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Using molecular dynamics to probe the structural basis for enhanced stability in thermal stable cytochromes P450.

ABSTRACT: High-temperature molecular dynamics (MD) has been used to assess if MD can be employed as a useful tool for probing the structural basis for enhanced stability in thermal stable cytochromes P450. CYP119, the most thermal stable P450 known, unfolds more slowly during 500 K MD simulations than P450s that melt at lower temperatures, P450cam and P450cin. A comparison of the 500 K MD trajectories shows that the Cys ligand loop, a critically important structural feature just under the heme, in both P450cin and P450cam completely unfolds while this region is quite stable in CYP119. In CYP119, this region is stabilized by tight nonpolar interactions involving Tyr26 and Leu308. The corresponding residues in P450cam are Gly and Thr, respectively. The in silico generated Y26A/L308A CYP119 double mutant is substantially less stable than wild-type CYP119, and the Cys ligand loop unfolds in a manner similar to that of P450cam. The MD thus has identified a potential "hot spot" important for stability. As an experimental test of the MD results, the Y26A/L308A double mutant was prepared, and thermal melting curves show that the double mutant exhibits a melting temperature (T(m)) 16 degrees C lower than that of wild-type CYP119. Control mutations that were predicted by MD not to destabilize the protein were also generated, and the experimental melting temperature was not significantly different from that of the wild-type enzyme. Therefore, high-temperature MD is a useful tool in predicting the structural underpinnings of thermal stability in P450s.

SUBMITTER: Meharenna YT

PROVIDER: S-EPMC3193997 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Using molecular dynamics to probe the structural basis for enhanced stability in thermal stable cytochromes P450.

Meharenna Yergalem T YT Poulos Thomas L TL

Biochemistry 20100801 31

High-temperature molecular dynamics (MD) has been used to assess if MD can be employed as a useful tool for probing the structural basis for enhanced stability in thermal stable cytochromes P450. CYP119, the most thermal stable P450 known, unfolds more slowly during 500 K MD simulations than P450s that melt at lower temperatures, P450cam and P450cin. A comparison of the 500 K MD trajectories shows that the Cys ligand loop, a critically important structural feature just under the heme, in both P4 ...[more]

PMID: 20593793

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Project description:This Account summarizes recent findings centered on the role that redox partner binding, allostery, and conformational dynamics plays in cytochrome P450 proton coupled electron transfer. P450s are one of Nature's largest enzyme families and it is not uncommon to find a P450 wherever substrate oxidation is required in the formation of essential molecules critical to the life of the organism or in xenobiotic detoxification. P450s can operate on a remarkably large range of substrates from the very small to the very large, yet the overall P450 three-dimensional structure is conserved. Given this conservation of structure, it is generally assumed that the basic catalytic mechanism is conserved. In nearly all P450s, the O2 O-O bond must be cleaved heterolytically enabling one oxygen atom, the distal oxygen, to depart as water and leave behind a heme iron-linked O atom as the powerful oxidant that is used to activate the nearby substrate. For this process to proceed efficiently, externally supplied electrons and protons are required. Two protons must be added to the departing O atom while an electron is transferred from a redox partner that typically contains either a Fe2S2 or FMN redox center. The paradigm P450 used to unravel the details of these mechanisms has been the bacterial CYP101A1 or P450cam. P450cam is specific for its own Fe2S2 redox partner, putidaredoxin or Pdx, and it has long been postulated that Pdx plays an effector/allosteric role by possibly switching P450cam to an active conformation. Crystal structures, spectroscopic data, and direct binding experiments of the P450cam-Pdx complex provide some answers. Pdx shifts the conformation of P450cam to a more open state, a transition that is postulated to trigger the proton relay network required for O2 activation. An essential part of this proton relay network is a highly conserved Asp (sometimes Glu) that is known to be critical for activity in a number of P450s. How this Asp and proton delivery networks are connected to redox partner binding is quite simple. In the closed state, this Asp is tied down by salt bridges, but these salt bridges are ruptured when Pdx binds, leaving the Asp free to serve its role in proton transfer. An alternative hypothesis suggests that a specific proton relay network is not really necessary. In this scenario, the Asp plays a structural role in the open/close transition and merely opening the active site access channel is sufficient to enable solvent protons in for O2 protonation. Experiments designed to test these various hypotheses have revealed some surprises in both P450cam and other bacterial P450s. Molecular dynamics and crystallography show that P450cam can undergo rather significant conformational gymnastics that result in a large restructuring of the active site requiring multiple cis/trans proline isomerizations. It also has been found that X-ray driven substrate hydroxylation is a useful tool for better understanding the role that the essential Asp and surrounding residues play in catalysis. Here we summarize these recent results which provide a much more dynamic picture of P450 catalysis.

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Using molecular dynamics to probe the structural basis for enhanced stability in thermal stable cytochromes P450.

Publications

Using molecular dynamics to probe the structural basis for enhanced stability in thermal stable cytochromes P450.

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