A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation

Donovan C. Haines, Amita Hegde, Baozhi Chen, Weiqiang Zhao, Muralidhar Bondlela, John M. Humphreys, David A. Mullin, Diana R. Tomchick, Mischa MacHius, Julian A. Peterson

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Identifying key structural features of cytochromes P450 is critical in understanding the catalytic mechanism of these important drug-metabolizing enzymes. Cytochrome P450BM-3 (BM-3), a structural and mechanistic P450 model, catalyzes the regio- and stereoselective hydroxylation of fatty acids. Recent work has demonstrated the importance of water in the mechanism of BM-3, and site-specific mutagenesis has helped to elucidate mechanisms of substrate recognition, binding, and product formation. One of the amino acids identified as playing a key role in the active site of BM-3 is alanine 328, which is located in the loop between the K helix and β 1-4. In the A328V BM-3 mutant, substrate affinity increases 5-10-fold and the turnover number increases 2-8-fold compared to wild-type enzyme. Unlike wild-type enzyme, this mutant is purified from E. coli with endogenous substrate bound due to the higher binding affinity. Close examination of the crystal structures of the substrate-bound native and A328V mutant BMPs indicates that the positioning of the substrate is essentially identical in the two forms of the enzyme, with the two valine methyl groups occupying voids present in the active site of the wild-type substrate-bound structure.

Original languageEnglish (US)
Pages (from-to)8333-8341
Number of pages9
JournalBiochemistry
Volume50
Issue number39
DOIs
StatePublished - Oct 4 2011

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Catalytic Domain
Mutation
Substrates
Enzymes
Valine
Cytochromes
Hydroxylation
Site-Directed Mutagenesis
Alanine
Cytochrome P-450 Enzyme System
Mutagenesis
Fatty Acids
Escherichia coli
Amino Acids
Water
Crystal structure
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biochemistry

Cite this

Haines, D. C., Hegde, A., Chen, B., Zhao, W., Bondlela, M., Humphreys, J. M., ... Peterson, J. A. (2011). A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation. Biochemistry, 50(39), 8333-8341. https://doi.org/10.1021/bi201099j

A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation. / Haines, Donovan C.; Hegde, Amita; Chen, Baozhi; Zhao, Weiqiang; Bondlela, Muralidhar; Humphreys, John M.; Mullin, David A.; Tomchick, Diana R.; MacHius, Mischa; Peterson, Julian A.

In: Biochemistry, Vol. 50, No. 39, 04.10.2011, p. 8333-8341.

Research output: Contribution to journalArticle

Haines, DC, Hegde, A, Chen, B, Zhao, W, Bondlela, M, Humphreys, JM, Mullin, DA, Tomchick, DR, MacHius, M & Peterson, JA 2011, 'A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation', Biochemistry, vol. 50, no. 39, pp. 8333-8341. https://doi.org/10.1021/bi201099j
Haines, Donovan C. ; Hegde, Amita ; Chen, Baozhi ; Zhao, Weiqiang ; Bondlela, Muralidhar ; Humphreys, John M. ; Mullin, David A. ; Tomchick, Diana R. ; MacHius, Mischa ; Peterson, Julian A. / A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation. In: Biochemistry. 2011 ; Vol. 50, No. 39. pp. 8333-8341.
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