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The isomeric mixture of cis- and trans-1,3-dichloropropene constitutes the active component of a widely used nematocide known as Telone II®. The mixture is processed by various soil bacteria to acetaldehyde through the 1,3-dichloropropene catabolic pathway. The pathway relies on an isomer-specific hydrolytic dehalogenation reaction catalyzed by cis- or trans-3-chloroacrylic acid dehalogenase, known respectively as cis-CaaD and CaaD. Previous sequence analysis and crystallographic studies of the native and covalently modified enzymes identified Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114 as key binding and catalytic residues in cis-CaaD. Mutagenesis of these residues confirmed their importance to the dehalogenation reaction. Crystal structures of the native enzyme (2.01Å resolution) and the enzyme covalently modified at the Pro-1 nitrogen by 2-hydroxypropanoate (1.65Å resolution) are reported here. Both structures are at a resolution higher than previously reported (2.75Å and 2.1Å resolution, respectively). The conformation of the covalent adduct is strikingly different from that previously reported due to its interaction with a 7-residue loop (Thr-32 to Leu-38). The participation of another active site residue, Arg-117, in catalysis and inactivation was also examined. The implications of the combined findings for the mechanisms of catalysis and inactivation are discussed., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Macrophage migration inhibitory factor (MIF) is an important immunoregulatory protein that has been implicated in several inflammatory diseases. MIF also has a phenylpyruvate tautomerase (PPT) activity, the role of which remains elusive in these biological activities. The acetylene compound, 2-oxo-4-phenyl-3-butynoate (2-OPB), has been synthesized and tested as a potential irreversible inhibitor of its enzymatic activity. Incubation of the compound with MIF results in the rapid and irreversible loss of the PPT activity. Mass spectral analysis established that the amino-terminal proline, previously implicated as a catalytic base in the PPT-catalyzed reaction, is the site of covalent modification. Inactivation of the PPT activity likely occurs by a Michael addition of Pro-1 to C-4 of the inhibitor. Attempts to crystallize the inactivated complex to confirm the structure of the adduct on the covalently modified Pro-1 by X-ray crystallography were not successful. Nor was it possible to unambiguously interpret electron density observed in the active sites of the native crystals soaked with the inhibitor. This may be due to crystal packing in that the side chain of Glu-16 from an adjacent trimer occupies one active site. However, this crystal contact may be partially responsible for the high-resolution quality of these MIF crystals. Nonetheless, because MIF is a member of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta structural motif and a catalytic Pro-1, 2-OPB may find general use as a probe of tautomerase superfamily members that function as PPTs.