Decoupled Roles for the Atypical, Bifurcated Binding Pocket of the ybfF Hydrolase

Abstract

Serine hydrolases have diverse intracellular substrates, biological functions, and structural plasticity, and are thus important for biocatalyst design. Amongst serine hydrolases, the recently described ybfF enzyme family are promising novel biocatalysts with an unusual bifurcated substrate-binding cleft and the ability to recognize commercially relevant substrates. We characterized in detail the substrate selectivity of a novel ybfF enzyme from Vibrio cholerae (Vc-ybfF) by using a 21-member library of fluorogenic ester substrates. We assigned the roles of the two substrate-binding clefts in controlling the substrate selectivity and folded stability of Vc-ybfF by comprehensive substitution analysis. The overall substrate preference of Vc-ybfF was for short polar chains, but it retained significant activity with a range of cyclic and extended esters. This broad substrate specificity combined with the substitutional analysis demonstrates that the larger binding cleft controls the substrate specificity of Vc-ybfF. Key selectivity residues (Tyr116, Arg120, Tyr209) are also located at the larger binding pocket and control the substrate specificity profile. In the structure of ybfF the narrower binding cleft contains water molecules prepositioned for hydrolysis, but based on substitution this cleft showed only minimal contribution to catalysis. Instead, the residues surrounding the narrow binding cleft and at the entrance to the binding pocket contributed significantly to the folded stability of Vc-ybfF. The relative contributions of each cleft of the binding pocket to the catalytic activity and folded stability of Vc-ybfF provide a valuable map for designing future biocatalysts based on the ybfF scaffold.

Structural Basis for the Strict Substrate Selectivity of the Mycobacterial Hydrolase LipW

“…38–40 Fluorogenic substrates were synthesized as previously described 38;41;42 . Fluorogenic substrates were prepared as stock solutions in DMSO (10 mM) and were diluted into PBS containing acetylated BSA (Sigma; 0.1 mg/mL) to starting concentrations between 100–1000 μM, depending on the K m value of Mm LipW for the substrate.…”

“…All of the substrates were synthesized as described previously. 38;42;51 C) Global comparison of the catalytic specificity ( k cat / K M ) of LipW against each of the 21 substrates (structures and numbering given in Fig. 3B) with ester classes labeled.…”

Implementation of a Collaborative Series of Classroom-Based Undergraduate Research Experiences Spanning Chemical Biology, Biochemistry, and Neurobiology

“…Introduction of a serine hydrolase to these protected fluorogenic substrates catalyzes their activation, with the relative efficiency of enzyme-catalyzed hydrolysis dependent upon the specific recognition of the acyl-chain structure of these fluorogenic esters by the serine hydrolase (Lavis et al ., 2011; Hedge et al ., 2012; Tian et al ., 2012). Thus, a series of bis-acyloxymethyl ether derivatives of fluorescein can serve as a tool for characterizing the substrate specificity of mycobacterial serine hydrolases (Hedge et al ., 2012; Ellis et al ., 2013; Filippova et al ., 2013; Johnson et al ., 2014a; Lukowski et al ., 2014) and for developing novel neural-signaling sensors (Tian et al ., 2012). …”

Distinct Substrate Selectivity of a Metabolic Hydrolase from Mycobacterium tuberculosis

“…All of the substrates were synthesized as described previously. 18 (∼16−20 h) at 16°C. Bacterial cultures were collected by centrifugation at 6000g for 10 min at 4°C.…”