Structural Basis for the Activity and Substrate Specificity of Fluoroacetyl-CoA Thioesterase FlK

Dias, M.V.B.; Huang, Fanglu; Chirgadze, Dimitri Y.; Tosin, Manuela; Spiteller, Dieter; Dry, Emily F. V.; Leadlay, Peter F.; Spencer, Jonathan B.; Blundell, Tom L.

doi:10.1074/jbc.m110.107177

Cited by 24 publications

(46 citation statements)

References 39 publications

(53 reference statements)

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“…Sequence homologues of the Arthrobacter 4-HB-CoA thioesterase which have known physiological substrates include the bacterial phenylacetyl-CoA thioesterase (34), benzoyl-CoA thioesterase (35), enterobactin biosynthetic pathway “housekeeper” thioesterase EntH (8, 36, 37), long chain acyl-CoA thioesterase II and III (37, 38) and fluoroacetyl-CoA thioesterase (39-41). Sequence alignments of these homologues show conservation of Thr77 yet substitutions of Glu73 with an Asp residue and Gln58 with either an Asn or Glu residue.…”

Section: Resultsmentioning

confidence: 99%

The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU

et al. 2012

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The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden J. B., Zhuang Z., Dunaway-Mariano D., Holden H. M. J. Biol. Chem. 2003 278, 43709-43716) a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the Native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to 18O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from 31P-NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120 and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64 and Glu78 site directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.

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Section: Resultsmentioning

confidence: 99%

The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU

et al. 2012

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“…These two hotdog domains associate together with the two central -helices aligning together and the two -sheets associating through strands 3 and 9 to form an extended -sheet. In other thioesterase families (TE2, TE6 and TE7), these hotdog folds self-associate into higher order configurations depending on the thioesterase class, ranging from dimers (Dias et al, 2010) and tetramers (back to back or face to face; Tilton et al, 2004) to hexamers (trimer of dimers configuration; Forwood et al, 2007;Pidugu et al, 2009). Based on the structure of the E. coli TesB structure, the TesB enzymes are believed to function as a double-hotdog protomer (Li et al, 2000;Pidugu et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Structural and functional characterization of TesB fromYersinia pestisreveals a unique octameric arrangement of hotdog domains

Swarbrick

Perugini

Cowieson

et al. 2015

Acta Cryst D Biol Crystallogr

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“…Consequently, there exists a natural selective pressure on S. cattleya to meet this challenge, and it has evolved a fluoroacetyl-CoA thioesterase (FlK) that catalyzes the breakdown of fluoroacetyl-CoA to prevent lethal synthesis of fluorocitrate (Fig. 1A) (22)(23)(24). We have recently shown that FlK demonstrates an extremely high, 10 6 -fold selectivity for hydrolysis of fluoroacetyl-CoA over acetyl-CoA based on a decrease in K m (10 2 ) and increase in k cat (10 4 ) for the fluorinated substrate (24).…”

mentioning

confidence: 99%

Catalytic control of enzymatic fluorine specificity

Weeks¹,

Chang

2012

Proc. Natl. Acad. Sci. U.S.A.

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The investigation of unique chemical phenotypes has led to the discovery of enzymes with interesting behaviors that allow us to explore unusual function. The organofluorine-producing microbe Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that demonstrates a surprisingly high level of discrimination for a single fluorine substituent on its substrate compared with the cellularly abundant hydrogen analog, acetyl-CoA. In this report, we show that the high selectivity of FlK is achieved through catalysis rather than molecular recognition, where deprotonation at the C α position to form a putative ketene intermediate only occurs on the fluorinated substrate, thereby accelerating the rate of hydrolysis 10 4 -fold compared with the nonfluorinated congener. These studies provide insight into mechanisms of catalytic selectivity in a native system where the existence of two reaction pathways determines substrate rather than product selection.enzyme mechanism | substrate selectivity L iving organisms have solved some of the most difficult challenges in catalysis by harnessing the exquisite selectivity and reactivity of enzyme active sites to build complex chemical behaviors (1-5). Indeed, the plasticity of enzymes toward evolution of new function has allowed life to flourish in diverse biospheres by conferring a selective advantage to hosts that can demonstrate catalytic innovation and use unusual resources. The enzymes that form the molecular basis for these chemical phenotypes are a rich source of molecular diversity and provide an experimental platform for the continual search to gain insight into the mechanisms and dynamics of protein and organismal evolution (6-9). One of the salient features of enzymes is their extremely high substrate selectivity, which allows them to choose the correct substrate over the thousands of other small molecules that are present in cells at any given time. Thus, the exploration of substrate specificity and its evolution is key for understanding both enzyme catalysis and the expansion of biodiversity at the molecular level (7)(8)(9)(10)(11)(12).In this context, a singular chemical trait found in the soil bacterium Streptomyces cattleya is its ability to catalyze the formation of carbon-fluorine bonds (13,14). Fluorine resides at one corner of the periodic table, and its unique elemental properties make it highly effective as a design element for drug discovery but also quite challenging for the synthesis of fluorinecontaining molecules (15)(16)(17). Although fluorine has emerged as a common motif in synthetic compounds, including top-selling drugs such as atorvastatin and fluticasone, it has been underexploited in nature, and only a few biogenic organofluorine compounds (<20) have been identified to date despite the thousands of known natural products containing chlorine and bromine (∼5,000) (18,19). In fact, the only fully characterized organofluorine natural products are derived from a single pathway that produces the deceptively simple poison fluoroacetate (1). T...

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Structural Basis for the Activity and Substrate Specificity of Fluoroacetyl-CoA Thioesterase FlK

Cited by 24 publications

References 39 publications

The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU

The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU

Structural and functional characterization of TesB fromYersinia pestisreveals a unique octameric arrangement of hotdog domains

Catalytic control of enzymatic fluorine specificity

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