EVOLUTION OF AN ENZYME ACTIVE-SITE - THE STRUCTURE OF A NEW CRYSTAL FORM OF MUCONATE LACTONIZING ENZYME COMPARED WITH MANDELATE RACEMASE AND ENOLASE

Muconate lactonizing enzyme (MLE), a component of the beta-ketoadipate pathway of Pseudomonas putida, is a member of a family of related enz ymes (the ''enolase superfamily'') that catalyze the abstraction of th e alpha-proton of a carboxylic acid in the context of different overal l reactions. New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0- Angstrom resolution. The packing o f the octameric enzyme in this crystal form is unusual, because the as ymmetric unit contains three subunits, The structure of MLE presented here contains no bound metal ion, but is very similar to a recently de termined Mn2+-bound structure. Thus, absence of the metal ion does not perturb the structure of the active site. The structures of enolase, mandelate racemase, and MLE were superimposed. A comparison of metal l igands suggests that enolase may retain some characteristics of the an cestor of this enzyme family. Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (i) that th e position of catalytic atoms remains constant, although the residues that contain them are located at different points in the protein fold; and (ii) that the positions of catalytic residues in the protein scaf fold are conserved, whereas their identities and roles in catalysis va ry.