TY - JOUR
T1 - Evolution of enzymatic activities in the enolase superfamily
T2 - D-tartrate dehydratase from Bradyrhizobium japonicum
AU - Yew, Wen Shan
AU - Fedorov, Alexander A.
AU - Fedorov, Elena V.
AU - Wood, Bryant Mc Kay
AU - Almo, Steven C.
AU - Gerlt, John A.
PY - 2006/12/12
Y1 - 2006/12/12
N2 - 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 (kcat = 7.3 s -1; kcat/KM = 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.
AB - 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 (kcat = 7.3 s -1; kcat/KM = 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.
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U2 - 10.1021/bi061688g
DO - 10.1021/bi061688g
M3 - Article
C2 - 17144653
AN - SCOPUS:33845398832
SN - 0006-2960
VL - 45
SP - 14598
EP - 14608
JO - Biochemistry
JF - Biochemistry
IS - 49
ER -