Evolution of Enzymatic Activity in the Enolase Superfamily:  Structural Studies of the Promiscuous o-Succinylbenzoate Synthase from Amycolatopsis^,

Abstract

Divergent evolution of enzyme function is commonly explained by a gene duplication event followed by mutational changes that allow the protein encoded by the copy to acquire a new function. An alternate hypothesis is that this process is facilitated when the progenitor enzyme acquires a second function while maintaining the original activity. This phenomenon has been suggested to occur in the o-succinylbenzoate synthase (OSBS) from a species of Amycolatopsis that catalyzes not only the physiological syn-dehydration reaction of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate but also an accidental racemization of N-acylamino acids [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258]. To understand the molecular basis of this promiscuity, three-dimensional structures of liganded complexes of this enzyme have been determined, including the product of the OSBS reaction and three N-acylamino acid substrates for the N-acylamino acid racemase (NAAAR) reaction, N-acetylmethionine, N-succinylmethionine, and N-succinylphenylglycine, to 2.2, 2.3, 2.1, and 1.9 A resolution, respectively. These structures show how the active-site cavity can accommodate both the hydrophobic substrate for the OSBS reaction and the substrates for the accidental NAAAR reaction. As expected, the N-acylamino acid is sandwiched between lysines 163 and 263, which function as the catalytic bases for the abstraction of the alpha-proton in the (R)- and (S)-racemization reactions, respectively [Taylor Ringia, E. A., Garrett, J. B, Thoden, J. B., Holden, H. M., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 42, 224-229]. Importantly, the protein forms specific favorable interactions with the hydrophobic amino acid side chain, alpha-carbon, carboxylate, and the polar components of the N-acyl linkage. Accommodation of the components of the N-acyl linkage appears to be the reason that this enzyme is capable of a racemization reaction on these substrates, whereas the orthologous OSBS from Escherichia coli lacks this functionality.

A structural model of latent evolutionary potentials underlying neutral networks in proteins

“…As reported in the parallel experimental study (Amitai et al, 2007), the reactivity of serum paraoxonase with at least five substrates were well demonstrated. For several members of the very divergent enolase superfamily, it was recently argued that the evolution from an enzyme with one specificity toward another enzyme happened via promiscuous intermediates (Matsumura and Ellington, 2001;Glasner et al, 2006;Thoden et al, 2004). Taken together, these data support the idea that conformational intermediates between two or more different structures can correspond to evolutionary transitional forms that bridge different dominant biological functions.…”

Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

“…S1). Like most other members of the enolase superfamily, the five enzymes from the NSAR/OSBS subfamily are multimers (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). The three previously characterized NSAR/OSBS subfamily enzymes are (53).…”

“…octamers, and the NSAR/OSBS subfamily enzymes from E. faecalis and L. innocua are dimers (22)(23)(24). In contrast, the OSBSs from other subfamilies are all monomers.…”

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

“…25 This structure includes the OSB substrate and the magnesium ion. According to experimental data, OSBS from Amycolatopsis assembles as an octamer.…”

“…According to experimental data, OSBS from Amycolatopsis assembles as an octamer. 25 Active sites are found inside each subunit, with residues of the closer subunit placed at 15 Å from the active site chosen to model the reaction (chain B). Our model consisted of two subunits, chains A and B and the distance between the active sites of these two subunits is about 30 Å.…”