Serine hydrolases with their diverse intracellular substrates, biological functions, and structural plasticity are important targets for biocatalyst design. Amongst serine hydrolases, the recently described ybfF enzyme family are promising novel biocatalysts due to an unusual bifurcated substrate-binding cleft and the ability to recognize commercially relevant substrates. Herein, we characterized the detailed substrate selectivity of a novel ybfF enzyme from Vibrio cholerae using a versatile 21-member library of fluorogenic ester substrates. The diverse functionalities and low background reactivity of the fluorogenic substrate library allow us to precisely define the broad substrate selectivity profile of ybfF. We then assigned the roles of the dual substrate binding clefts in controlling the substrate selectivity and folded stability of ybfF. The substrate selectivity of ybfF is mainly enforced by the more voluminous substrate-binding cleft and by key substrate selectivity residues localized to the larger binding cleft. In contrast, the narrow binding pocket lacks a defined role in the catalytic activity of ybfF, but provides essential contributions to the folded stability of ybfF. The combined contributions of each cleft of this unusual binding pocket to the catalytic activity and substrate selectivity of ybfF provides a unique mechanism for serine hydrolases to differentiate between their diverse biological substrates. The relative contributions of each cleft of the binding pocket to the catalytic activity and folded stability of ybfF also provides a valuable map for designing future biocatalysts based on the ybfF scaffold.

Decoupled roles for the atypical, bifurcated binding pocket of the ybfF hydrolase

246th National Meeting (2013)

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