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 -strands, respectively, as well as ligands for an essential Mg{sup 2+}, 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 (k{sub cat} = 7.3 s{sup -1}; k{sub cat}/K{sub M} = 8.5 x 10{sup 4} M{sup -1} s{sup -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 Mg{sup 2+}-stabilized enediolate intermediate, and the vinylogous -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 {sup 1}H 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 Mg{sup 2+}-stabilized enediolate intermediate followed by acid-catalyzed decomposition of that intermediate to yield the product.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 930150
- Report Number(s):
- BNL-80808-2008-JA; TRN: US200822%%1350
- Journal Information:
- Biochemistry, Vol. 45; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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