The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2

Coleman, Tom, Chao, Rebecca R., de Voss, James J. and Bell, Stephen G. (2016) The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2. Biochimica et Biophysica Acta, 1864 6: 667-675. doi:10.1016/j.bbapap.2016.03.006


Author Coleman, Tom
Chao, Rebecca R.
de Voss, James J.
Bell, Stephen G.
Title The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2
Formatted title
The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2
Journal name Biochimica et Biophysica Acta   Check publisher's open access policy
ISSN 1570-9639
Publication date 2016-06
Year available 2016
Sub-type Article (original research)
DOI 10.1016/j.bbapap.2016.03.006
Open Access Status Not Open Access
Volume 1864
Issue 6
Start page 667
End page 675
Total pages 9
Place of publication Amsterdam, Netherlands
Publisher Elsevier BV
Collection year 2017
Language eng
Formatted abstract
Background: The cytochrome P450 enzyme CYP199A4 can efficiently demethylate 4-methoxybenzoic cid. The substrate is positioned in the enzyme active site with the methoxy group ideally positioned for demethylation. This ccurs through interactions of hydrophobic benzene ring with aromatic phenylalanine residues and the charged carboxylate group with polar and basic amino acids.

Methods: In vitro substrate binding and kinetic turnover assays coupled with HPLC and GC-MS analysis and whole-cell oxidation turnovers.

Results: Modification of the carboxylate group to an amide or aldehyde resulted in substrate binding, as judged by the almost total shift of the spin state to the high-spin form, but binding was three orders of magnitude weaker. Changing the carboxylate to phenol alcohol, ketone, ester and nitro groups and boronic, sulfinic and sulfonic acids resulted in a dramatic reduction in the binding affinity. Even phenylacetic acids were mediocre substrates for CYP199A4, despite maintaining a carboxylate group. The weaker binding of all of these substrates results in lower levels of turnover activity and product formation compared to 4-methoxybenzoic acid.

Conclusion: Substrate binding to CYP199A4 is tightly regulated by interactions between the 4-methoxybenzoic acid and the amino acids in the active site. The benzoic acid carboxylate moiety is critical for optimal substrate binding and turnover activity with CYP199A4.

General significance: An understanding of how the CYP199A4 enzyme has evolved to be highly selective for para-substituted benzoic acids. This provides valuable insight into how other, as yet structurally uncharacterised, monooxygenase enzymes may bind benzoic acid substrates.
Keyword Benzoic acids
Cytochrome P450 enzymes
Oxidative demethylation
Rhodopseudomonas palustris
Substrate recognition
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
School of Chemistry and Molecular Biosciences
 
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Created: Fri, 18 Mar 2016, 14:37:53 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences