We assigned
l-talarate dehydratase (TalrD) and galactarate
dehydratase (GalrD) functions to a group of orthologous
proteins in the mechanistically diverse
enolase superfamily, focusing our characterization on the
protein encoded by the Salmonella typhimurium LT2 genome (GI:16766982; STM3697). Like the homologous
mandelate racemase,
l-fuconate dehydratase, and
d-tartrate dehydratase, the active site of TalrD/GalrD contains a general
acid/base Lys 197 at the end of the second beta-strand in the (beta/alpha)7beta-barrel domain, Asp 226, Glu 252, and Glu 278 as
ligands for the essential Mg2+ at the ends of the third, fourth, and fifth beta-strands, a general
acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth beta-strands, and an electrophilic Glu 348 at the end of the eighth beta-strand. We discovered the function of STM3697 by screening a library of
acid sugars; it catalyzes the efficient
dehydration of both
l-talarate (kcat = 2.1 s-1, kcat/Km = 9.1 x 10(3) M-1 s-1) and galactarate (kcat = 3.5 s-1, kcat/Km = 1.1 x 10(4) M-1 s-1). Because
l-talarate is a previously unknown metabolite, we demonstrated that S. typhimurium LT2 can utilize
l-talarate as
carbon source. Insertional disruption of the gene encoding STM3697 abolishes this phenotype; this disruption also diminishes, but does not eliminate, the ability of the organism to utilize galactarate as
carbon source. The
dehydration of
l-talarate is accompanied by competing epimerization to galactarate; little epimerization to
l-talarate is observed in the
dehydration of galactarate. On the basis of (1) structures of the wild type
enzyme complexed with l-lyxarohydroxamate, an analogue of the enolate intermediate, and of the K197A mutant complexed with l-glucarate, a substrate for exchange of the alpha-
proton, and (2) incorporation of
solvent deuterium into galactarate in competition with
dehydration, we conclude that Lys 197 functions as the galactarate-specific base and His 328 functions as the
l-talarate-specific base. The epimerization of
l-talarate to galactarate that competes with
dehydration can be rationalized by partitioning of the enolate intermediate between
dehydration (departure of the 3-
OH group catalyzed by the conjugate
acid of His 328) and epimerization (protonation on C2 by the conjugate
acid of Lys 197). The promiscuous catalytic activities discovered for STM3697 highlight the evolutionary potential of a "conserved" active site architecture.