The members of the me
chanisti
cally diverse, (
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8-barrel fold-
containing enolase superfamilyevolved from a
common progenitor but
catalyze different rea
ctions using a
conserved partial rea
ction.The mole
cular pathway for natural divergent evolution of fun
ction in the superfamily is unknown. Wehave identified single-site mutants of the (
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8-barrel domains in both the
L-Ala-
D/
L-Glu epimerase from
Escherichia coli (AEE) and the mu
conate la
ctonizing enzyme II from
Pseudomonas sp. P51 (MLE II)that
catalyze the
o-su
ccinylbenzoate synthase (OSBS) rea
ction as well as the wild-type rea
ction. Theseenzymes are members of the MLE subgroup of the superfamily, share
conserved lysines on oppositesides of their a
ctive sites, but
catalyze a
cid- and base-mediated rea
ctions with different me
chanisms. A
comparison of the stru
ctures of AEE and the OSBS from
E. coli was used to design the D297G mutantof AEE; the E323G mutant of MLE II was isolated from dire
cted evolution experiments. Although neitherwild-type enzyme
catalyzes the OSBS rea
ction, both mutants
complement an
E. coli OSBS auxotrophand have measurable levels of OSBS a
ctivity. The analogous mutations in the D297G mutant of AEEand the E323G mutant of MLE II are ea
ch lo
cated at the end of the eighth
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8-barreland alter the ability of AEE and MLE II to bind the substrate of the OSBS rea
ction.
The substitutionsrelax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reactionwith the assistance of the active site lysines. The generation of fun
ctionally promis
cuous and me
chanisti
callydiverse enzymes via single-amino a
cid substitutions likely mimi
cs the natural divergent evolution ofenzymati
c a
ctivities and also highlights the utility of the (
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8-barrel as a s
caffold for new fun
ction.