An evaluation of molecular models of the cytochrome P450 Streptomyces griseolus enzymes P450SU1 and P450SU2

Abstract

P450SU1 and P450SU2 are herbicide-inducible bacterial cytochrome P450 enzymes from Streptomyces griseolus. They have two of the highest sequence indentities to camphor hydroxylase (P450cam from Pseudomonas putida), the cytochrome P450 with the first known crystal structure. We have built several models of these two proteins to investigate the variability in the structures that can occur from using different modeling protocols. We looked at variability due to alignment methods, backbone loop conformations and refinement methods. We have constructed two models for each protein using two alignment algorithms, and then an additional model using an identical alignment but different loop conformations for both buried and surface loops. The alignments used to build the models were created using the Needleman-Wunsch method, adapted for multiple sequences, and a manual method that utilized both a dot-matrix search matrix and the Needleman-Wunsch method. After constructing the initial models, several energy minimization methods were used to explore the variability in the final models caused by the choice of minimization techniques. Features of cytochrome P450cam and the cytochrome P450 superfamily, such as the ferredoxin binding site, the heme binding site and the substrate binding site were used to evaluate the validity of the models. Although the final structures were very similar between the models with different alignments, active-site residues were found to be dependent on the conformations of buried loops and early stages of energy minimization. We show which regions of the active site are the most dependent on the particular methods used, and which parts of the structures seem to be independent of the methods.

Site-Directed Mutagenesis as a Tool for Molecular Modeling of Cytochrome P450 2B1

“…In modeling a new structure, the sequence alignment as well as the choice of reference proteins is of crucial importance. Since until recently only one P450 structure, that of P450cam, was available, all earlier models used P450cam as the only reference protein (Zvelebil et al, 1991;Vijayakumar & Salerno, 1992;Koymans et al, 1993;Laughton et al, 1993;Braatz et al, 1994;Szklarz et al, 1994). Recently, the crystal structures of P450 BM-3 and P450cam were utilized to model thromboxane synthase, and the former structure proved to be a better template for the synthase (Ruan et al, 1994).…”

Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function

“…Given their associations with compound degradation and steroid biosynthesis, researchers have utilized P450s from Streptomyces , as well as other sources, as a tool to convert target compounds into compounds of interest. Notable examples include (i) a systematic screening of P450s for the regio- and stereoselective hydroxylation of testosterone, 131 (ii) the biosynthetic conversion of avermectin to 4´-oxo-avermectin for the agriculturally important insecticide emamectin, 44,132,133 and (iii) the multipurpose P450 SU-1 (CYP105A1), which, among other reactions, catalyzes the conversion of vitamin D 3 into its active form 1α,25-dihydroxyvitamin D 3 , 134–136 O - and N -dealkylations of coumarin and sulfonylurea herbicides, 36,37,137,138 and the hydroxylation and epoxidation of diterpenoid-based resin acids. 139 In many cases, P450s with “xenobiotic” activities have unknown endogenous substrates and functions (i.e., orphan P450s).…”

Modeling Kinetics of Subcellular Disposition of Chemicals

“…The long absence of the mammalian CYP structures resulted in numerous ligand-based 3D-QSAR models of the CYP binding for both substrates [1416][1417][1418][1419][1420][1421][1422] and inhibitors. [1423][1424][1425][1426][1427][1428] The 3D-QSAR models were combined with comparative structural models, which used bacterial, 1425,[1429][1430][1431][1432] rabbit, [1433][1434][1435][1436][1437] and human 1438 CYP structures as templates. Peculiar properties of the CYP binding sites, including sizes that often exceed the size of common ligands, conformational changes, and the possibility of accommodation of two bound molecules, led to less-predictive docking than for those with other receptors.…”