The members of the mecha
nistically diverse, (
![](/images/gifchars/beta2.gif)
/
![](/images/gifchars/alpha.gif)
)
8-barrel fold-co
ntai
ni
ng e
nolase superfamilyevolved from a commo
n proge
nitor but catalyze differe
nt reactio
ns usi
ng a co
nserved partial reactio
n.The molecular pathway for
natural diverge
nt evolutio
n of fu
nctio
n i
n the superfamily is u
nk
now
n. Wehave ide
ntified si
ngle-site muta
nts of the (
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/
![](/images/gifchars/alpha.gif)
)
8-barrel domai
ns i
n both the
L-Ala-
D/
L-Glu epimerase from
Escherichia coli (AEE) a
nd the muco
nate lacto
nizi
ng e
nzyme II from
Pseudomonas sp. P51 (MLE II)that catalyze the
o-succi
nylbe
nzoate sy
nthase (OSBS) reactio
n as well as the wild-type reactio
n. Thesee
nzymes are members of the MLE subgroup of the superfamily, share co
nserved lysi
nes o
n oppositesides of their active sites, but catalyze acid- a
nd base-mediated reactio
ns with differe
nt mecha
nisms. Acompariso
n of the structures of AEE a
nd the OSBS from
E. coli was used to desig
n the D297G muta
ntof AEE; the E323G muta
nt of MLE II was isolated from directed evolutio
n experime
nts. Although
neitherwild-type e
nzyme catalyzes the OSBS reactio
n, both muta
nts compleme
nt a
n E. coli OSBS auxotropha
nd have measurable levels of OSBS activity. The a
nalogous mutatio
ns i
n the D297G muta
nt of AEEa
nd the E323G muta
nt of MLE II are each located at the e
nd of the eighth
![](/images/gifchars/beta2.gif)
-stra
nd of the (
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/
![](/images/gifchars/alpha.gif)
)
8-barrela
nd alter the ability of AEE a
nd MLE II to bi
nd the substrate of the OSBS reactio
n.
The substitutionsrelax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reactionwith the assistance of the active site lysines. The ge
neratio
n of fu
nctio
nally promiscuous a
nd mecha
nisticallydiverse e
nzymes via si
ngle-ami
no acid substitutio
ns likely mimics the
natural diverge
nt evolutio
n ofe
nzymatic activities a
nd also highlights the utility of the (
![](/images/gifchars/beta2.gif)
/
![](/images/gifchars/alpha.gif)
)
8-barrel as a scaffold for
new fu
nctio
n.