The Enolase Superfamily:  A General Strategy for Enzyme-Catalyzed Abstraction of the α-Protons of Carboxylic Acids

Abstract

We have discovered a superfamily of enzymes related by their ability to catalyze the abstraction of the alpha-proton of a carboxylic acid to form an enolic intermediate. Although each reaction catalyzed by these enzymes is initiated by this common step, their overall reactions (including racemization, beta-elimination of water, beta-elimination of ammonia, and cycloisomerization) as well as the stereochemical consequences (syn vs anti) of the beta-elimination reactions are diverse. Analysis of sequence and structural similarities among these proteins suggests that all of their chemical reactions are mediated by a common active site architecture modified through evolution to allow the enolic intermediates to partition to different products in their respective active sites via different overall mechanisms. All of these enzymes retain the ability to catalyze the thermodynamically difficult step of proton abstraction. These homologous proteins, designated the "enolase superfamily", include enolase as well as more metabolically specialized enzymes: mandelate racemase, galactonate dehydratase, glucarate dehydratase, muconate-lactonizing enzymes, N-acylamino acid racemase, beta-methylaspartate ammonia-lyase, and o-succinylbenzoate synthase. Comparative analysis of structure-function relationships within the superfamily suggests that carboxyphosphonoenolpyruvate synthase, another member of the superfamily, does not catalyze the reaction proposed in the literature but catalyzes an enolase-like reaction instead. The established and deduced structure-function relationships in the superfamily allow the prediction that other apparent members of the family for which no catalytic functions have yet been assigned will also perform chemistry involving abstraction of the alpha-protons of carboxylic acids.

A mechanism for the association of amino acids with their codons and the origin of the genetic code

“…The reaction is proposed to occur in a step-wise fashion, with stabilization of an initially formed enolate by Mg ϩ2 associated with the diphosphate moiety of the dinucleotide, followed by attack of the enolate on the ␤ phosphate. Stabilization of an enolate by metal ions is a common strategy in the enolase superfamily of protein enzymes (43). The reaction could be further catalyzed by hydrogen bonding between either C or G in the second position and the ␤ phosphate that is to be transferred (not shown).…”

Divergent Evolution of (??)8-Barrel Enzymes

“…However, their similar three-dimensional structures and catalytic mechanisms indicate a common evolutionary origin (Babbitt and Gerlt, 2000). Members of the enolase superfamiliy consist of two domains: a larger (βα) 7 β barrel domain, which is a modified version of the (βα) 8 -barrel, and a mixed α/β domain that is formed by the N-and C-terminal parts of the sequence (Babbitt et al, 1996). The mixed α/β domain is an important determinant of the substrate specificity and caps the barrel domain at the C-terminal ends of the β-strands, where the residues that are essential for catalysis are located.…”

Erratum: correction: Plant diversity enhances ecosystem responses to elevated CO2 and nitrogen deposition

“…7, reactions 14-16) and enolase superfamily (Fig. 7, reactions 17-19) [30][31][32] indicate that the active sites all generate carbanionic transition states from bound substrates and then use carbanion chemistry for directed fluxes and distinct product outcomes. These families should be fruitful starting points for directed enzyme evolution to elicit new fluxes, based on the knowledge that carbanion chemistry will be facilitated insight overview NATURE in one of the co-substrates and that binding sites can be reengineered for electrophilic substrate components.…”

“…1) has remained a seismic gap for at least the past two centuries 30 . At the higher end of our estimated slip rates, the faults bounding the Shillong plateau could absorb one-third of the inferred Himalayan contraction rate of 18 mm yr -1 (ref.…”