R. Jeremy Johnson

The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima.

Acetyl esterases from carbohydrate esterase family 7 exhibit unusual substrate specificity. These proteins catalyze the cleavage of disparate acetate esters with high efficiency, but are unreactive to larger acyl groups. The structural basis for this distinct selectivity profile is unknown. Here, we investigate a thermostable acetyl esterase (TM0077) from Thermotoga maritima using evolutionary relationships, structural information, fluorescent kinetic measurements, and site directed mutagenesis. We measured the kinetic and structural determinants for this specificity using a diverse series of small molecule enzyme substrates, including novel fluorogenic esters. These experiments identified two hydrophobic plasticity residues (Pro228, and Ile276) surrounding the nucleophilic serine that impart this specificity of TM0077 for small, straight-chain esters. Substitution of these residues with alanine imparts broader specificity to TM0077 for the hydrolysis of longer and bulkier esters. Our results suggest the specificity of acetyl esterases have been finely tuned by evolution to catalyze the removal of acetate groups from diverse substrates, but can be modified by focused amino acid substitutions to yield enzymes capable of cleaving larger ester functionalities.

Large-scale Structural Rearrangement of a Serine Hydrolase from Francisella Tularensis Facilitates Catalysis

Background: Acyl protein thioesterases control protein S-acylation at cellular membranes. Results: FTT258 is a serine hydrolase with broad substrate specificity that binds to bacterial membranes and exists in two distinct conformations. Conclusion: Conformational changes in FTT258 are correlated with catalytic activity. Significance: Structural rearrangement dually regulates the membrane binding and catalytic activity of acyl protein thioesterases. Tularemia is a deadly, febrile disease caused by infection by the Gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues.

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

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.

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

This paper describes the design, implementation, and assessment of three collaborative classroom undergraduate research experiences (CUREs) integrating faculty research interests across chemical biology, biochemistry, and neurobiology. Benefits of CURE participation included increased faculty productivity, generation of novel scientific data, and the expected CURE benefits for student learning.

Ester-prodrugs of ethambutol control its antibacterial activity and provide rapid screening for mycobacterial hydrolase activity

M. tuberculosis contains an unusually high number of serine hydrolases by proteome percentage compared to other common bacteria or humans. This letter describes a method to probe the global substrate specificity of mycobacterial serine hydrolases with ester-protected prodrugs of ethambutol, a first-line antibiotic treatment for TB. These compounds were synthesized directly from ethambutol using a selective o-acylation to yield products in high yield and purity with minimal workup. A library of derivatives was screened against M. smegmatis, a non-infectious model for M. tuberculosis, which displayed significantly lowered biological activity compared to ethambutol. Incubation with a general serine hydrolase reactivated each derivative to near-ethambutol levels, demonstrating that esterification of ethambutol should provide a simple screen for mycobacterial hydrolase activity.

The unusual substrate specificity of a virulence associated serine hydrolase from the highly toxic bacterium, Francisella tularensis

Francisella tularensis is the causative agent of the highly, infectious disease, tularemia. Amongst the genes identified as essential to the virulence of F. tularensis was the proposed serine hydrolase FTT0941c. Herein, we purified FTT0941c to homogeneity and then characterized the folded stability, enzymatic activity, and substrate specificity of FTT0941c. Based on phylogenetic analysis, FTT0941c was classified within a divergent Francisella subbranch of the bacterial hormone sensitive lipase (HSL) superfamily, but with the conserved sequence motifs of a bacterial serine hydrolase. FTT0941c showed broad hydrolase activity against diverse libraries of ester substrates, including significant hydrolytic activity across alkyl ester substrates from 2 to 8 carbons in length. Among a diverse library of fluorogenic substrates, FTT0941c preferred α-cyclohexyl ester substrates, matching with the substrate specificity of structural homologues and the broad open architecture of its modeled binding pocket. By substitutional analysis, FTT0941c was confirmed to have a classic catalytic triad of Ser115, His278, and Asp248 and to remain thermally stable even after substitution. Its overall substrate specificity profile, divergent phylogenetic homology, and preliminary pathway analysis suggested potential biological functions for FTT0941c in diverse metabolic degradation pathways in F. tularensis.

Teaching Foundational Topics and Scientific Skills in Biochemistry within the Conceptual Framework of HIV Protease.

HIV protease has served as a model protein for understanding protein structure, enzyme kinetics, structure‐based drug design, and protein evolution. Inhibitors of HIV protease are also an essential part of effective HIV/AIDS treatment and have provided great societal benefits. The broad applications for HIV protease and its inhibitors make it a perfect framework for integrating foundational topics in biochemistry around a big picture scientific and societal issue. Herein, I describe a series of classroom exercises that integrate foundational topics in biochemistry around the structure, biology, and therapeutic inhibition of HIV protease. These exercises center on foundational topics in biochemistry including thermodynamics, acid/base properties, protein structure, ligand binding, and enzymatic catalysis. The exercises also incorporate regular student practice of scientific skills including analysis of primary literature, evaluation of scientific data, and presentation of technical scientific arguments. Through the exercises, students also gain experience accessing computational biochemical resources such as the protein data bank, Proteopedia, and protein visualization software. As these HIV centered exercises cover foundational topics common to all first semester biochemistry courses, these exercises should appeal to a broad audience of undergraduate students and should be readily integrated into a variety of teaching styles and classroom sizes.

A dynamic loop provides dual control over the catalytic and membrane binding activity of a bacterial serine hydrolase

The bacterial acyl protein thioesterase (APT) homologue FTT258 from the gram-negative pathogen Francisella tularensis exists in equilibrium between a closed and open state. Interconversion between these two states is dependent on structural rearrangement of a dynamic loop overlapping its active site. The dynamics and structural properties of this loop provide a simple model for how the catalytic activity of FTT258 could be spatiotemporally regulated within the cell. Herein, we characterized the dual roles of this dynamic loop in controlling its catalytic and membrane binding activity. Using a comprehensive library of loop variants, we determined the relative importance of each residue in the loop to these two biological functions. For the catalytic activity, a centrally located tryptophan residue (Trp66) was essential, with the resulting alanine variant showing complete ablation of enzyme activity. Detailed analysis of Trp66 showed that its hydrophobicity in combination with spatial arrangement defined its essential role in catalysis. Substitution of other loop residues congregated along the N-terminal side of the loop also significantly impacted catalytic activity, indicating a critical role for this loop in controlling catalytic activity. For membrane binding, the centrally located hydrophobic residues played a surprising minor role in membrane binding. Instead general electrostatic interactions regulated membrane binding with positively charged residues bracketing the dynamic loop controlling membrane binding. Overall for FTT258, this dynamic loop dually controlled its biological activities through distinct residues within the loop and this regulation provides a new model for the spatiotemporal control over FTT258 and potentially homologous APT function.

In Vivo Delivery and Activation of Masked Fluorogenic Hydrolase Substrates by Endogenous Hydrolases in C. elegans

Protein expression and localization are often studied in vivo by tagging molecules with green fluorescent protein (GFP), yet subtle changes in protein levels are not easily detected. To develop a sensitive in vivo method to amplify fluorescence signals and allow cell‐specific quantification of protein abundance changes, we sought to apply an enzyme‐activated cellular fluorescence system in vivo by delivering ester‐masked fluorophores to Caenorhabditis elegans neurons expressing porcine liver esterase (PLE). To aid uptake into sensory neuron membranes, we synthesized two novel fluorogenic hydrolase substrates with long hydrocarbon tails. Recombinant PLE activated these fluorophores in vitro. In vivo activation occurred in sensory neurons, along with potent activation in intestinal lysosomes quantifiable by imaging and microplate and partially attributable to gut esterase 1 (GES‐1) activity. These data demonstrate the promise of biorthogonal hydrolases and their fluorogenic substrates as in vivo neuronal imaging tools and for characterizing endogenous C. elegans hydrolase substrate specificities.

Teaching argumentation and scientific discourse using the ribosomal peptidyl transferase reaction

Argumentation and discourse are two integral parts of scientific investigation that are often overlooked in undergraduate science education. To address this limitation, the story of peptide bond formation by the ribosome can be used to illustrate the importance of evidence, claims, arguments, and counterarguments in scientific discourse. With the determination of the first structure of the large ribosomal subunit bound to a transition state inhibitor came an initial hypothesis about the role of the ribosome in peptide bond formation. This initial hypothesis was based on a few central assumptions about the transition state mimic and acid–base catalysis by serine proteases. The initial proposed mechanism started a flurry of scientific discourse in experimental articles and commentaries that tested the validity of the initial proposed mechanism. Using this civil argumentation as a guide, class discussions, assignments, and a debate were designed that allow students to analyze and question the claims and evidence about the mechanism of peptide bond synthesis. In the end, students develop a sense of critical skepticism, and an understanding of scientific discourse, while learning about the current consensus mechanism for peptide bond synthesis. Biochemistry and Molecular Biology Education Vol. 39, No. 3, pp. 185‐190, 2011