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Divergence of substrate specificity and function in the Escherichia coli hotdog-fold thioesterase paralogs YdiI and YbdB.

Latham JA, Chen D, Allen KN, Dunaway-Mariano D - Biochemistry (2014)

Bottom Line: These results were interpreted as evidence for substrate promiscuity that facilitates YbdB and YdiI evolvability, and divergence in substrate preference, which correlates with their assumed biological function.YdiI support of the menaquinone biosynthetic pathway was confirmed by demonstrating reduced anaerobic growth of the E. coli ydiI-knockout mutant (vs wild-type E. coli) on glucose in the presence of the electron acceptor fumarate.Bioinformatic analysis revealed that a small biological range exists for YbdB orthologs (i.e., limited to Enterobacteriales) relative to that of YdiI orthologs.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry & Chemical Biology, University of New Mexico , Albuquerque, New Mexico 87131, United States.

ABSTRACT
The work described in this paper, and its companion paper (Wu, R., Latham, J. A., Chen, D., Farelli, J., Zhao, H., Matthews, K. Allen, K. N., and Dunaway-Mariano, D. (2014) Structure and Catalysis in the Escherichia coli Hotdog-fold Thioesterase Paralogs YdiI and YbdB. Biochemistry, DOI: 10.1021/bi500334v), focuses on the evolution of a pair of paralogous hotdog-fold superfamily thioesterases of E. coli, YbdB and YdiI, which share a high level of sequence identity but perform different biological functions (viz., proofreader of 2,3-dihydroxybenzoyl-holoEntB in the enterobactin biosynthetic pathway and catalyst of the 1,4-dihydoxynapthoyl-CoA hydrolysis step in the menaquinone biosynthetic pathway, respectively). In vitro substrate activity screening of a library of thioester metabolites showed that YbdB displays high activity with benzoyl-holoEntB and benzoyl-CoA substrates, marginal activity with acyl-CoA thioesters, and no activity with 1,4-dihydoxynapthoyl-CoA. YdiI, on the other hand, showed a high level of activity with its physiological substrate, significant activity toward a wide range of acyl-CoA thioesters, and minimal activity toward benzoyl-holoEntB. These results were interpreted as evidence for substrate promiscuity that facilitates YbdB and YdiI evolvability, and divergence in substrate preference, which correlates with their assumed biological function. YdiI support of the menaquinone biosynthetic pathway was confirmed by demonstrating reduced anaerobic growth of the E. coli ydiI-knockout mutant (vs wild-type E. coli) on glucose in the presence of the electron acceptor fumarate. Bioinformatic analysis revealed that a small biological range exists for YbdB orthologs (i.e., limited to Enterobacteriales) relative to that of YdiI orthologs. The divergence in YbdB and YdiI substrate specificity detailed in this paper set the stage for their structural analyses reported in the companion paper.

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Summary of the E. coli enterobactin biosynthesticpathway. (A) Ordering of the clustered genes encoding the steps ofthe biosynthetic pathway (blue) and the genes encoding proteins involvedin enterobactin transport and function (black). The proofreading hotdog-foldthioesterase YbdB gene is colored red. The chemical steps of the biosyntheticpathway catalyzed by the enzymes isochorismate synthase (EntC), bifunctionalisochorismate lyase/aryl carrier protein (EntB), 2,3-dihydro-2,3-dihydroxybenzoatedehydrogenase (EntA), phosphopantetheinyltransferase component ofentobacterin synthase multienzyme complex (EntD), enterobactin synthasecomponent F (EntF), and 2,3 dihydroxybenzoate-AMP ligase (EntE). (B)Depiction of the mischarging of EntB catalyzed by EntD or holoEntB catalyzed EntE, and the EntB-regenerating thioesterhydrolysis reaction (rescue) catalyzed by YbdB (aka EntH). In thefigure, “R” can represent any organic group.
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fig2: Summary of the E. coli enterobactin biosynthesticpathway. (A) Ordering of the clustered genes encoding the steps ofthe biosynthetic pathway (blue) and the genes encoding proteins involvedin enterobactin transport and function (black). The proofreading hotdog-foldthioesterase YbdB gene is colored red. The chemical steps of the biosyntheticpathway catalyzed by the enzymes isochorismate synthase (EntC), bifunctionalisochorismate lyase/aryl carrier protein (EntB), 2,3-dihydro-2,3-dihydroxybenzoatedehydrogenase (EntA), phosphopantetheinyltransferase component ofentobacterin synthase multienzyme complex (EntD), enterobactin synthasecomponent F (EntF), and 2,3 dihydroxybenzoate-AMP ligase (EntE). (B)Depiction of the mischarging of EntB catalyzed by EntD or holoEntB catalyzed EntE, and the EntB-regenerating thioesterhydrolysis reaction (rescue) catalyzed by YbdB (aka EntH). In thefigure, “R” can represent any organic group.

Mentions: The steady-staterate constants kcat and Km were determined for YbdB- and YdiI-catalyzed hydrolysisof a structurally diverse library of thioesters for the purpose ofevaluating selectivity toward the thiol moiety (CoA vs holoACP)2 and toward the acyl or aroyl moiety(see Chart 1 for chemical structures). TheCoA thioesters represent various classes of metabolites, differingin the size, shape, and polarity of the acyl/aroyl group. The acylcarrier protein (ACP)-based thioesters were used to test recognitionof the holoACP of the E. coli fattyacid synthetic (FAS) pathway28 and the holoACP domain of EntB of the E. coli enterobactinsynthetic pathway29 (Figure 2A).


Divergence of substrate specificity and function in the Escherichia coli hotdog-fold thioesterase paralogs YdiI and YbdB.

Latham JA, Chen D, Allen KN, Dunaway-Mariano D - Biochemistry (2014)

Summary of the E. coli enterobactin biosynthesticpathway. (A) Ordering of the clustered genes encoding the steps ofthe biosynthetic pathway (blue) and the genes encoding proteins involvedin enterobactin transport and function (black). The proofreading hotdog-foldthioesterase YbdB gene is colored red. The chemical steps of the biosyntheticpathway catalyzed by the enzymes isochorismate synthase (EntC), bifunctionalisochorismate lyase/aryl carrier protein (EntB), 2,3-dihydro-2,3-dihydroxybenzoatedehydrogenase (EntA), phosphopantetheinyltransferase component ofentobacterin synthase multienzyme complex (EntD), enterobactin synthasecomponent F (EntF), and 2,3 dihydroxybenzoate-AMP ligase (EntE). (B)Depiction of the mischarging of EntB catalyzed by EntD or holoEntB catalyzed EntE, and the EntB-regenerating thioesterhydrolysis reaction (rescue) catalyzed by YbdB (aka EntH). In thefigure, “R” can represent any organic group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4116150&req=5

fig2: Summary of the E. coli enterobactin biosynthesticpathway. (A) Ordering of the clustered genes encoding the steps ofthe biosynthetic pathway (blue) and the genes encoding proteins involvedin enterobactin transport and function (black). The proofreading hotdog-foldthioesterase YbdB gene is colored red. The chemical steps of the biosyntheticpathway catalyzed by the enzymes isochorismate synthase (EntC), bifunctionalisochorismate lyase/aryl carrier protein (EntB), 2,3-dihydro-2,3-dihydroxybenzoatedehydrogenase (EntA), phosphopantetheinyltransferase component ofentobacterin synthase multienzyme complex (EntD), enterobactin synthasecomponent F (EntF), and 2,3 dihydroxybenzoate-AMP ligase (EntE). (B)Depiction of the mischarging of EntB catalyzed by EntD or holoEntB catalyzed EntE, and the EntB-regenerating thioesterhydrolysis reaction (rescue) catalyzed by YbdB (aka EntH). In thefigure, “R” can represent any organic group.
Mentions: The steady-staterate constants kcat and Km were determined for YbdB- and YdiI-catalyzed hydrolysisof a structurally diverse library of thioesters for the purpose ofevaluating selectivity toward the thiol moiety (CoA vs holoACP)2 and toward the acyl or aroyl moiety(see Chart 1 for chemical structures). TheCoA thioesters represent various classes of metabolites, differingin the size, shape, and polarity of the acyl/aroyl group. The acylcarrier protein (ACP)-based thioesters were used to test recognitionof the holoACP of the E. coli fattyacid synthetic (FAS) pathway28 and the holoACP domain of EntB of the E. coli enterobactinsynthetic pathway29 (Figure 2A).

Bottom Line: These results were interpreted as evidence for substrate promiscuity that facilitates YbdB and YdiI evolvability, and divergence in substrate preference, which correlates with their assumed biological function.YdiI support of the menaquinone biosynthetic pathway was confirmed by demonstrating reduced anaerobic growth of the E. coli ydiI-knockout mutant (vs wild-type E. coli) on glucose in the presence of the electron acceptor fumarate.Bioinformatic analysis revealed that a small biological range exists for YbdB orthologs (i.e., limited to Enterobacteriales) relative to that of YdiI orthologs.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry & Chemical Biology, University of New Mexico , Albuquerque, New Mexico 87131, United States.

ABSTRACT
The work described in this paper, and its companion paper (Wu, R., Latham, J. A., Chen, D., Farelli, J., Zhao, H., Matthews, K. Allen, K. N., and Dunaway-Mariano, D. (2014) Structure and Catalysis in the Escherichia coli Hotdog-fold Thioesterase Paralogs YdiI and YbdB. Biochemistry, DOI: 10.1021/bi500334v), focuses on the evolution of a pair of paralogous hotdog-fold superfamily thioesterases of E. coli, YbdB and YdiI, which share a high level of sequence identity but perform different biological functions (viz., proofreader of 2,3-dihydroxybenzoyl-holoEntB in the enterobactin biosynthetic pathway and catalyst of the 1,4-dihydoxynapthoyl-CoA hydrolysis step in the menaquinone biosynthetic pathway, respectively). In vitro substrate activity screening of a library of thioester metabolites showed that YbdB displays high activity with benzoyl-holoEntB and benzoyl-CoA substrates, marginal activity with acyl-CoA thioesters, and no activity with 1,4-dihydoxynapthoyl-CoA. YdiI, on the other hand, showed a high level of activity with its physiological substrate, significant activity toward a wide range of acyl-CoA thioesters, and minimal activity toward benzoyl-holoEntB. These results were interpreted as evidence for substrate promiscuity that facilitates YbdB and YdiI evolvability, and divergence in substrate preference, which correlates with their assumed biological function. YdiI support of the menaquinone biosynthetic pathway was confirmed by demonstrating reduced anaerobic growth of the E. coli ydiI-knockout mutant (vs wild-type E. coli) on glucose in the presence of the electron acceptor fumarate. Bioinformatic analysis revealed that a small biological range exists for YbdB orthologs (i.e., limited to Enterobacteriales) relative to that of YdiI orthologs. The divergence in YbdB and YdiI substrate specificity detailed in this paper set the stage for their structural analyses reported in the companion paper.

Show MeSH
Related in: MedlinePlus