(127d) Cross-Catalytic Activity Across Two Subgroups of the Enolase Superfamily

The members of the enolase superfamily share a conserved bi-domain structure and a common partial catalytic mechanism, although members catalyze more than a dozen different reactions. Muconate lactonizing enzyme (MLE), for example, has been identified as a component of the beta-ketoadipate pathway of Pseudomonas putida. Evidence for divergent evolution within the mandelate racemase (MR) or muconate lactonizing enzyme (MLE) subfamilies has been demonstrated. Since MR itself and o-succinylbenzoate synthase/N-acetyl amino acid racemase (OSBS/NAAR) from Amycolatopsis (MLE subfamily) possess nearly superimposable crystal structures, we hypothesized that both subfamilies have a common ancestor. Both subfamilies contain members with racemase functionality employing similar substrates; however, the orientation of substrate binding vis-à-vis the divalent metal seems to be different. By introducing just one or three mutations into MR from Ps. putida, we have obtained variants with promiscuous MR, NAAR, and N-succinylamino acid racemase (NSAR) activity and thus for the first time demonstrated cross-reactivity across two enolase superfamily subgroups, the MR and MLE subgroups. Inhibition of promiscuous NAAR activity in MR with (R)-phenylglycine by MR inhibitor cupferron demonstrates that the same active site architecture is involved in both MR and NAAR catalysis. We provide further evidence for divergent evolution across both subgroups by introducing the active site residues from the MLE subgroup into the MR scaffold. By exchanging the histidine-aspartic acid (H297/D270) catalytic base dyad of MR to the structural homologs in NAAR, we obtained a quintuple variant F52V/E317D/L319A/H297G/D270K, which retains, albeit low, promiscuous MR and NAAR activity. While change from wild-type MR to the E317D variant involved a strong negative trade-off with the increase in the new (NAAR) function from immeasurable levels in the wild-type MR accompanied by a drastic decrease in the original (MR) function of three orders of magnitude, both functions improved 102-103-fold upon the change from the E317D to the F52V/E317D/L319A variant.