Distinct Substrate Selectivity of a Metabolic Hydrolase from Mycobacterium tuberculosis

Abstract

The transition between dormant and active Mycobacterium tuberculosis infection requires reorganization of its lipid metabolism and activation of a battery of serine hydrolase enzymes. Among these serine hydrolases, Rv0045c is a mycobacterial-specific serine hydrolase with limited sequence homology outside mycobacteria but structural homology to divergent bacterial hydrolase families. Herein, we determined the global substrate specificity of Rv0045c against a library of fluorogenic hydrolase substrates, constructed a combined experimental and computational model for its binding pocket, and performed comprehensive substitutional analysis to develop a structural map of its binding pocket. Rv0045c showed strong substrate selectivity toward short, straight chain alkyl esters with the highest activity toward four atom chains. This strong substrate preference was maintained through the combined action of residues in a flexible loop connecting the cap and α/β hydrolase domains and in residues close to the catalytic triad. Two residues bracketing the substrate-binding pocket (Gly90 and His187) were essential to maintaining the narrow substrate selectivity of Rv0045c toward various acyl ester substituents, as independent conversion of these residues significantly increased its catalytic activity and broadened its substrate specificity. Focused saturation mutagenesis of position 187 implicated this residue, as the differentiation point between the substrate specificity of Rv0045c and the structurally homologous ybfF hydrolase family. Insertion of the analogous tyrosine residue from ybfF hydrolases into Rv0045c increased the catalytic activity of Rv0045 by over 20-fold toward diverse ester substrates. The unique binding pocket structure and selectivity of Rv0045c provide molecular indications of its biological role and evidence for expanded substrate diversity in serine hydrolases from M. tuberculosis.

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

“…The collaborative experimental goals included the development and use of a class of biological imaging agents known as fluorogenic hydrolase substrates to characterize the activity of a class of enzymes known as serine hydrolases from M. tuberculosis (TB) and to investigate the molecular control of neuronal signaling in Caenorhabditis elegans (Lavis and Raines, 2008; Long and Cravatt, 2011; Dedieu et al ., 2013). Specifically, students synthesized the fluorogenic substrates in CHEMBIO, used these imaging agents to catalogue the substrate specificity of serine hydrolases from TB in BIOCHEM (Johnson et al ., 2014a,b; Lukowski et al ., 2014), and initially tried to combine the products of the two labs to screen for new components of neuronal signaling in C. elegans in NEURO (Figure 1A). Although the initial goal of having NEURO students use the newly characterized hydrolase–fluorophore substrate pairs as imaging tools to investigate neuronal signaling was limited by the permeability of the fluorophores to the C. elegans nervous system (Tian et al ., 2012), NEURO maintained similar methodologies and overlapping pedagogical design and goals.…”

“…A three-dimensional depiction of the binding pocket of the Rv0045c serine hydrolase from M. tuberculosis (PDB: 3P2M). Each of the residues shown in the ball-and stick-representation was individually substituted to alanine by different BIOCHEM students, and kinetic data from BIOCHEM students’ purified proteins were combined to create a coherent story for the overall course (Lukowski et al ., 2014). (E and F) BIOCHEM phase 2 independent project results.…”

“…CHEMBIO served as the starting point for the larger experimental goals of using the fluorogenic compound library to characterize mycobacterial serine hydrolases (BIOCHEM) and to identify novel ubiquitin ligases in C. elegans (NEURO). The synthetic target compounds for CHEMBIO were bis-acyloxymethyl ether derivatives of fluorescein, previously shown to be highly stable enzyme probes (Figure 1A; Lavis et al ., 2011; Tian et al ., 2012; Lukowski et al ., 2014). 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).…”

“…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). …”

“…Approximately 10–14 total mutations for the course were targeted per semester so that, with two mutations per eight students, a significant degree of redundancy was included in the course design. During phase 1, BIOCHEM students then combined the structural information about their assigned residues with the biochemical data about their resulting proteins to construct hypotheses about the roles of their amino acids in the structure and function of the mycobacterial serine hydrolase (Table 1; Figure 2D; Lukowski et al ., 2014). The pinnacle of phase 1 was the biochemical characterization of the final purified hydrolase variants using thermal stability and enzymatic catalysis measurements with the highest-activity fluorogenic substrate identified in each year of CHEMBIO (Table 1, weeks 5–9).…”