:Hydro-Lyases; D010751:Phosphopyruvate Hydratase; C021545:mannonate dehydratase