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Title: Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity

Abstract

A benzoate CoA ligase (BadA), isolated from the bacterium Rhodopseudomonas palustris, catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids. Herein, apparent Michaelis constants kcatapp and KMapp were determined for an expanded array of 31 substrates chosen to systematically probe the active site architecture of the enzyme and provide a baseline for expansion of wild-type substrate specificity. Acyl CoA products were observed for 25 of the 31 substrates; in general, BadA converted ortho-substituted substrates better than the corresponding meta and para regioisomers, and the turnover number was more affected by steric rather than electronic effects. The kinetic data are interpreted in relation to six crystal structures of BadA in complex with several substrates and a benzoyl-AMP reaction intermediate. In contrast to other known natural substrate-bound benzoate ligase structures, all substrate-bound BadA structures adopted the thiolation conformation instead of the adenylation conformation. We also observed all the aryl carboxylates to be uniquely oriented within the active site, relative to other structures. Together, the kinetics and structural data suggested a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation. Onmore » the basis of this hypothesis and the structural data, sterically demanding active site residues were mutated, and the substrate specificity was expanded substantially versus that of BadA. Novel activities were seen for substrates with larger substituents, including phenyl acetate. Additionally, the mutant Lys427Ala identified this nonconserved residue as essential for the thiolation step of BadA, but not adenylation. Furthermore, these variously acylated CoAs can serve as novel substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogues of bioactive natural products.« less

Authors:
 [1];  [1];  [1];  [1];  [1]
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  1. Michigan State Univ., East Lansing, MI (United States)
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division; USDOE Advanced Research Projects Agency - Energy (ARPA-E); National Institute of General Medical Sciences
OSTI Identifier:
1227505
Grant/Contract Number:  
AC02-06CH11357; P41-GM103311; 085P1000817
Resource Type:
Accepted Manuscript
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 54; Journal Issue: 40; Journal ID: ISSN 0006-2960
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; peptides and proteins; genetics; surface interactions; substituents; conformation

Citation Formats

Thornburg, Chelsea K., Wortas-Strom, Susan, Nosrati, Meisam, Geiger, James H., and Walker, Kevin D. Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity. United States: N. p., 2015. Web. doi:10.1021/acs.biochem.5b00899.
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Thornburg, Chelsea K., Wortas-Strom, Susan, Nosrati, Meisam, Geiger, James H., & Walker, Kevin D. Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity. United States. https://doi.org/10.1021/acs.biochem.5b00899
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Thornburg, Chelsea K., Wortas-Strom, Susan, Nosrati, Meisam, Geiger, James H., and Walker, Kevin D. Thu . "Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity". United States. https://doi.org/10.1021/acs.biochem.5b00899. https://www.osti.gov/servlets/purl/1227505.
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@article{osti_1227505,
title = {Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity},
author = {Thornburg, Chelsea K. and Wortas-Strom, Susan and Nosrati, Meisam and Geiger, James H. and Walker, Kevin D.},
abstractNote = {A benzoate CoA ligase (BadA), isolated from the bacterium Rhodopseudomonas palustris, catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids. Herein, apparent Michaelis constants kcatapp and KMapp were determined for an expanded array of 31 substrates chosen to systematically probe the active site architecture of the enzyme and provide a baseline for expansion of wild-type substrate specificity. Acyl CoA products were observed for 25 of the 31 substrates; in general, BadA converted ortho-substituted substrates better than the corresponding meta and para regioisomers, and the turnover number was more affected by steric rather than electronic effects. The kinetic data are interpreted in relation to six crystal structures of BadA in complex with several substrates and a benzoyl-AMP reaction intermediate. In contrast to other known natural substrate-bound benzoate ligase structures, all substrate-bound BadA structures adopted the thiolation conformation instead of the adenylation conformation. We also observed all the aryl carboxylates to be uniquely oriented within the active site, relative to other structures. Together, the kinetics and structural data suggested a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation. On the basis of this hypothesis and the structural data, sterically demanding active site residues were mutated, and the substrate specificity was expanded substantially versus that of BadA. Novel activities were seen for substrates with larger substituents, including phenyl acetate. Additionally, the mutant Lys427Ala identified this nonconserved residue as essential for the thiolation step of BadA, but not adenylation. Furthermore, these variously acylated CoAs can serve as novel substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogues of bioactive natural products.},
doi = {10.1021/acs.biochem.5b00899},
journal = {Biochemistry},
number = 40,
volume = 54,
place = {United States},
year = {Thu Sep 17 00:00:00 EDT 2015},
month = {Thu Sep 17 00:00:00 EDT 2015}
}
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https://doi.org/10.1021/acs.biochem.5b00899
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