Heterotropic and homotropic cooperativity by a drug-metabolising mutant of cytochrome P450 BM3

Vugt-Lussenburg, Barbara M.A. van; Damsten, Micaela C.; Maasdijk, Dianne M.; Vermeulen, Nico; Commandeur, Jan N. M.

doi:10.1016/j.bbrc.2006.05.179

Cited by 91 publications

(96 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Two substrate binding sites have been established on the distal side of the heme in CYP3A4, but the binding of up to six substrate molecules, together with surface binding sites, open conformations and equilibration between oligomers, have been proposed to model the complex behavior (Davydov and Halpert, 2008). The effect of changes at sites remote from the active site on the product selectivity of P450 BM3 was noted in the oxidation of non-natural substrates by the R47L/Y51F mutant (Carmichael and Wong, 2001), and allosteric effects as well as space-filling mechanisms have been proposed for atypical kinetics in drug oxidation by the R47L/F87A/L188Q mutant (van Vugt-Lussenburg et al, 2006). Since substrate binding is detected by changes in the heme electronic spectrum, a plausible mechanism for the behavior at low ionic strength is that the second and third palmitate molecules bind outside the active site and cause conformational (allosteric) changes that reduce the heme spin state shift and hence ΔA max .…”

Section: Discussionmentioning

confidence: 99%

“…No sigmoidal behavior has been reported for fatty acid binding or oxidation by P450 BM3 (Gustafsson et al, 2004), though isotope effect studies suggested that both laurate and palmitate could be simultaneously present in the active site (Rock et al, 2003). On the other hand, both homotropic and heterotropic cooperativity have been reported for the oxidation of non-natural substrates such as indole by P450 BM3 (Li et al, 2005;Maurer et al, 2005;Huang et al, 2007), and for drug metabolism by the R47L/ F87V/L188Q mutant in which allosteric effects were also proposed (van Vugt-Lussenburg et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

et al. 2011

View full text Add to dashboard Cite

Fatty acid binding and oxidation kinetics for wild type P450 BM3 (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than k cat . The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450 BM3 could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

et al. 2011

View full text Add to dashboard Cite

show abstract

“…[13a, 21,26,28] Although the scaleability of those biohydroxylations on the screening scale was predicted repeatedly, literature reports of preparative metabolite syntheses using bifuncional CYPs are practically non-existent.…”

Section: Resultsmentioning

confidence: 99%

A Diversified Library of Bacterial and Fungal Bifunctional Cytochrome P450 Enzymes for Drug Metabolite Synthesis

et al. 2009

View full text Add to dashboard Cite

Innovative biohydroxylation catalysts for the preparation of drug metabolites were developed from scratch. A set of bacterial and fungal sequences of putative and already known bifunctional P450 enzymes was identified by protein sequence alignments, expressed in Escherichia coli and characterised. Notably, a fungal self-sufficient cytochrome P450 (CYP) from Aspergillus fumigatus turned out to be especially stable during catalyst preparation and application and also in presence of organic co-solvents. To enhance the catalytic activity and broaden the substrate specificity of those variants with high expression levels prominent single mutations were introduced. Selected improved variants were then used as lyophilised bacterial lysates for the synthesis of 4'-hydroxydiclofenac and 6-hydroxychlorzoxazone, the two metabolites of active pharmaceutical compounds diclofenac and chlorzoxazone representing the same metabolites as generated by human P450s.

show abstract

“…Here again, molecules that are not fatty acids are typically not accepted as substrates because the carboxylate group is required for catalytic activity. Although many mutagenic studies have been performed with P450 BM3 with respect to broadening the substrate scope [34][35][36][37][38][39][40][41][42][43][44], this study demonstrated that perfluorocarboxylic acids (PFCs) can be used as decoy molecules to expand the substrate scope of P450 BM3 without resorting to mutagenesis. PFCs are a great alternative to fatty acids because many of the physical properties are maintained, while the greater bond energy of C-F (116 kcal mol −1 ) compared to that of C-H (95-99 kcal mol −1 ) renders them inert toward oxidation [30].…”

Section: Perfluorinated Fatty Acidsmentioning

confidence: 92%

Use of Chemical Auxiliaries to Control P450 Enzymes for Predictable Oxidations at Unactivated C-H Bonds of Substrates

Auclair

Polic

2015

Advances in Experimental Medicine and Biology

View full text Add to dashboard Cite

Cytochrome P450 enzymes (P450s) have the ability to oxidize unactivated C-H bonds of substrates with remarkable regio-and stereoselectivity. Comparable selectivity for chemical oxidizing agents is typically difficult to achieve. Hence, there is an interest in exploiting P450s as potential biocatalysts. Despite their impressive attributes, the current use of P450s as biocatalysts is limited. While bacterial P450 enzymes typically show higher activity, they tend to be highly selective for one or a few substrates. On the other hand, mammalian P450s, especially the drugmetabolizing enzymes, display astonishing substrate promiscuity. However, product prediction continues to be challenging. This review discusses the use of small molecules for controlling P450 substrate specificity and product selectivity. The focus will be on two approaches in the area: (1) the use of decoy molecules, and (2) the application of substrate engineering to control oxidation by the enzyme.

show abstract

Heterotropic and homotropic cooperativity by a drug-metabolising mutant of cytochrome P450 BM3

Cited by 91 publications

References 33 publications

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

A Diversified Library of Bacterial and Fungal Bifunctional Cytochrome P450 Enzymes for Drug Metabolite Synthesis

Use of Chemical Auxiliaries to Control P450 Enzymes for Predictable Oxidations at Unactivated C-H Bonds of Substrates

Contact Info

Product

Resources

About