The functional diversity of the Enolase Superfamily (ENS) continues to expand. This study summarizes the discovery of yet another unique function: cis-3-hydroxy-L-proline dehydratase. The prevailing strategy of the EFI was employed: bioinformatics-driven predictions refined the boundaries of an in vitro screening assay, and enzymatic activity was validated as a physiological function using microbiological techniques. In this way, the potential of an uncharacterized gene sequenced in a genome project is efficiently realized.
The genome of Labrenzia aggregata IAM 12614 encodes an uncharacterized member of the muconate lactonizing enzyme (MLE) subgroup of the enolase superfamily (UniProt ID A0NXQ8 ). The gene encoding A0NXQ8 is located between genes that encode members of the proline racemase superfamily, 4R-hydroxyproline 2-epimerase (UniProt ID A0NXQ7 ; 4HypE) and trans-3-hydroxy-l-proline dehydratase (UniProt ID A0NXQ9 ; t3LHypD). A0NXQ8 was screened with a library of proline analogues; two reactions were observed with cis-3-hydroxy-l-proline (c3LHyp), competing 2-epimerization to trans-3-hydroxy-d-proline (1,1-proton transfer) and dehydration to Δ1-pyrroline-2-carboxylate (β-elimination; c3LHyp dehydratase), with eventual total dehydration. The genome context encoding A0NXQ8 both (1) confirms its novel c3LHyp dehydratase function and (2) provides evidence for metabolic pathways that allow L. aggregata to utilize several isomeric 3- and 4-hydroxyprolines as sole carbon sources.
Figure 4: Initial progress of the A0NXQ8-catalyzed reaction with c3LHyp during the initial of the reaction showing formation of t3DHyp (epimerization) and Pyr2C (dehydration) monitiored by 1H NMR; the 1H NMR assay contained 10 mM c3LHyp, 10 mM MgCl2, 50 mM sodium phosphate, pH 8.0, and 0.14 μM A0NXQ8. Eventually, only Pyr2C is observed.
Reprinted with permission from Zhang et al. JACS. © 2015 American Chemical Society.