Evolution of an enzyme active site: The structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase

Abstract

Muconate lactonizing enzyme (MLE), a component of the ␤-ketoadipate pathway of Pseudomonas putida, is a member of a family of related enzymes (the ''enolase superfamily'') that catalyze the abstraction of the ␣-proton of a carboxylic acid in the context of different overall reactions. New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0-Å resolution. The packing of the octameric enzyme in this crystal form is unusual, because the asymmetric unit contains three subunits. The structure of MLE presented here contains no bound metal ion, but is very similar to a recently determined Mn 2؉ -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 ligands suggests that enolase may retain some characteristics of the ancestor of this enzyme family. Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (i) that the 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 scaffold are conserved, whereas their identities and roles in catalysis vary.

Directed evolution of a (βα) 8 -barrel enzyme to catalyze related reactions in two different metabolic pathways

“…In addition, two of the catalytically important residues are located at equivalent positions in ␤-strand 1 (Asp-8 in tHisA and Cys-7 in tTrpF) and in ␤-strand 6 (Thr-164 in tHisA and Asp-126 in tTrpF), as are residues that bind the phosphate moieties of the substrates between ␤-strand 7 and ␣-helix 8Ј. A similar ''structural conservation'' of catalytically essential residues has been found for several members from the superfamily of enolases, which comprises (␤␣) 8 -barrel enzymes that catalyze the abstraction of the ␣-proton from a carboxylic acid (26).…”

Expanding protein universe and its origin from the biological Big Bang

“…General models of protein evolution that are based solely on the requirement of protein stability reproduced observed conservation of amino acids in proteins with dissimilar sequences with reasonable accuracy (4,7). Several authors have also pointed out that diverse functions can be carried out by proteins of the same fold (8)(9)(10)(11). However, a striking observation is that different folds are represented to a different degree in genomes: some folds are represented by many nonhomologous and functionally diverse proteins, whereas other folds are uniquely represented by a single sequence (orphans, i.e., domains that are not structurally similar to any other domains; refs.…”

Untitled

“…For example, proteins of the TIM-barrel and flavodoxin folds have "super-sites", i.e. active sites in the same spatial location, although amino acids forming the sites and the biochemical function of these proteins have widely diverged [38,39]. This extreme conservation of the spatial location of functionally crucial residues supports the assumption of common location of the specificity determinants.…”