Cytochrome P450cin (CYP176A1) D241N: Investigating the role of the conserved acid in the active site of cytochrome P450s

Stok, Jeanette E.; Yamada, Sean; Farlow, Anthony; Slessor, Kate E.; Voss, James J. De

doi:10.1016/j.bbapap.2012.12.022

Cited by 23 publications

(22 citation statements)

References 57 publications

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“…If so then recent work with P450cin places into question the mechanical model. Asp241 in P450cin corresponds to Asp251 in P450cam and the D241N mutant in P450cin shows that Asp241 also is essential for activity 227 but is not tied down by ion pairs and should be free to rotate into the active site. A way out of this apparent conundrum is if the open conformation of P450cam observed in P450cam-Pdx structure represents the active conformational state.…”

Section: Cytochrome P450mentioning

confidence: 99%

Heme Enzyme Structure and Function

2014

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Section: Cytochrome P450mentioning

confidence: 99%

Heme Enzyme Structure and Function

2014

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“…The P450 cin in vitro GC‐MS assay was performed as outlined previously employing either (1 R )‐or (1 S )‐thiocamphor 3 as the substrate . The P450 cam in vitro GC‐MS assay with camphane 7 or (1 R ) or (1 S )‐thiocamphor 3 was performed as follows.…”

Section: Methodsmentioning

confidence: 99%

“…Eur.J. 2019,25,[4149][4150][4151][4152][4153][4154][4155] www.chemeurj.org epi-isoborneol 8 [22] and epi-camphor 19 [23] were synthesised according to previously published protocols. All other compounds were commercially available.…”

Section: Experimental Section Experimental Detailsmentioning

confidence: 99%

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Significance of Protein–Substrate Hydrogen Bonding for the Selectivity of P450‐Catalysed Oxidations

Slessor

Stok

Chow

et al. 2019

Chemistry A European J

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P450cin and P450cam are bacterial cytochromes P450 that specifically hydroxylate bicyclic monoterpenes. Protein–substrate H bonding has been previously proposed as crucial in the selectivity of P450cin oxidations, but not as essential for P450cam. To examine the difference in importance of H bonds in these two model P450s, the P450‐catalysed oxidation products from thiocamphor were compared. Surprisingly, both P450s oxidised thiocamphor predominantly to the corresponding S‐oxides, in contrast to previous reports, and this is the first report of P450‐catalysed sulfine generation from a thioketone. Additionally, the result emphasised the importance of the protein–substrate H bond to selectivity in both P450cin and P450cam. The H bonding in P450cam was re‐examined using camphane, another substrate for which the protein–substrate H bond is absent. The results indicated that both H bonding and hydrophobic interactions between substrate and protein play a role in selectivity. Interestingly, the protein–substrate H bond was not a factor in substrate affinity for the enzyme.

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“…Mutagenesis studies have demonstrated that a range of hydroxycineole isomers can be obtained from a number of single amino acid mutations of CYP176A1 around the heme containing active site ( Fig. 2) [45][46][47]. In addition, CYP176A1…”

Section: A N U S C R I P Tmentioning

confidence: 99%

Selective hydroxylation of 1,8- and 1,4-cineole using bacterial P450 variants

Lee

Wong

Stok

et al. 2019

Archives of Biochemistry and Biophysics

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This study has evaluated the use of the P450 metalloenzymes CYP176A1, CYP101A1 and CYP102A1, together with engineered protein variants of CYP101A1 and CYP102A1, to alter the regioselectivity of 1,8-and 1,4-cineole hydroxylation. CYP176A1 was less selective for 1,4-cineole oxidation when compared to its preferred substrate, 1,8-cineole. The CYP102A1 variants significantly improved the activity over the WT enzyme for oxidation of 1,4-and 1,8-cineole. The CYP102A1 R47L/Y51F/A74G/F87V/L188Q mutant generated predominantly (1S)-6α-hydroxy-1,8-cineole (78% e.e.) from 1,8-cineole. Oxidation of 1,4-cineole by the CYP102A1 R47L/Y51F/F87A/I401P variant generated the 3α product in >90% yield. WT CYP101A1 formed a mixture metabolites with 1,8-cineole and very little product was generated with 1,4-cineole. In contrast the F87W/Y96F/L244A/V247L and F87W/Y96F/L244A variants of CYP101A1 favoured formation of 5α-hydroxy-1,8-cineole (>88%, 1S 86% e.e.) while the F87V/Y96F/L244A variant generated (1S)-6α-hydroxy-1,8-cineole in excess (90% regioselective, >99% e.e.). The CYP101A1 F87W/Y96F/L244A/V247L and F87W/Y96F/L244A mutants improved the oxidation of 1,4-cineole generating an excess of the 3α metabolite (1S >99% e.e. with the latter). The CYP101A1 F87L/Y96F variant also improved the oxidation of this substrate but shifted the site of oxidation to the isopropyl group, (8-hydroxy-1,4-cineole). When this 8hydroxy metabolite was generated in significant quantities desaturation of C8-C9 to the corresponding alkene was also detected. Highlights CYP176A1 was less selective for 1,4-cineole oxidation compared to 1,8-cineole. CYP102A1 and CYP101A1 variants were used to generate (1S)-6α-hydroxy-1,8-cineole in excess (>90%). Other mutant forms of CYP101A1 were selective for 5α-hydroxy-1,8-cineole (>88%, 86% e.e.). Oxidation of 1,4-cineole by CYP102A1 and CYP101A1 mutants generated the 3α product in >90% yield. The CYP101A1 F87L/Y96F variant favoured 8-hydroxy-1,4-cineole formation. The formation of 8-hydroxy-1,4cineole was accompanied by desaturation to a C8-C9 alkene.

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Cytochrome P450cin (CYP176A1) D241N: Investigating the role of the conserved acid in the active site of cytochrome P450s

Cited by 23 publications

References 57 publications

Heme Enzyme Structure and Function

Heme Enzyme Structure and Function

Significance of Protein–Substrate Hydrogen Bonding for the Selectivity of P450‐Catalysed Oxidations

Selective hydroxylation of 1,8- and 1,4-cineole using bacterial P450 variants

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