| The cytochrome P450s (CYPs) are a superfamily of enzymes found in bacteria, fungi, plants and animals. They all contain a molecule of heme and range in size from 46 to 60 KiloDaltons (KDa). CYPs are the major xenobiotic metabolizing enzymes found in mammals.;CYP2A5 is a mouse P450 that catalyzes specifically coumarin 7-hydroxylase activity. While working with this enzyme, Lindberg and Negishi discovered that by mutating phenylalanine at residue position 209 to leucine (F209L), they converted the enzyme from a coumarin hydroxylase to a steroid hydroxylase. They also discovered that the activity and/or specificity of the enzyme was dependent on the amino acids at positions 117, 209, 365 and 481, which are therefore critical residues for enzymatic activity.;In the absence of a crystal structure for mouse P4502A5, this study examined the possibility of constructing a model to understand and to predict the regio- and stereo-selectivity of this mammalian enzyme using dehydroepiandrosterone (DHEA) as the ligand. P4502A5 mutants were made (experimentally) and there regio- and stereo-selectivity for DHEA was assessed experimentally. A model was built using a multiple sequence alignment that included the bacterial P450s Cam, Terp and BM-3, which have solved x-ray crystal structures. The critical residues of P4502A5 were mapped into these bacterial P450s. These mapped positions were mutated to reflect the experimental work. An automated side chain placement program (MVP) was used to place these mutated side chains in low energy conformations relative to their neighboring residues. MVP was also used to automate the docking of DHEA and find its low energy conformations. The total energy of these conformations were computed and compared for each of the mutated structures. The results show that through the use of sequence alignments, secondary and tertiary structure prediction, and automated docking, it is possible to use the bacterial P450s to model (qualitatively) what is seen in a mammalian P450. These results also show promise for the potential use of this type of modeling in the field of risk assessment. |
