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Cytochrome P450SPα (CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H2O2-dependent P450. P450SPα hydroxylates fatty acids with high α-regioselectivity. Herein we report the crystal structure of P450SPα with palmitic acid as a substrate at a resolution of 1.65 Å. The structure revealed that the Ca of the bound palmitic acid in one of the alternative conformations is 4.5 Å from the heme iron. This conformation explains the highly selective α-hydroxylation of fatty acid observed in P450SPα. Mutations at the active site and the F-G loop of P450SPα did not impair its regioselectivity. The crystal structures of mutants (L78F and F288G) revealed that the location of the bound palmitic acid was essentially the same as that in the WT, although amino acids at the active site were replaced with the corresponding amino acids of cytochrome P450BSβ (CYP152A1), which shows β-regioselectivity. This implies that the high regioselectivity of P450SPα is caused by the orientation of the hydrophobic channel, which is more perpendicular to the heme plane than that of P450BSβ. [ABSTRACT FROM AUTHOR]

Poly[ N 5 -( R and S )-1-(1-pyrenyl)ethyl- l -glutamines] ( 1 and 2 ) were prepared by condensation of poly( l -glutamic acid) with optically resolved amines. In solution, these polymers, 2 in particular, gave large circular dichroism (CD) indicative of exciton coupling among the side-chain pyrene chromophores. When compared with the corresponding polymer with achiral side groups, i.e. poly(1-pyrenylmethyl- l -glutamine) ( 3 ), 1 and 2 not only gave much stronger CD, but also gave much reduced excimer emission with a significant hypsochromic shift of emission maximum. The highly controlled orientation of the side-chain chromophores is apparently brought about by the specific steric interactions among the bulky chiral side chains along the helical main chain. [ABSTRACT FROM AUTHOR]

Abstract: The enantioselectivity in the sulfoxidation of thioanisole catalyzed by cytochrome P450BSβ with a decoy molecule, a dummy molecule of the natural substrate, can be inverted by changing the structure of the decoy molecule. The methodology demonstrated herein shows the potential for controlling the stereoselectivity of biocatalysts without any mutagenesis. [Copyright &y& Elsevier]

The wild-type cytochrome P450 (CYP) monooxygenase enzyme CYP102A1 (P450Bm3) has low activity for cycloalkane oxidation. The oxidation of these substrates by variants of this enzyme in combination with perfluorinated decoy molecules (PFCs) was investigated to improve productivity. The use of rate accelerating variants, which have mutations located outside of the substrate binding pocket as well as an active site variant of CYP102A1 (A74G/F87V/L188Q) all enhanced cycloalkane oxidation (C5 to C10). The addition of the decoy molecules to the wild-type and the rate accelerating mutants of CYP102A1 boosted the substrate oxidation rates even further. However, the levels of cycloalkanol product decreased with the larger alkanes when the decoy molecules were used with the variant A74G/F87V/L188Q, which contained mutations within the substrate binding pocket. For the majority of the enzymes and PFC decoy molecule combinations the highest levels of oxidation were obtained with cyclooctane. When larger second generation decoy molecules, based on modified amino acids were utilised there was a significant improvement in the oxidation of the smaller cycloalkanes by the wild-type enzyme and one other variant. This resulted in significant improvements in biocatalytic oxidation of cyclopentane and cyclohexane. However, the use of these optimised decoy molecules did not significantly improve cycloalkane oxidation over the fluorinated fatty acid derivatives when combined with the best rate accelerating variant, R47L/Y51F/I401P. Overall our approach enabled the cycloalkanes to be oxidised 300- to 8000-fold more efficiently than the wild-type enzyme at product formation rates in excess of 500 and up to 1700 nmol·nmol-CYP −1 ·min −1 . [ABSTRACT FROM AUTHOR]

Cytochrome P450BM3 has long been regarded as a promising candidate for use as a biocatalyst, owing to its excellent efficiency for the hydroxylation of unactivated C–H bonds. However, because of its high substrate specificity, its possible applications have been severely limited. Consequently, various approaches have been proposed to overcome the enzyme's natural limitations, thereby expanding its substrate scope to encompass non-native substrates, evoking chemoselectivity, regioselectivity and stereoselectivity and enabling previously inaccessible chemical conversions. Herein, these approaches will be classified into three categories: (1) mutagenesis including directed evolution, (2) haem substitution with artificial cofactors and (3) use of substrate mimics, 'decoy molecules'. Herein, we highlight the representative work that has been conducted in above three categories for discussion of the future outlook of P450BM3 in green chemistry. [ABSTRACT FROM AUTHOR]

Abstract 2,3′,4,4′,5-Pentachlorobiphenyl (CB118) is one of the most abundant polychlorinated biphenyl (PCB) congeners in the environment, and perfluoroalkyl acids, including perfluorocarboxylic acids (PFCAs), are widely distributed in the environment. Although CB118 and perfluoroalkyl acids are present in all humans and biota, effects in the metabolic fate of CB118 leading to toxicity change are unclear. P450BM3, which is isolated from the soil bacterium Bacillus megaterium , metabolized CB118 to three different hydroxylated pentachlorobiphenyls (M1-M3). M2 was identified as 4′-OH-2,3′,4,5,5′-pentachlorobiphenyl. These reactions were promoted by the presence of PFCAs, and perfluorooctanoic acid (PFCA-C8) was the most effective for accelerating these reactions among PFCAs with different carbon chain length. The production rate of M2 was accelerated by 25-times using PFCA-C8. Furthermore, the docking models of P450BM3 with CB118 and PFCAs revealed that the conformational changes of the substrate-binding cavity of P450BM3 after binding of PFCAs to P450BM3 were important for selective production of CB118 metabolites. This study leads to the clarification of the different metabolic fates of PCBs under complex contamination with PFCAs. Graphical abstract Image 1 Highlights • Bacterial P450BM3 metabolized CB118 to hydroxylated metabolites. • Hydroxylation of CB118 was promoted by the existence of PFCAs. • The size of the substrate-binding cavity of P450BM3 was changed by binding PFCAs. • Metabolic fates of PCBs under complex contamination with PFCAs may be different. [ABSTRACT FROM AUTHOR]

Cytochrome P450 enzymes (CYPs) have attracted much promise as biocatalysts in a push for cleaner and more environmentally friendly catalytic systems. However, changing the substrate specificity of CYPs, such as CYP102A1, can be a challenging task, requiring laborious mutagenesis. An alternative approach is the use of decoy molecules that "trick" the enzyme into becoming active by impersonating the native substrate. Whilst the decoy molecule system has been extensively developed for CYP102A1, its general applicability for other CYP102-family enzymes has yet to be shown. Herein, we demonstrate that decoy molecules can "trick" CYP102A5 and A7 into becoming active and hydroxylating non-native substrates. Furthermore, significant differences in decoy molecule selectivity as well as decoy molecule binding were observed. The X-ray crystal structure of the CYP102A5 haem domain was solved at 2.8 Å, delivering insight into a potential substate-binding site that differs significantly from CYP102A1. Decoy molecules were shown to be compatible for use with both CYP102A5 and CYP102A7. Both CYP102A5 and CYP102A7 displayed a unique and unexpected decoy molecule preference. The crystal structure of CYP102A5 was solved at a resolution of 2.8 Å. [Display omitted] • Decoy molecules were shown to be compatible for use with both CYP102A5 and A7. • Both CYP102A5 and A7 displayed a unique and unexpected decoy molecule preference. • Decoy molecules were found to bind in a two-step fashion to CYP102A5 and A7 that was not observed for A1. • We report the first structure of CYP102A5 at a resolution of 2.8 Å. • The structure of CYP102A5 reveals a potential binding site that differs significantly from the CYP102A1 reference system. [ABSTRACT FROM AUTHOR]