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 (bll6730; 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 beta-strand, and a His 322-Asp 292 dyad at the ends of the seventh and sixth beta-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 beta-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 x 10(4) 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 alpha-proton to generate a Mg2+-stabilized enediolate intermediate, and the vinylogous beta-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.
Yew
Wen Shan
WS
Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Fedorov
Alexander A
AA
Fedorov
Elena V
EV
Wood
Bryant McKay
BM
Almo
Steven C
SC
Gerlt
John A
JA
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D020369:Bradyrhizobium / Q000201:enzymology*; D017931:DNA Primers; D019143:Evolution, Molecular; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
D001426:Bacterial Proteins; D017931:DNA Primers; D009243:NAD; C034710:D-(-)-tartrate dehydratase; D006836:Hydro-Lyases; D010751:Phosphopyruvate Hydratase; D008274:Magnesium