Using molecular modeling and molecular dynamics simulation to predict P450 oxidation products.
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Binding Sites↗
Biomedical subjects
J I Manchester
Publications and source records attributed to J I Manchester.
Rational approach to improving reductive catalysis by cytochrome P450cam.
Although halogenated hydrocarbons are noted for low chemical reactivity, small amounts are toxic to humans. Cytochromes P450 have been implicated in transforming these compounds to more reactive species, under anaerobic conditions, through reduction at the heme. A significant amount of effort has been directed toward turning this catalytic ability to our advantage by engineering P450 variants than can efficiently remediate these compounds in situ, before they come in contact with the human population. We have taken a 'rational' approach to this problem, in which a combination of theory and molecular modeling is applied to identify which properties of the enzyme have the greatest influence over reductive dehalogenation. Recent progress in this area is briefly reviewed. Two novel mutants, incorporating tryptophan (positions 87 and 396) and histidine (position 96, neutral and protonated) amino acid substitutions in the active site, are proposed and evaluated using molecular dynamics simulations. The upper bound on rate enhancement relative to wild-type is estimated in each mutant using electron transfer theory. The most significant rate enhancement is predicted for the His 96 mutant in the protonated state; while some His residues of certain proteins exhibit a pKa high enough to support a large protonated population, such information is not presently available for this proposed mutant.
Camphor 5-Monooxygenase↗
Molecular dynamics simulations indicate that F87W,T185F-cytochrome P450cam may reductively dehalogenate 1,1,1-trichloroethane.
Cytochrome P450cam is capable of reductively dehalogenating several chlorinated alkanes at low, but measurable, rates. In previous investigations of structure-function relationships in this enzyme using molecular dynamics simulations, we noticed that 1,1,1-trichloroethane (TCA) exhibits a very high degree of mobility in the active site due to its smaller molecular volume relative to the native substrate, camphor(1,2). Several amino acid sidechains lining the active site also exhibit significant dynamic fluctuations, possibly as a result of poor steric complementary to TCA. Guided by these results, we modeled double (F87W, T185F) and triple (F87W, T185F, V295I) mutants of P450cam, which provide additional bulk in the active site and increase the frequency of heme-substrate collision. Molecular dynamics simulations (300 ps on each protein) indicate that these mutants do not significantly perturb the three-dimensional fold of the enzyme, or local structure in the region of the active site. Both mutants bind the substrate more stably near the heme than the wild-type. Interestingly, however, the bulkier triple mutant seems to actually inhibit heme-substrate interactions relative to the double mutant. Over the final 200 ps of simulation, TCA is within 1 A of nonbonded contact with the heme 25% more often in the double mutant versus the wild-type. The triple mutant, on the other hand, binds TCA within 1 A of the heme only 15% as often as the wild-type. These results indicate that the double mutant may reductively dehalogenate TCA, a property not observed for the native protein. Implications for other experimentally measurable parameters are discussed.
Binding Sites↗
Enzyme-catalyzed dehalogenation of pentachloroethane: why F87W-cytochrome P450cam is faster than wild type.
Under anaerobic conditions, cytochromes P450 can reductively dehalogenate heavily halogenated hydrocarbons, such as one- and two-carbon organic solvents. This catalytic capacity has drawn attention to the potential use of engineered forms of P450s in the remediation of contaminated deep subsurface ecosystems. Loida (1994, PhD Thesis, University of Illinois at Urbana-Champaign, IL) and S.G. Sligar (personal communication) have observed recently that an active-site variant of cytochrome P450cam (F87W) dechlorinates pentachloroethane approximately three times faster than the wild-type enzyme. Molecular dynamics simulations have revealed that the mutant enzyme binding pocket remains smaller, and that pentachloroethane assumes configurations closer to the heme-Fe in the F87W mutant twice as often as in the wild-type enzyme. This result is consistent with a collisional model of dehalogenation, which agrees with experimental observations [Li and Wackett (1993) Biochemistry, 32, 9355-9361] that solutions containing wild-type P450cam dehalogenate pentachloroethane 100 times faster than those containing free heme. The simulations suggest that it is unlikely that Trp87 significantly stabilizes the developing negative charge on the substrate during carbon-halogen bond reduction. The design of improved microbial enzymes that incorporate both steric and electronic effects continues for use in remediating halogenated contaminants in situ.
Binding Sites↗
Investigation of domain motions in bacteriophage T4 lysozyme.
Hinge-bending in T4 lysozyme has been inferred from single amino acid mutant crystalline allomorphs by Matthews and coworkers. This raises an important question: are the different conformers in the unit cell artifacts of crystal packing forces, or do they represent different solution state structures? The objective of this theoretical study is to determine whether domain motions and hinge-bending could be simulated in T4 lysozyme using molecular dynamics. An analysis of a 400 ps molecular dynamics simulation of the 164 amino acid enzyme T4 lysozyme is presented. Molecular dynamics calculations were computed using the Discover software package (Biosym Technologies). All hydrogen atoms were modeled explicitly with the inclusion of all 152 crystallographic waters at a temperature of 300 K. The native T4 lysozyme molecular dynamics simulation demonstrated hinge-bending in the protein. Relative domain motions between the N-terminal and C-terminal domains were evident. The enzyme hinge bending sites resulted from small changes in backbone atom conformations over several residues rather than rotation about a single bound. Two hinge foci were found in the simulation. One locus comprises residues 8-14 near the C-terminal of the A helix; the other site, residues 77-83 near the C-terminal of the C helix. Comparison of several snapshot structures from the dynamics trajectory clearly illustrates domain motions between the two lysozyme lobes. Time correlated atomic motions in the protein were analyzed using a dynamical cross-correlation map. We found a high degree of correlated atomic motions in each of the domains and, to a lesser extent, anticorrelated motions between the two domains. We also found that the hairpin loop in the N-terminal lobe (residues 19-24) acted as a mobile 'flap' and exhibited highly correlated dynamic motions across the cleft of the active site, especially with residue 142.
Bacteriophage T4↗
Applicability of PM3 to transphosphorylation reaction path: toward designing a minimal ribozyme.
A growing body of evidence shows that RNA can catalyze many of the reactions necessary both for replication of genetic material and the possible transition into the modern protein-based world. However, contemporary ribozymes are too large to have self-assembled from a prebiotic oligonucleotide pool. Still, it is likely that the major features of the earliest ribozymes have been preserved as molecular fossils in the catalytic RNA of today. Therefore, the search for a minimal ribozyme has been aimed at finding the necessary structural features of a modern ribozyme (Beaudry and Joyce, 1990). Both a three-dimensional model and quantum chemical calculations are required to quantitatively determine the effects of structural features of the ribozyme on the reaction it catalyzes. Using this model, quantum chemical calculations must be performed to determine quantitatively the effects of structural features on catalysis. Previous studies of the reaction path have been conducted at the ab initio level, but these methods are limited to small models due to enormous computational requirements. Semiempirical methods have been applied to large systems in the past; however, the accuracy of these methods depends largely on the system under investigation. In the present study we assess the validity of the MNDO/PM3 method on a simple model of the ribozyme-catalyzed reaction, or hydrolysis of phosphoric acid. We find that the results are qualitatively similar to ab initio results using large basis sets. Therefore, PM3 is suitable for studying the reaction path of the ribozyme-catalyzed reaction.
Models, Biological↗
Modeling study on the cleavage step of the self-splicing reaction in group I introns.
A three-dimensional model of the Tetrahymena thermophila group I intron is used to further explore the catalytic mechanism of the transphosphorylation reaction of the cleavage step. Based on the coordinates of the catalytic core model proposed by Michel and Westhof (Michel, F., Westhof, E. J. Mol. Biol. 216, 585-610 (1990)), we first converted their ligation step model into a model of the cleavage step by the substitution of several bases and the removal of helix P9. Next, an attempt to place a trigonal bipyramidal transition state model in the active site revealed that this modified model for the cleavage step could not accommodate the transition state due to insufficient space. A lowering of P1 helix relative to surrounding helices provided the additional space required. Simultaneously, it provided a better starting geometry to model the molecular contacts proposed by Pyle et al. (Pyle, A. M., Murphy, F. L., Cech, T. R. Nature 358, 123-128. (1992)), based on mutational studies involving the J8/7 segment. Two hydrated Mg2+ complexes were placed in the active site of the ribozyme model, using the crystal structure of the functionally similar Klenow fragment (Beese, L.S., Steitz, T.A. EMBO J. 10, 25-33 (1991)) as a guide. The presence of two metal ions in the active site of the intron differs from previous models, which incorporate one metal ion in the catalytic site to fulfill the postulated roles of Mg2+ in catalysis. The reaction profile is simulated based on a trigonal bipyramidal transition state, and the role of the hydrated Mg2+ complexes in catalysis is further explored using molecular orbital calculations.
Animals↗