Structural basis for selective oxidation of phosphorylated ethylphenols by cytochrome P450 monooxygenase CreJ

Selective oxidation of C-H bonds in alkylphenols holds great significance for not only structural derivatization in pharma- and bio-manufacturing but also biological degradation of these toxic chemicals in environmental protection. A unique chemomimetic biocatalytic system using enzymes from a p-cresol biodegradation pathway has recently been developed. As the central biocatalyst, the cytochrome P450 monooxygenase CreJ oxidizes diverse p- and m-alkylphenyl phosphates with perfect stereoselectivity at different efficiencies. However, the mechanism of regio- and stereoselectivity of this chemomimetic biocatalytic system remained unclear. Here, using p- and m-ethylphenyl substrates, we elucidate the CreJ-catalyzed key steps for selective oxidations. The crystal structure of CreJ in complex with m-ethylphenyl phosphate was solved and compared with its complex structure with p-ethylphenyl phosphate isomer. The results indicate that the conformational changes of substrate-binding residues are slight, while the substrate promiscuity is achieved mainly by the available space in the catalytic cavity. Moreover, the catalytic preferences of regio- and stereoselective hydroxylation for the two ethylphenyl substrates is explored by molecular dynamics simulations. The ethyl groups in the complexes display different flexibility, and the distances of the active oxygen to Hpro-S and Hpro-R of methylene agree with the experimental stereoselectivity. The regioselectivity can be explained by the distances and bond dissociation energy. These results provide not only the mechanistic insights of CreJ into its regio- and stereoselectivity but also the structural basis for further P450 enzyme design and engineering. Importance The key cytochrome P450 monooxygenase CreJ showed excellent regio- and stereoselectivity in the oxidation of various alkylphenol substrates. C-H bond functionalization of these toxic alkylphenols holds great significance both for biological degradation of these environmental chemicals and production of value-added structural derivatives in pharmaceutical and biochemical industries. Our results, combined with in vitro enzymatic assays, crystal structure determination of enzyme-substrate complex, and molecular dynamics simulations, provide not only significant mechanism elucidation of the regio- and stereoselective catalyzation mediated by CreJ, but also the promising directions for the future engineering efforts of this enzyme towards more useful products. It also has great extendable potentials to couple this multifunctional P450 biocatalyst with other post modifying enzymes (e.g. hydroxyl based glycosylase) to access more alkylphenol derived high-value chemicals through environment-friendly biocatalysis and biotransformation.

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Molecular Dynamics Simulations of a Cytochrome P450 from Tepidiphilus thermophilus (P450-TT) Reveal How Its Substrate-Binding Channel Opens

Molecules ◽

10.3390/molecules26123614 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3614

Author(s):

Abayomi S. Faponle ◽

Anupom Roy ◽

Ayodeji A. Adelegan ◽

James W. Gauld

Keyword(s):

Crystal Structure ◽

Molecular Dynamics ◽

Cytochrome P450 ◽

Amino Acid ◽

Molecular Dynamics Simulations ◽

Organic Molecules ◽

Side Chain ◽

Relative Solvent Accessibility ◽

Amino Acid Residues ◽

Dynamics Simulations

Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl’s side chain tightens and relaxes the opening to the channel in conjunction with the arginyl’s, though the latter’s side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel.

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Structural basis for the hyperthermostability of an archaeal glutaminase induced by post-translational succinimide formation

10.1101/2021.03.02.433506 ◽

2021 ◽

Author(s):

Aparna Vilas Dongre ◽

Sudip Das ◽

Asutosh Bellur ◽

Sanjeev Kumar ◽

Anusha Chandrashekarmath ◽

...

Keyword(s):

Crystal Structure ◽

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Torsion Angle ◽

Conformational Flexibility ◽

Structural Basis ◽

Post Translational Modification ◽

Backbone Amide ◽

Conformationally Restrained ◽

Dynamics Simulations

AbstractStability of proteins from hyperthermophiles enabled by reduction of conformational flexibility is realized through various mechanisms. Presence of a stable, hydrolysis-resistant succinimide arising from cyclization of the side chains of aspartyl/asparaginyl residues with backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ. Here, we describe the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase (MjGATase) and address the mechanism of a succinimide-induced increased thermostability using molecular dynamics simulations. This study reveals the interplay of negatively charged electrostatic shield and n→π* interactions in preventing succinimide hydrolysis. The stable succinimidyl residue induces formation of a ‘conformational-lock’, reducing protein flexibility. Protein destabilization upon replacement with the Φ-restricted prolyl residue highlights the specificity of the conformationally restrained succinimidyl residue in imparting hyperthermostability. The conservation of succinimide-forming tripeptide sequence (E(N/D)(E/D)) in a group of archaeal GATases suggests an adaptation of this otherwise detrimental post-translational modification as an inducer of thermostability.

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Structural Basis for Selectivity in Flavin-Dependent Monooxygenase-Catalyzed Oxidative Dearomatization

10.26434/chemrxiv.7297766 ◽

2018 ◽

Author(s):

Attabey Rodríguez Benítez ◽

Sara Tweedy ◽

Summer A. Baker Dockrey ◽

April L. Lukowski ◽

Troy Wymore ◽

...

Keyword(s):

Crystal Structure ◽

Experimental Data ◽

Molecular Dynamics ◽

Protein Engineering ◽

Molecular Dynamics Simulations ◽

Substrate Binding ◽

Structural Basis ◽

Oxidative Dearomatization ◽

Dynamics Simulations

Herein, we disclose the structural basis for substrate binding in TropB, which performs a synthetically challenging asymmetric oxidative dearomatization reaction with exquisite site- and stereoselectivity across a range of substrates, providing a foundation for future protein engineering and reaction development efforts. Our hypothesis for substrate binding is informed by the first crystal structure of TropB and molecular dynamics simulations with the corresponding computational TropB model and is supported by experimental data.

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Structural Basis for Selectivity in Flavin-Dependent Monooxygenase-Catalyzed Oxidative Dearomatization

10.26434/chemrxiv.7297766.v1 ◽

2018 ◽

Author(s):

Attabey Rodríguez Benítez ◽

Sara Tweedy ◽

Summer A. Baker Dockrey ◽

April L. Lukowski ◽

Troy Wymore ◽

...

Keyword(s):

Crystal Structure ◽

Experimental Data ◽

Molecular Dynamics ◽

Protein Engineering ◽

Molecular Dynamics Simulations ◽

Substrate Binding ◽

Structural Basis ◽

Oxidative Dearomatization ◽

Dynamics Simulations

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Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway

10.26434/chemrxiv.12710159.v1 ◽

2020 ◽

Author(s):

Sean A. Newmister ◽

Kinshuk Raj Srivastava ◽

Rosa V. Espinoza ◽

Kersti Caddell Haatveit ◽

Yogan Khatri ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Molecular Basis ◽

Hydrophobic Interactions ◽

Cytochromes P450 ◽

Targeted Mutagenesis ◽

P450 Monooxygenase ◽

Bond Functionalization ◽

Dynamics Simulations

Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C-H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C-H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C-H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM/MM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.<br>

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Combined Use of Structure Analysis, Studies of Molecular Association in Solution, and Molecular Modelling to Understand the Different Propensities of Dihydroxybenzoic Acids to Form Solid Phases

Pharmaceutics ◽

10.3390/pharmaceutics13050734 ◽

2021 ◽

Vol 13 (5) ◽

pp. 734

Author(s):

Aija Trimdale ◽

Anatoly Mishnev ◽

Agris Bērziņš

Keyword(s):

Crystal Structure ◽

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Structure Analysis ◽

Crystal Structure Analysis ◽

Molecular Association ◽

Hydroxyl Groups ◽

Solid Phases ◽

Solvent Molecules ◽

Dynamics Simulations

The arrangement of hydroxyl groups in the benzene ring has a significant effect on the propensity of dihydroxybenzoic acids (diOHBAs) to form different solid phases when crystallized from solution. All six diOHBAs were categorized into distinctive groups according to the solid phases obtained when crystallized from selected solvents. A combined study using crystal structure and molecule electrostatic potential surface analysis, as well as an exploration of molecular association in solution using spectroscopic methods and molecular dynamics simulations were used to determine the possible mechanism of how the location of the phenolic hydroxyl groups affect the diversity of solid phases formed by the diOHBAs. The crystal structure analysis showed that classical carboxylic acid homodimers and ring-like hydrogen bond motifs consisting of six diOHBA molecules are prominently present in almost all analyzed crystal structures. Both experimental spectroscopic investigations and molecular dynamics simulations indicated that the extent of intramolecular bonding between carboxyl and hydroxyl groups in solution has the most significant impact on the solid phases formed by the diOHBAs. Additionally, the extent of hydrogen bonding with solvent molecules and the mean lifetime of solute–solvent associates formed by diOHBAs and 2-propanol were also investigated.

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