Author Report for: Johnson RJNo contact information in database for Johnson RJ
Schemenauer DE, Pool EH, Raynor SN, Ruiz GP, Goehring LM, Koelper AJ, Wilson MA, Durand AJ, Jr., Kourtoglou EC and Johnson RJ <4 more author(s)> Schemenauer DE, Pool EH, Raynor SN, Ruiz GP, Goehring LM, Koelper AJ, Wilson MA, Durand AJ, Jr., Kourtoglou EC, Larsen EM, Lavis LD, Esteb JJ, Hoops GC, Johnson RJ (- 4) Ref: ACS Omega, 8:13252, 2023 : PubMed ESTHER: Schemenauer_2023_ACS.Omega_8_13252 PubMedSearch: Schemenauer 2023 ACS.Omega 8 13252 PubMedID: 37065048 Gene_locus related to this paper: mycmm-b2h1k1 Abstract Mycobacterium tuberculosis has a complex life cycle transitioning between active and dormant growth states depending on environmental conditions. LipN (Rv2970c) is a conserved mycobacterial serine hydrolase with regulated catalytic activity at the interface between active and dormant growth conditions. LipN also catalyzes the xenobiotic degradation of a tertiary ester substrate and contains multiple conserved motifs connected with the ability to catalyze the hydrolysis of difficult tertiary ester substrates. Herein, we expanded a library of fluorogenic ester substrates to include more tertiary and constrained esters and screened 33 fluorogenic substrates for activation by LipN, identifying its unique substrate signature. LipN preferred short, unbranched ester substrates, but had its second highest activity against a heteroaromatic five-membered oxazole ester. Oxazole esters are present in multiple mycobacterial serine hydrolase inhibitors but have not been tested widely as ester substrates. Combined structural modeling, kinetic measurements, and substitutional analysis of LipN showcased a fairly rigid binding pocket preorganized for catalysis of short ester substrates. Substitution of diverse amino acids across the binding pocket significantly impacted the folded stability and catalytic activity of LipN with two conserved motifs (HGGGW and GDSAG) playing interconnected, multidimensional roles in regulating its substrate specificity. Together this detailed substrate specificity profile of LipN illustrates the complex interplay between structure and function in mycobacterial hormone-sensitive lipase homologues and indicates oxazole esters as promising inhibitor and substrate scaffolds for mycobacterial hydrolases. Title: Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C Bowles IE, Pool EH, Lancaster BS, Lawson EK, Savas CP, Kartje ZJ, Severinac L, Cho DH, Macbeth MR and Hoops GC <1 more author(s)> Bowles IE, Pool EH, Lancaster BS, Lawson EK, Savas CP, Kartje ZJ, Severinac L, Cho DH, Macbeth MR, Johnson RJ, Hoops GC (- 1) Ref: Protein Science, :, 2021 : PubMed ESTHER: Bowles_2021_Protein.Sci__ PubMedSearch: Bowles 2021 Protein.Sci PubMedID: 33914998 Gene_locus related to this paper: myctu-RV0045C Abstract Mycobacterium tuberculosis virulence is highly metal-dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu(2+) <= Zn(2+) > Ni(2+) > Co(2+) ) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for k(cat) rather than K(M) . Removal of the artificial N-terminal 6xHis-tag did not change the metal-dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 A and 2.0 A resolution in the presence and absence of Zn(2+) with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal-dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals. This article is protected by copyright. All rights reserved. Title: Structure-guided microbial targeting of antistaphylococcal prodrugs Miller JJ, Shah IT, Hatten J, Barekatain Y, Mueller EA, Moustafa AM, Edwards RL, Dowd CS, Hoops GC and Odom John AR <4 more author(s)> Miller JJ, Shah IT, Hatten J, Barekatain Y, Mueller EA, Moustafa AM, Edwards RL, Dowd CS, Hoops GC, Johnson RJ, Planet PJ, Muller FL, Jez JM, Odom John AR (- 4) Ref: Elife, 10:, 2021 : PubMed ESTHER: Miller_2021_Elife_10_e66657 PubMedSearch: Miller 2021 Elife 10 e66657 PubMedID: 34279224 Gene_locus related to this paper: staau-SA2422 Abstract Carboxy ester prodrugs are widely employed to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can mask problematic chemical features that prevent cellular uptake and may enable tissue-specific compound delivery. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, limiting their therapeutic potential. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes as microbial esterase-specific promoieties have not been described. Here we identify the bacterial esterases, GloB and FrmB, that activate carboxy ester prodrugs in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, ultimately identifying several promoieties likely to be serum esterase-resistant and microbially labile. These studies will enable structure-guided design of antistaphylococcal promoieties and expand the range of molecules to target staphylococcal pathogens. Title: Comparative analysis of the human serine hydrolase OVCA2 to the model serine hydrolase homolog FSH1 from S. cerevisiae Bun JS, Slack MD, Schemenauer DE, Johnson RJ Ref: PLoS ONE, 15:e0230166, 2020 : PubMed ESTHER: Bun_2020_PLoS.One_15_e0230166 PubMedSearch: Bun 2020 PLoS.One 15 e0230166 PubMedID: 32182256 Gene_locus related to this paper: human-OVCA2, yeast-FSH1 Abstract Over 100 metabolic serine hydrolases are present in humans with confirmed functions in metabolism, immune response, and neurotransmission. Among potentially clinically-relevant but uncharacterized human serine hydrolases is OVCA2, a serine hydrolase that has been linked with a variety of cancer-related processes. Herein, we developed a heterologous expression system for OVCA2 and determined the comprehensive substrate specificity of OVCA2 against two ester substrate libraries. Based on this analysis, OVCA2 was confirmed as a serine hydrolase with a strong preference for long-chain alkyl ester substrates (>10-carbons) and high selectivity against a variety of short, branched, and substituted esters. Substitutional analysis was used to identify the catalytic residues of OVCA2 with a Ser117-His206-Asp179 classic catalytic triad. Comparison of the substrate specificity of OVCA2 to the model homologue FSH1 from Saccharomyces cerevisiae illustrated the tighter substrate selectivity of OVCA2, but their overlapping substrate preference for extended straight-chain alkyl esters. Conformation of the overlapping biochemical properties of OVCA2 and FSH1 was used to model structural information about OVCA2. Together our analysis provides detailed substrate specificity information about a previously, uncharacterized human serine hydrolase and begins to define the biological properties of OVCA2. Title: Microbial esterases and ester prodrugs: An unlikely marriage for combating antibiotic resistance Larsen EM, Johnson RJ Ref: Drug Dev Res, 80:33, 2019 : PubMed ESTHER: Larsen_2019_Drug.Dev.Res_80_33 PubMedSearch: Larsen 2019 Drug.Dev.Res 80 33 PubMedID: 30302779 Inhibitor(s) related to this paper: EZ120P, EZ120 Abstract The rise of antibiotic resistance necessitates the search for new platforms for drug development. Prodrugs are common tools for overcoming drawbacks typically associated with drug formulation and delivery, with ester prodrugs providing a classic strategy for masking polar alcohol and carboxylic acid functionalities and improving cell permeability. Ester prodrugs are normally designed to have simple ester groups, as they are expected to be cleaved and reactivated by a wide spectrum of cellular esterases. However, a number of pathogenic and commensal microbial esterases have been found to possess significant substrate specificity and can play an unexpected role in drug metabolism. Ester protection can also introduce antimicrobial properties into previously nontoxic drugs through alterations in cell permeability or solubility. Finally, mutation to microbial esterases is a novel mechanism for the development of antibiotic resistance. In this review, we highlight the important pathogenic and xenobiotic functions of microbial esterases and discuss the development and application of ester prodrugs for targeting microbial infections and combating antibiotic resistance. Esterases are often overlooked as therapeutic targets. Yet, with the growing need to develop new antibiotics, a thorough understanding of the specificity and function of microbial esterases and their combined action with ester prodrug antibiotics will support the design of future therapeutics. Title: Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates Bassett B, Waibel B, White A, Hansen H, Stephens D, Koelper A, Larsen EM, Kim C, Glanzer A and Johnson RJ <2 more author(s)> Bassett B, Waibel B, White A, Hansen H, Stephens D, Koelper A, Larsen EM, Kim C, Glanzer A, Lavis LD, Hoops GC, Johnson RJ (- 2) Ref: ACS Infect Dis, 4:904, 2018 : PubMed ESTHER: Bassett_2018_ACS.Infect.Dis_4_904 PubMedSearch: Bassett 2018 ACS.Infect.Dis 4 904 PubMedID: 29648787 Abstract Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases. Title: A dynamic loop provides dual control over the catalytic and membrane binding activity of a bacterial serine hydrolase Smith MA, Phillips WK, Rabin PL, Johnson RJ Ref: Biochimica & Biophysica Acta, 1866:925, 2018 : PubMed ESTHER: Smith_2018_Biochim.Biophys.Acta_1866_925 PubMedSearch: Smith 2018 Biochim.Biophys.Acta 1866 925 PubMedID: 29857162 Gene_locus related to this paper: fratt-q5ni32 Abstract 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. Title: Fluorogenic structure activity library pinpoints molecular variations in substrate specificity of structurally homologous esterases White A, Koelper A, Russell A, Larsen EM, Kim C, Lavis LD, Hoops GC, Johnson RJ Ref: Journal of Biological Chemistry, 293:13851, 2018 : PubMed ESTHER: White_2018_J.Biol.Chem_293_13851 PubMedSearch: White 2018 J.Biol.Chem 293 13851 PubMedID: 30006352 Gene_locus related to this paper: myctu-RV0045C, vibch-VC2097 Abstract Cellular esterases catalyze many essential biological functions by performing hydrolysis reactions on diverse substrates. The promiscuity of esterases complicates assignment of their substrate preferences and biological functions. To identify universal factors controlling esterase substrate recognition, we designed a 32-member structure-activity relationship (SAR) library of fluorogenic ester substrates and used this library to systematically interrogate esterase preference for chain length, branching patterns, and polarity to differentiate common classes of esterase substrates. Two structurally homologous bacterial esterases were screened against this library, refining their previously broad overlapping substrate specificity. Vibrio cholerae esterase ybfF displayed a preference for gamma-position thioethers and ethers, whereas Rv0045c from Mycobacterium tuberculosis displayed a preference for branched substrates with and without thioethers. We determined that this substrate differentiation was partially controlled by individual substrate selectivity residues Tyr-119 in ybfF and His-187 in Rv0045c; reciprocal substitution of these residues shifted each esterase's substrate preference. This work demonstrates that the selectivity of esterases is tuned based on transition state stabilization, identifies thioethers as an underutilized functional group for esterase substrates, and provides a rapid method for differentiating structural isozymes. This SAR library could have multifaceted future applications, including in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design. Title: In Vivo Delivery and Activation of Masked Fluorogenic Hydrolase Substrates by Endogenous Hydrolases in C. elegans Dube S, Dube H, Green NB, Larsen EM, White A, Johnson RJ, Kowalski JR Ref: Chembiochem, 18:1807, 2017 : PubMed ESTHER: Dube_2017_Chembiochem_18_1807 PubMedSearch: Dube 2017 Chembiochem 18 1807 PubMedID: 28703362 Abstract 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. Title: The unusual substrate specificity of a virulence associated serine hydrolase from the highly toxic bacterium, Francisella tularensis Farberg AM, Hart WK, Johnson RJ Ref: Biochem Biophys Rep, 7:415, 2016 : PubMed ESTHER: Farberg_2016_Biochem.Biophys.Rep_7_415 PubMedSearch: Farberg 2016 Biochem.Biophys.Rep 7 415 Gene_locus related to this paper: fratt-q5nga8 Abstract 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 alpha-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. Title: Structural Basis for the Strict Substrate Selectivity of the Mycobacterial Hydrolase LipW McKary MG, Abendroth J, Edwards TE, Johnson RJ Ref: Biochemistry, 55:7099, 2016 : PubMed ESTHER: McKary_2016_Biochemistry_55_7099 PubMedSearch: McKary 2016 Biochemistry 55 7099 PubMedID: 27936614 Gene_locus related to this paper: myctu-Rv0217c Abstract The complex life cycle of Mycobacterium tuberculosis requires diverse energy mobilization and utilization strategies facilitated by a battery of lipid metabolism enzymes. Among lipid metabolism enzymes, the Lip family of mycobacterial serine hydrolases is essential to lipid scavenging, metabolic cycles, and reactivation from dormancy. On the basis of the homologous rescue strategy for mycobacterial drug targets, we have characterized the three-dimensional structure of full length LipW from Mycobacterium marinum, the first structure of a catalytically active Lip family member. LipW contains a deep, expansive substrate-binding pocket with only a narrow, restrictive active site, suggesting tight substrate selectivity for short, unbranched esters. Structural alignment reinforced this strict substrate selectivity of LipW, as the binding pocket of LipW aligned most closely with the bacterial acyl esterase superfamily. Detailed kinetic analysis of two different LipW homologues confirmed this strict substrate selectivity, as each homologue selected for unbranched propionyl ester substrates, irrespective of the alcohol portion of the ester. Using comprehensive substitutional analysis across the binding pocket, the strict substrate selectivity of LipW for propionyl esters was assigned to a narrow funnel in the acyl-binding pocket capped by a key hydrophobic valine residue. The polar, negatively charged alcohol-binding pocket also contributed to substrate orientation and stabilization of rotameric states in the catalytic serine. Together, the structural, enzymatic, and substitutional analyses of LipW provide a connection between the structure and metabolic properties of a Lip family hydrolase that refines its biological function in active and dormant tuberculosis infection. Title: Distinct Substrate Selectivity of a Metabolic Hydrolase from Mycobacterium tuberculosis Lukowski JK, Savas CP, Gehring AM, McKary MG, Adkins CT, Lavis LD, Hoops GC, Johnson RJ Ref: Biochemistry, 53:7386, 2014 : PubMed ESTHER: Lukowski_2014_Biochemistry_53_7386 PubMedSearch: Lukowski 2014 Biochemistry 53 7386 PubMedID: 25354081 Gene_locus related to this paper: myctu-RV0045C 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 alpha/beta 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. Title: Decoupled roles for the atypical, bifurcated binding pocket of the ybfF hydrolase Ellis EE, Adkins CT, Galovska NM, Lavis LD, Johnson RJ Ref: Chembiochem, 14:1134, 2013 : PubMed ESTHER: Ellis_2013_Chembiochem_14_1134 PubMedSearch: Ellis 2013 Chembiochem 14 1134 PubMedID: 23670977 Gene_locus related to this paper: vibch-VC2097 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. Title: Large-scale structural rearrangement of a serine hydrolase from Francisella tularensis facilitates catalysis Filippova EV, Weston LA, Kuhn ML, Geissler B, Gehring AM, Armoush N, Adkins CT, Minasov G, Dubrovska I and Johnson RJ <6 more author(s)> Filippova EV, Weston LA, Kuhn ML, Geissler B, Gehring AM, Armoush N, Adkins CT, Minasov G, Dubrovska I, Shuvalova L, Winsor JR, Lavis LD, Satchell KJ, Becker DP, Anderson WF, Johnson RJ (- 6) Ref: Journal of Biological Chemistry, 288:10522, 2013 : PubMed ESTHER: Filippova_2013_J.Biol.Chem_288_10522 PubMedSearch: Filippova 2013 J.Biol.Chem 288 10522 PubMedID: 23430251 Gene_locus related to this paper: fratt-q5ni32 Inhibitor(s) related to this paper: 2-allyl-4-oxocyclobutyl-4-methylbenzenesulfonamide Abstract 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 amongst 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. Title: The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima Hedge MK, Gehring AM, Adkins CT, Weston LA, Lavis LD, Johnson RJ Ref: Biochimica & Biophysica Acta, 1824:1024, 2012 : PubMed ESTHER: Hedge_2012_Biochim.Biophys.Acta_1824_1024 PubMedSearch: Hedge 2012 Biochim.Biophys.Acta 1824 1024 PubMedID: 22659119 Gene_locus related to this paper: thema-TM0077 Abstract 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. | |||||||
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