Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from computer simulations studies

Roccatano, Danilo

doi:10.1088/0953-8984/27/27/273102

Cited by 31 publications

(10 citation statements)

References 90 publications

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“…Hence, we additionally introduced the HighSP sequence in a loop region near the FAD pocket exchanging the amino acids 855–859 from WSGYG to (RTHRK) 2 . This led to a loss of activity in set‐up I and II, which was attributed to a steric distortion of the NADPH binding pocket by length‐divergent loop alteration (Figure a) . By application of −0.6 V, the distorted NADPH binding is avoided, leading to equal activity with respect to HSP.…”

Section: Resultsmentioning

confidence: 99%

Surface‐Binding Peptide Facilitates Electricity‐Driven NADPH‐Free Cytochrome P450 Catalysis

et al. 2018

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Industrial biotechnology aims to exploit cytochrome P450 enzymes to access their sophisticated catalytic activity for challenging chemical reactions on inert C−H bonds. Limited by the need for NADPH, approaches to bind P450 enzymes to electrode surfaces for an artificial electron supply are promising. Here, we demonstrate that a recombinant fusion of an indium tin oxide binding peptide and the multi‐domain class VIII cytochrome P450 BM3 can be used in electrically driven catalysis. Bioelectrocatalytic activity is analyzed by direct product quantification resulting in superior activity of the specifically immobilized P450 BM3 in contrast to unspecifically adsorbed enzyme. Spacer and anchor point studies imply that enzyme flexibility and alignment are crucial factors to achieve high activity on the electrode. Furthermore, we demonstrate that our approach is also feasible for pharmaceutical application using naringenin as substrate.

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Section: Resultsmentioning

confidence: 99%

Surface‐Binding Peptide Facilitates Electricity‐Driven NADPH‐Free Cytochrome P450 Catalysis

et al. 2018

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“…[40] Recent molecular dynamics (MD) simulations using the crystal structure as starting point identified putative electron transfer pathways from the FMN cofactor to the heme. [42, 43]…”

Section: Discussionmentioning

confidence: 99%

Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies

Spradlin

Lee

Mahadevan

et al. 2016

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Background In order to perform selective C-H functionalization upon visible light irradiation, Ru(II)-diimine functionalized P450 heme enzymes have been developed. The sL407C-1 enzyme containing the Ru(bpy)2PhenA (bpy = 2,2′-bipyridine and PhenA = 5-acetamido-1,10-phenanthroline) photosensitizer (1) covalently attached to the non-native single cysteine L407C of the P450BM3 heme domain mutant, displays high photocatalytic activity in the selective C-H bond hydroxylation of several substrates. Methods A combination of X-ray crystallography, site-directed mutagenesis, transient absorption measurements and enzymatic assays was used to gain insights into its photocatalytic activity and electron transfer pathway. Results The crystal structure of the sL407C-1 enzyme was solved in the open and closed conformations revealing a through-space electron transfer pathway involving highly conserved, F393 and Q403, residues. Several mutations of these residues (F393A, F393W or Q403W) were introduced to probe their roles in the overall reaction. Transient absorption measurements confirm rapid electron transfer as heme reduction is observed in all four hybrid enzymes. Compared to the parent sL407C-1, photocatalytic activity was negligible in the dF393A-1 enzyme while 60% increase in activity with total turnover numbers of 420 and 90% product conversion was observed with the dQ403W-1 mutant. Conclusions In the sL407C-1 enzyme, the photosensitizer is ideally located to rapidly deliver electrons, using the naturally occurring electron transfer pathway, to the heme center in order to activate molecular dioxygen and sustain photocatalytic activity. General Significance The results shed light on the design of efficient light-driven biocatalysts and the approach can be generalized to other members of the P450 superfamily.

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“…The protonation state of protein residues was assumed to be the same as that of the corresponding isolated amino acids in solution at pH 7. The GROMOS96 43a1 force field, used for all simulations, was adopted for consistency with the previous simulation studies of the same enzyme . Parameters of the heme cofactor were the same as in our previous paper …”

Section: Computational Methodsmentioning

confidence: 99%

“…45 Parameters of heme cofactor were the same as in our previous paper. 31 The MD simulations were set up for HEME in water and aqueous Hence, the starting confirmation was solvated by stacking an equilibrated box of water molecules to fill the empty space in the simulation box.…”

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confidence: 99%

Unraveling Binding Effects of Cobalt(II) Sepulchrate with the Monooxygenase P450 BM-3 Heme Domain Using Molecular Dynamics Simulations

Verma

Schwaneberg

Holtmann

et al. 2015

J. Chem. Theory Comput.

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One of the major limitations to exploit enzymes in industrial processes is their dependence on expensive reduction equivalents like NADPH to drive their catalytic cycle. Soluble electron transfer (ET) mediators like Cobalt(II)Sepulchrate have been proposed as a cost-effective alternative to shuttle electrons between an inexpensive electron source and enzyme redox center.The interactions of these molecules with enzymes are not elucidated at molecular level yet.

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Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from computer simulations studies

Cited by 31 publications

References 90 publications

Surface‐Binding Peptide Facilitates Electricity‐Driven NADPH‐Free Cytochrome P450 Catalysis

Surface‐Binding Peptide Facilitates Electricity‐Driven NADPH‐Free Cytochrome P450 Catalysis

Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies

Unraveling Binding Effects of Cobalt(II) Sepulchrate with the Monooxygenase P450 BM-3 Heme Domain Using Molecular Dynamics Simulations

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