2dw6
From Proteopedia
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Crystal structure of the mutant K184A of D-Tartrate Dehydratase from Bradyrhizobium japonicum complexed with Mg++ and D-tartrate
Overview
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.
About this Structure
2DW6 is a Single protein structure of sequence from Bradyrhizobium japonicum with , and as ligands. Active as D(-)-tartrate dehydratase, with EC number 4.2.1.81 Full crystallographic information is available from OCA.
Reference
Evolution of enzymatic activities in the enolase superfamily: D-tartrate dehydratase from Bradyrhizobium japonicum., Yew WS, Fedorov AA, Fedorov EV, Wood BM, Almo SC, Gerlt JA, Biochemistry. 2006 Dec 12;45(49):14598-608. PMID:17144653
Page seeded by OCA on Tue Jan 29 19:13:22 2008
