Cytochrome P450 1A2 (P450 1A2, CYP1A2) is a membrane-bound enzyme that oxidizes a broad range of hydrophobic substrates. The structure and dynamics of both the catalytic and trans-membrane (TM) domains of this enzyme in the membrane/water environment were investigated using a multiscale computational approach, including coarse-grained and all-atom molecular dynamics. Starting from the spontaneous self-assembly of the system containing the TM or soluble domain immersed in randomized dilauroyl phosphatidylcholine (DLPC)/water mixture into their respective membrane-bound forms, we reconstituted the membrane-bound structure of the full-length P450 1A2. This structure includes a TM helix that spans the membrane, while being connected to the catalytic domain by a short flexible loop. Furthermore, in this model, the upper part of the TM helix interacts directly with a conserved and highly hydrophobic N-terminal proline-rich segment of the catalytic domain; this segment and the FG loop are immersed in the membrane, whereas the remaining portion of the catalytic domain remains exposed to aqueous solution. The shallow membrane immersion of the catalytic domain induces a depression in the opposite intact layer of the phospholipids. This structural effect may help in stabilizing the position of the TM helix directly beneath the catalytic domain. The partial immersion of the catalytic domain also allows for the enzyme substrates to enter the active site from either aqueous solution or phospholipid environment via several solvent- and membrane-facing tunnels in the full-length P450 1A2. The calculated tunnel dynamics indicated that the opening probability of the membrane-facing tunnels is significantly enhanced when a DLPC molecule spontaneously penetrates into the membrane-facing tunnel 2d. The energetics of the lipid penetration process were assessed by the linear interaction energy (LIE) approximation, and found to be thermodynamically feasible.

• MeSH
• cytochrom P-450 CYP1A2 chemie   MeSH
• fosfatidylcholiny MeSH
• fosfolipidy chemie   MeSH
• katalytická doména MeSH
• katalýza MeSH
• lidé MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 1,2-dilauroylphosphatidylcholine MeSH Prohlížeč
• cytochrom P-450 CYP1A2 MeSH
• fosfatidylcholiny MeSH
• fosfolipidy MeSH

• MeSH
• enzymy chemie   genetika   metabolismus   MeSH
• koenzymy chemie   metabolismus   MeSH
• konformace proteinů MeSH
• proteinové inženýrství metody   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• rozpouštědla MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• enzymy MeSH
• koenzymy MeSH
• rekombinantní proteiny MeSH
• rozpouštědla MeSH

Cytochrome P450 2C9 (CYP2C9) is a membrane-anchored human microsomal protein involved in the drug metabolism in liver. CYP2C9 consists of an N-terminal transmembrane anchor and a catalytic cytoplasmic domain. While the structure of the catalytic domain is well-known from X-ray experiments, the complete structure and its incorporation into the membrane remains unsolved. We constructed an atomistic model of complete CYP2C9 in a dioleoylphosphatidylcholine membrane and evolved it by molecular dynamics simulations in explicit water on a 100+ ns time-scale. The model agrees well with known experimental data about membrane positioning of cytochromes P450. The entry to the substrate access channel is proposed to be facing the membrane interior while the exit of the product egress channel is situated above the interface pointing toward the water phase. The positions of openings of the substrate access and product egress channels correspond to free energy minima of CYP2C9 substrate ibuprofen and its metabolite in the membrane, respectively.

• MeSH
• aromatické hydroxylasy chemie   metabolismus   MeSH
• cytochrom P450 CYP2C9 MeSH
• fosfatidylcholiny chemie   metabolismus   MeSH
• ibuprofen chemie   metabolismus   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• membrány umělé * MeSH
• molekulární modely MeSH
• povrchové vlastnosti MeSH
• simulace molekulární dynamiky MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2-oleoylphosphatidylcholine MeSH Prohlížeč
• aromatické hydroxylasy MeSH
• CYP2C9 protein, human MeSH Prohlížeč
• cytochrom P450 CYP2C9 MeSH
• fosfatidylcholiny MeSH
• ibuprofen MeSH
• membrány umělé * MeSH

Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.

• MeSH
• biodegradace MeSH
• cirkulární dichroismus MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• látky znečišťující životní prostředí chemie   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• počítačová simulace MeSH
• propan analogy a deriváty   chemie   MeSH
• proteinové inženýrství * MeSH
• Rhodococcus enzymologie   genetika   růst a vývoj   MeSH
• řízená evoluce molekul MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2,3-trichloropropane MeSH Prohlížeč
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• látky znečišťující životní prostředí MeSH
• propan MeSH