@article {49324, title = {Fluorogenic structure activity library pinpoints molecular variations in substrate specificity of structurally homologous esterases.}, journal = {The Journal of Biological Chemistry}, volume = {293}, year = {2018}, month = {2018 Jul 13}, pages = {13851-62}, 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. esterase ybfF displayed a preference for γ-position thioethers and ethers, whereas Rv0045c from displayed a preference for branched substrates with and without thioethers. We determined that this substrate differentiation was partially controlled by individual substrate selectivity residues Tyr119 in ybfF and His187 in Rv0045c; reciprocal substitution of these residues shifted each esterase{\textquoteright}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 multi-faceted future applications including in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design.

}, issn = {1083-351X}, doi = {10.1074/jbc.RA118.003972}, author = {White, Alex and Koelper, Andrew and Russell, Arielle and Larsen, Erik M and Kim, Charles and Lavis, Luke D and Hoops, Geoffrey C and Johnson, R Jeremy} } @article {49153, title = {Measuring the global substrate specificity of mycobacterial serine hydrolases using a library of fluorogenic ester substrates.}, journal = {ACS Infectious Diseases}, volume = {4}, year = {2018}, month = {2018 Jun 8}, pages = {904-11}, 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.

}, issn = {2373-8227}, doi = {10.1021/acsinfecdis.7b00263}, author = {Bassett, Braden and Waibel, Brent and White, Alex and Hansen, Heather and Stephens, Dominique and Koelper, Andrew and Larsen, Erik M and Kim, Charles and Glanzer, Adam and Lavis, Luke D and Hoops, Geoffrey C and Johnson, R Jeremy} } @article {45779, title = {Distinct substrate selectivity of a metabolic hydrolase from Mycobacterium tuberculosis.}, journal = {Biochemistry}, volume = {53}, year = {2014}, month = {2014 Oct 29}, pages = {7386-95}, 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.

}, issn = {1520-4995}, doi = {10.1021/bi501108u}, author = {Lukowski, Jessica K and Savas, Christopher P and Gehring, Alexandra M and McKary, Magy G and Adkins, Chinessa T and Lavis, Luke D and Hoops, Geoffrey C and Johnson, R Jeremy} } @article {6586, title = {Decoupled roles for the atypical, bifurcated binding pocket of the ybfF hydrolase.}, journal = {Chembiochem : A European Journal of Chemical Biology}, volume = {14}, year = {2013}, month = {2013 Jun 17}, pages = {1134-44}, 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.

}, issn = {1439-7633}, doi = {10.1002/cbic.201300085}, author = {Ellis, Elizabeth E and Adkins, Chinessa T and Galovska, Natalie M and Lavis, Luke D and Johnson, R Jeremy} } @article {65022, title = {Large scale structural rearrangement of a serine hydrolase from Francisella tularensis facilitates catalysis.}, journal = {J Biol Chem}, volume = {288}, year = {2013}, month = {2013 Apr 12}, pages = {10522-35}, 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 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.

}, keywords = {Bacterial Proteins, Cell Membrane, Francisella tularensis, Humans, Mutagenesis, Site-Directed, Serine Endopeptidases, Structural Homology, Protein, Structure-Activity Relationship, Thiolester Hydrolases}, issn = {1083-351X}, doi = {10.1074/jbc.M112.446625}, author = {Filippova, Ekaterina V and Weston, Leigh A and Kuhn, Misty L and Geissler, Brett and Gehring, Alexandra M and Armoush, Nicola and Adkins, Chinessa T and Minasov, George and Dubrovska, Ievgeniia and Shuvalova, Ludmilla and Winsor, James R and Lavis, Luke D and Satchell, Karla J F and Becker, Daniel P and Anderson, Wayne F and Johnson, R Jeremy} } @article {6299, title = {The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima.}, journal = {Biochimica et Biophysica Acta}, volume = {1824}, year = {2012}, month = {2012 Sep}, pages = {1024-30}, 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.

}, keywords = {Acetylesterase, Bacterial Proteins, Binding Sites, Catalysis, Esters, Fluoresceins, Fluorescent Dyes, Hydrolysis, Kinetics, Models, Molecular, Mutagenesis, Site-Directed, Substrate Specificity, Thermotoga maritima}, issn = {0006-3002}, doi = {10.1016/j.bbapap.2012.05.009}, author = {Hedge, Matthew K and Gehring, Alexandra M and Adkins, Chinessa T and Weston, Leigh A and Lavis, Luke D and Johnson, R Jeremy} } @article {1103, title = {Cytotoxic ribonucleases: the dichotomy of Coulombic forces.}, journal = {Biochemistry}, volume = {46}, year = {2007}, month = {2007 Sep 11}, pages = {10308-16}, abstract = {

Cells tightly regulate their contents. Still, nonspecific Coulombic interactions between cationic molecules and anionic membrane components can lead to adventitious endocytosis. Here, we characterize this process in a natural system. To do so, we create variants of human pancreatic ribonuclease (RNase 1) that differ in net molecular charge. By conjugating a small-molecule latent fluorophore to these variants and using flow cytometry, we are able to determine the kinetic mechanism for RNase 1 internalization into live human cells. We find that internalization increases with solution concentration and is not saturable. Internalization also increases with time to a steady-state level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t1/2 = 2 h) is independent of charge. We conclude that internalization involves an extracellular equilibrium complex between the cationic proteins and abundant anionic cell-surface molecules, followed by rate-limiting internalization. The enhanced internalization of more cationic variants of RNase 1 is, however, countered by their increased affinity for the cytosolic ribonuclease inhibitor protein, which is anionic. Thus, Coulombic forces mediate extracellular and intracellular equilibria in a dichotomous manner that both endangers cells and defends them from the potentially lethal enzymatic activity of ribonucleases.

}, keywords = {Biomechanics, Cell Death, Endocytosis, Fluorescent Dyes, Humans, K562 Cells, Kinetics, Mutant Proteins, Pancreatic, Protein Structure, Ribonuclease, Secondary}, issn = {0006-2960}, doi = {10.1021/bi700857u}, author = {Johnson, R Jeremy and Chao, Tzu-Yuan and Lavis, Luke D and Raines, Ronald T} } @article {1102, title = {Intraspecies regulation of ribonucleolytic activity.}, journal = {Biochemistry}, volume = {46}, year = {2007}, month = {2007 Nov 13}, pages = {13131-40}, abstract = {

The evolutionary rate of proteins involved in obligate protein-protein interactions is slower and the degree of coevolution higher than that for nonobligate protein-protein interactions. The coevolution of the proteins involved in certain nonobligate interactions is, however, essential to cell survival. To gain insight into the coevolution of one such nonobligate protein pair, the cytosolic ribonuclease inhibitor (RI) proteins and secretory pancreatic-type ribonucleases from cow (Bos taurus) and human (Homo sapiens) were produced in Escherichia coli and purified, and their physicochemical properties were analyzed. The two intraspecies complexes were found to be extremely tight (bovine Kd = 0.69 fM; human Kd = 0.34 fM). Human RI binds to its cognate ribonuclease (RNase 1) with 100-fold greater affinity than to the bovine homologue (RNase A). In contrast, bovine RI binds to RNase 1 and RNase A with nearly equal affinity. This broader specificity is consistent with there being more pancreatic-type ribonucleases in cows (20) than humans (13). Human RI (32 cysteine residues) also has 4-fold less resistance to oxidation by hydrogen peroxide than does bovine RI (29 cysteine residues). This decreased oxidative stability of human RI, which is caused largely by Cys74, implies a larger role for human RI as an antioxidant. The conformational and oxidative stabilities of both RIs increase upon complex formation with ribonucleases. Thus, RI has evolved to maintain its inhibition of invading ribonucleases, even when confronted with extreme environmental stress. That role appears to take precedence over its role in mediating oxidative damage.

}, keywords = {Amino Acid Sequence, Animals, Biological Evolution, Cattle, Enzyme Inhibitors, Humans, Hydrogen Peroxide, Models, Molecular, Molecular Sequence Data, Oxidation-Reduction, Pancreatic, Placental Hormones, Protein Conformation, Protein Interaction Domains and Motifs, Protein Interaction Mapping, Ribonuclease, Sequence Alignment}, issn = {0006-2960}, doi = {10.1021/bi701521q}, author = {Johnson, R Jeremy and Lavis, Luke D and Raines, Ronald T} }