Biophysical Techniques for Distinguishing Ligand Binding Modes in Cytochrome P450 Monooxygenases

Podgorski, Matthew N.; Harbort, Joshua S.; Coleman, Tom; Stok, Jeanette E.; Yorke, Jake A.; Wong, Luet‐Lok; Bruning, John B.; Bernhardt, Paul V.; Voss, James J. De; Harmer, Jeffrey; Bell, Stephen

doi:10.1021/acs.biochem.0c00027

Cited by 23 publications

(44 citation statements)

References 75 publications

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“…According to one review report [ 3 , 51 , 52 ], all CYPs were different with different activities, and their functional properties were substrate-dependent. The user can swap the active sites to get the desired function from CYPs [ 53 ]. Rowland et al [ 54 ] reported the crystal structure of CYP2D 6 at the resolution of 3A° and noted that the primary structure of the CYP2D 6 is resampled with other CYPs .…”

Section: Discussionmentioning

confidence: 99%

[Retracted] In Silico Structural, Functional, and Phylogenetic Analysis of Cytochrome (CYPD) Protein Family

Ahmad

Afzal

Jamal

et al. 2021

BioMed Research International

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Cytochrome (CYP) enzymes catalyze the metabolic reactions of endogenous and exogenous compounds. The superfamily of enzymes is found across many organisms, regardless of type, except for plants. Information was gathered about CYP2D enzymes through protein sequences of humans and other organisms. The secondary structure was predicted using the SOPMA. The structural and functional study of human CYP2D was conducted using ProtParam, SOPMA, Predotar 1.03, SignalP, TMHMM 2.0, and ExPASy. Most animals shared five central motifs according to motif analysis results. The tertiary structure of human CYP2D, as well as other animal species, was predicted by Phyre2. Human CYP2D proteins are heavily conserved across organisms, according to the findings. This indicates that they are descended from a single ancestor. They calculate the ratio of alpha-helices to extended strands to beta sheets to random coils. Most of the enzymes are alpha-helix, but small amounts of the random coil were also found. The data were obtained to provide us with a better understanding of mammalian proteins’ functions and evolutionary relationships.

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Section: Discussionmentioning

confidence: 99%

[Retracted] In Silico Structural, Functional, and Phylogenetic Analysis of Cytochrome (CYPD) Protein Family

Ahmad

Afzal

Jamal

et al. 2021

BioMed Research International

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“…In addition, there was a spectral difference in the band between substratebound and unbound forms. Papaverine-bound spectra showed a Soret band at 322 nm, which was not observed in the binding of 4-(pyridine-3-yl) benzoic acid and 4-(pyridine-2-yl) benzoic acid to CYP199A4 (Podgorski et al, 2020). This is the first report of N-oxidation of a compound by bacterial CYP.…”

Section: Functional Characterization Of Cyp105d18mentioning

confidence: 71%

“…Even though THP was catalyzed by CYP105D18 using H 2 O 2 as cosubstrate, there was no spin-shift observed. The binding of 4-(pyridine-3-yl) benzoic acid and 4-(pyridine-2-yl) benzoic acid to CYP199A4 exhibits type II UV-vis spectral changes showing a greater shift in the Soret wavelength (422 versus 424 nm) (Podgorski et al, 2020). Papaverine was also modified in the pyridine ring (N-oxidation) by CYP105D18 and exhibits a similar pattern of spectral shift at 422 nm.…”

Section: Functional Characterization Of Cyp105d18mentioning

confidence: 98%

Characterization of high-H₂O₂-tolerant bacterial cytochrome P450 CYP105D18: insights into papaverine N-oxidation

Pardhe

Jeong

et al. 2021

Int Union Crystallogr J

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The bacterial CYP105 family is involved in secondary metabolite biosynthetic pathways and plays essential roles in the biotransformation of xenobiotics. This study investigates the newly identified H2O2-mediated CYP105D18 from Streptomyces laurentii as the first bacterial CYP for N-oxidation. The catalytic efficiency of CYP105D18 for papaverine N-oxidation was 1.43 s−1 µM −1. The heme oxidation rate (k) was low (<0.3 min−1) in the presence of 200 mM H2O2. This high H2O2 tolerance capacity of CYP105D18 led to higher turnover prior to heme oxidation. Additionally, the high-resolution papaverine complexed structure and substrate-free structure of CYP105D18 were determined. Structural analysis and activity assay results revealed that CYP105D18 had a strong substrate preference for papaverine because of its bendable structure. These findings establish a basis for biotechnological applications of CYP105D18 in the pharmaceutical and medicinal industries.

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“…In the presence of VBA, there is a significant positive shift in the Fe III/II redox potential relative to the substrate-free redox potential, ΔE = +203 mV (Figures 1d and S2). This positive shift in redox potential is smaller than that measured for 4-methoxybenzoic acid [ΔE = +250 mV, 4-methoxybenzoic acid-bound CYP199A4 and substrate-free CYP199A4 have redox potentials of −188 and −438 mV, respectively, vs normal hydrogen electrode (NHE)], 63 which conforms with the greater spin state shift induced by 4-methoxybenzoic acid. ΔE has been linked to the extent of the displacement of the aqua ligand and associated outer sphere water molecules from the ferric heme, which favors reduction and has been proposed to gate the commencement of the catalytic cycle.…”

Section: ■ Resultsmentioning

confidence: 99%

“…Anisotropic proton hyperfine coupling ridges characteristic of aqua ligands are visible in the weak coupling quadrant (top right, +,+ quadrant). The HYSCORE spectrum for 4methoxybenzoic acid has been reported previously 63.…”

mentioning

confidence: 99%

Understanding the Mechanistic Requirements for Efficient and Stereoselective Alkene Epoxidation by a Cytochrome P450 Enzyme

et al. 2021

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The cytochrome P450 (CYP) family of heme monooxygenase enzymes commonly catalyzes enantioselective hydroxylation and epoxidation reactions. Epoxidation reactions have been hypothesized to proceed via multiple mechanisms involving different reactive intermediates. Here, we use activity, spectroscopic, structural, and molecular dynamics data to investigate the activity and stereoselectivity of 4-vinylbenzoic acid epoxidation by the bacterial enzyme CYP199A4 from Rhodopseudomonas palustris HaA2. The epoxidation of 4-vinylbenzoic acid by CYP199A4 proceeded with high enantioselectivity, giving the (S)-epoxide in 99% ee at an activity of 220 nmol nmol-CYP–1 min–1. Optical and EPR spectroscopy, redox potential measurements, and the crystal structure of 4-vinylbenzoic acid-bound CYP199A4 indicated the partial retention of an aqua ligand at the heme center in the presence of the substrate, providing a justification of the lower activity (∼20%) compared to the oxidative demethylation of 4-methoxybenzoic acid. Mutagenesis at the conserved acid–alcohol pair (D251/T252), which perturbs the generation of the reactive oxygen intermediates, was employed to investigate their role in epoxidation reactions. The T252A mutant increased the rate of turnover of the catalytic cycle, but an elevation in hydrogen peroxide generation via uncoupling resulted in a similar rate of epoxide formation. The activity of epoxidation significantly reduced with the D251N mutant. The chemoselectivity and stereoselectivity of the epoxidation reaction were maintained in the turnovers by these mutants. Overall, there was little evidence that other intermediates, aside from the archetypal reactive ferryl porphyrin cation radical, Compound I, contributed significantly to the epoxidation reaction. The observation of the high selectivity for the (S)-enantiomer was rationalized by molecular dynamics simulations. When the arrangement of the alkene and the active intermediate approached an ideal transition state structure for epoxidation, one face of the alkene was more often exposed to the iron oxo unit.

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Biophysical Techniques for Distinguishing Ligand Binding Modes in Cytochrome P450 Monooxygenases

Cited by 23 publications

References 75 publications

[Retracted] In Silico Structural, Functional, and Phylogenetic Analysis of Cytochrome (CYPD) Protein Family

[Retracted] In Silico Structural, Functional, and Phylogenetic Analysis of Cytochrome (CYPD) Protein Family

Characterization of high-H₂O₂-tolerant bacterial cytochrome P450 CYP105D18: insights into papaverine N-oxidation

Understanding the Mechanistic Requirements for Efficient and Stereoselective Alkene Epoxidation by a Cytochrome P450 Enzyme

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Biophysical Techniques for Distinguishing Ligand Binding Modes in Cytochrome P450 Monooxygenases

Cited by 23 publications

References 75 publications

[Retracted] In Silico Structural, Functional, and Phylogenetic Analysis of Cytochrome (CYPD) Protein Family

[Retracted] In Silico Structural, Functional, and Phylogenetic Analysis of Cytochrome (CYPD) Protein Family

Characterization of high-H2O2-tolerant bacterial cytochrome P450 CYP105D18: insights into papaverine N-oxidation

Understanding the Mechanistic Requirements for Efficient and Stereoselective Alkene Epoxidation by a Cytochrome P450 Enzyme

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Characterization of high-H₂O₂-tolerant bacterial cytochrome P450 CYP105D18: insights into papaverine N-oxidation