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1.
J Inorg Biochem ; 245: 112235, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37167731

RESUMO

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.


Assuntos
Proteínas de Bactérias , Sistema Enzimático do Citocromo P-450 , Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/metabolismo , Sítios de Ligação , Especificidade por Substrato , NADPH-Ferri-Hemoproteína Redutase/química
2.
Sci Total Environ ; 890: 164475, 2023 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-37257625

RESUMO

Bacterial cytochrome P450 monooxygenase P450BM3 is a promising enzyme to provide novel substrate specificity and enhanced enzymatic activity. The wild type (WT) has been shown to metabolize the widely distributed polychlorinated biphenyl (PCB) 2,3',4,4',5-pentachlorobiphenyl (CB118) to hydroxylated metabolites. However, this reaction requires the coexistence of perfluoroalkyl carboxylic acids (PFCAs). To locate P450BM3 mutants metabolizing CB118 without PFCAs, mutations were selected from amino acids comprising the substrate-binding cavity and the substrate entrance. The mutant A264G showed enhanced hydroxylation activities compared to the WT for the production of five hydroxylated metabolites. Perfluorooctanoic acid addition provided the highest activity, as found in the WT. The docking model of A264G and CB118 indicated that the enlargement of the space above the heme brought CB118 close to the heme, resulting in high activity. In contrast, the mutants L188Q, QG, LVQ, and GVQ, which contain the L188Q mutation, showed higher activity than WT even without PFCAs. Docking models revealed that the closed form found in substrate binding was induced by the L188Q mutation in the substrate non-binding state of the mutants. These mutants are promising for bioremediation of PCBs using enhanced metabolizing activities.


Assuntos
Bacillus megaterium , Bifenilos Policlorados , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Bifenilos Policlorados/metabolismo , Hidroxilação , Heme/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
3.
Angew Chem Int Ed Engl ; 62(13): e202215706, 2023 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-36519803

RESUMO

Catching the structure of cytochrome P450 enzymes in flagrante is crucial for the development of P450 biocatalysts, as most structures collected are found trapped in a precatalytic conformation. At the heart of P450 catalysis lies Cpd I, a short-lived, highly reactive intermediate, whose recalcitrant nature has thwarted most attempts at capturing catalytically relevant poses of P450s. We report the crystal structure of P450BM3 mimicking the state in the precise moment preceding epoxidation, which is in perfect agreement with the experimentally observed stereoselectivity. This structure was attained by incorporation of the stable Cpd I mimic oxomolybdenum mesoporphyrin IX into P450BM3 in the presence of styrene. The orientation of styrene to the Mo-oxo species in the crystal structures sheds light onto the dynamics involved in the rotation of styrene to present its vinyl group to Cpd I. This method serves as a powerful tool for predicting and modelling the stereoselectivity of P450 reactions.


Assuntos
Sistema Enzimático do Citocromo P-450 , Estirenos , Oxirredução , Sistema Enzimático do Citocromo P-450/metabolismo , Catálise
4.
Angew Chem Int Ed Engl ; 59(19): 7611-7618, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32157795

RESUMO

Despite CYP102A1 (P450BM3) representing one of the most extensively researched metalloenzymes, crystallisation of its haem domain upon modification can be a challenge. Crystal structures are indispensable for the efficient structure-based design of P450BM3 as a biocatalyst. The abietane diterpenoid derivative N-abietoyl-l-tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild-type P450BM3 haem domain, with visible crystals forming within 2 hours and diffracting to a near-atomic resolution of 1.22 Å. Using these crystals as seeds in a cross-microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoy molecules and diverse artificial metalloporphyrins binding various ligand molecules, as well as heavily tagged haem-domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle.


Assuntos
Proteínas de Bactérias/química , Cristalização/métodos , Sistema Enzimático do Citocromo P-450/química , NADPH-Ferri-Hemoproteína Redutase/química , Bacillus megaterium/química , Catálise , Heme/química , Indicadores e Reagentes , Metaloporfirinas/síntese química , Mutagênese Sítio-Dirigida , Ligação Proteica , Especificidade por Substrato , Difração de Raios X
5.
Chemosphere ; 210: 376-383, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30015128

RESUMO

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.


Assuntos
Caprilatos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Fluorocarbonos/farmacologia , Bifenilos Policlorados/metabolismo , Bactérias/enzimologia , Sítios de Ligação , Caprilatos/química , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Fluorocarbonos/química , Humanos , Hidroxilação , Simulação de Acoplamento Molecular , Bifenilos Policlorados/química , Bifenilos Policlorados/toxicidade , Ligação Proteica , Solo
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