Gehring AM

References (5)

Title : Distinct Substrate Selectivity of a Metabolic Hydrolase from Mycobacterium tuberculosis - Lukowski_2014_Biochemistry_53_7386
Author(s) : Lukowski JK , Savas CP , Gehring AM , McKary MG , Adkins CT , Lavis LD , Hoops GC , Johnson RJ
Ref : Biochemistry , 53 :7386 , 2014
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.
ESTHER : Lukowski_2014_Biochemistry_53_7386
PubMedSearch : Lukowski_2014_Biochemistry_53_7386
PubMedID: 25354081
Gene_locus related to this paper: myctu-RV0045C

Title : Large-scale structural rearrangement of a serine hydrolase from Francisella tularensis facilitates catalysis - Filippova_2013_J.Biol.Chem_288_10522
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
Ref : Journal of Biological Chemistry , 288 :10522 , 2013
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.
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

Title : The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima - Hedge_2012_Biochim.Biophys.Acta_1824_1024
Author(s) : Hedge MK , Gehring AM , Adkins CT , Weston LA , Lavis LD , Johnson RJ
Ref : Biochimica & Biophysica Acta , 1824 :1024 , 2012
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.
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

Title : Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis - Gehring_1998_Chem.Biol_5_573
Author(s) : Gehring AM , DeMoll E , Fetherston JD , Mori I , Mayhew GF , Blattner FR , Walsh CT , Perry RD
Ref : Chemical Biology , 5 :573 , 1998
Abstract : BACKGROUND: Virulence in the pathogenic bacterium Yersinia pestis, causative agent of bubonic plague, has been correlated with the biosynthesis and transport of an iron-chelating siderophore, yersiniabactin, which is induced under iron-starvation conditions. Initial DNA sequencing suggested that this system is highly conserved among the pathogenic Yersinia. Yersiniabactin contains a phenolic group and three five-membered thiazole heterocycles that serve as iron ligands.
RESULTS: The entire Y. pestis yersiniabactin region has been sequenced. Sequence analysis of yersiniabactin biosynthetic regions (irp2-ybtE and ybtS) reveals a strategy for siderophore production using a mixed polyketide synthase/nonribosomal peptide synthetase complex formed between HMWP1 and HMWP2 (encoded by irp1 and irp2). The complex contains 16 domains, five of them variants of phosphopantetheine-modified peptidyl carrier protein or acyl carrier protein domains. HMWP1 and HMWP2 also contain methyltransferase and heterocyclization domains. Mutating ybtS revealed that this gene encodes a protein essential for yersiniabactin synthesis.
CONCLUSIONS: The HMWP1 and HMWP2 domain organization suggests that the yersiniabactin siderophore is assembled in a modular fashion, in which a series of covalent intermediates are passed from the amino terminus of HMWP2 to the carboxyl terminus of HMWP1. Biosynthetic labeling studies indicate that the three yersiniabactin methyl moieties are donated by S-adenosylmethionine and that the linker between the thiazoline and thiazolidine rings is derived from malonyl-CoA. The salicylate moiety is probably synthesized using the aromatic amino-acid biosynthetic pathway, the final step of which converts chorismate to salicylate. YbtS might be necessary for converting chorismate to salicylate.
ESTHER : Gehring_1998_Chem.Biol_5_573
PubMedSearch : Gehring_1998_Chem.Biol_5_573
PubMedID: 9818149
Gene_locus related to this paper: yerpe-IRP1 , yerpe-YBTT

Title : Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF - Gehring_1998_Biochemistry_37_2648
Author(s) : Gehring AM , Mori I , Walsh CT
Ref : Biochemistry , 37 :2648 , 1998
Abstract : The siderophore molecule enterobactin, a cyclic trimeric lactone of N-(2,3-dihydroxybenzoyl)serine, is synthesized and secreted by Escherichia coli in response to iron starvation. Here we report the first reconstitution of enterobactin synthetase activity from pure protein components: holo-EntB, EntE, and holo-EntF. Holo-EntB and holo-EntF were obtained by pretreatment of apo-EntB and apo-EntF with coenzyme A and EntD, thereby eliminating the requirement for EntD in the enterobactin synthetase. The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates with a turnover rate of 120-140 min-1. There is no evidence for a stable complex of the enterobactin synthetase components. Mutation of holo-EntF in the thioesterase domain at the putative active site serine residue (Ser1138 to Ala) eliminated enterobactin synthetase activity; however, the mutant holo-EntF retained the ability to adenylate serine and to autoacylate itself by thioester formation between serine and its attached phosphopantetheine cofactor. The mutant holo-EntF also appeared to slowly release N-(2, 3-dihydroxybenzoyl)serine.
ESTHER : Gehring_1998_Biochemistry_37_2648
PubMedSearch : Gehring_1998_Biochemistry_37_2648
PubMedID: 9485415
Gene_locus related to this paper: ecoli-entf