Insights into the structural basis for activity and selectivity of P450 BM3 monooxygenase

Insights into the Structural Basis for Activity and Selectivity of P450 BM3 Monooxygenase P450 BM3, as an important member of the cytochrome P450 superfamily of monooxygenases, preferentially catalyzes the subterminal hydroxylation of long chain fatty acids by sourcing electrons from the natural cofactor, NADPH. P450 BM3 variants which exhibit altered and inverted enantiopreference for styrene epoxidation have been previously reported. Furthermore, variants are known which can be catalytically driven based on alternative cofactor systems. In this thesis, crystal structures were determined to rationalize the function of P450 BM3 variants. Chapter I of this thesis has been dedicated to describing results originating from the crystallization of styrene epoxidation variants. For P450 BM3, it is known that an exchange of a single amino acid to a positively charged residue at position 184 in P450 BM3 variant 5F5 (heme domain substitutions: F87A, T235A) inverts the enantioselectivity of styrene epoxidation towards S-styrene oxide; even though Ala184 is located far from the active site. In order to explain mechanistic roles of individual substitutions in governing the enantiopreference, crystal structures of P450 BM3 heme domain variants have been successfully determined in complex with styrene, illustrating the bound substrate (styrene) in hitherto unobserved productive and non-productive substrate binding modes in the active site of P450 BM3. The structural data reveal the formation of a new inter-helical salt-bridge at the surface in S-selective variants, which causes the repositioning of the structural elements that are adjacent to the active site and, as a consequence, styrene binding geometries are altered as compared to the R-selective 5F5 variant. Chater II of this thesis describes results obtained from the crystallization of the P450 BM3 M7 heme domain variant (substitutions: F87A, V281G, and M354S) in complex with cobalt (III) sepluchrate (Co(III)Sep), the mediator of electronic transactions in the zinc (Zn) based alternative cofactor system. The mediator binds on the surface of the protein at the entrance of the substrate access channel, a position far from the active site, suggesting new routes for shuttling electrons to the active site heme-iron and implying that the electronic communication between the protein and the mediator is facilitated by the bound substrate in the active site. The structural evidence helps diagnose the molecular basis for ‘addiction’ of P450 BM3 variants to Co(III)Sep, and opens avenues for the future design of highly selective variants of P450 BM3 or structurally related monooxygenases towards alternative and cost-effective cofactor systems. In Chapter III, the crystal structure of the wild-type FAD/NADPH binding domain has been presented which sheds light on the cofactor selectivity in P450 BM3. The structure explains the role of individual residues which influence the crystallization process, and rationalizes the importance of substitutions previously reported to switch the cofactor specificity in P450 BM3. Towards the end are highlighted important structural features of the wild-type and T577G FMN domains, and the heme domains of aromatic hydroxylation variants of P450 BM3. Solved crystal structures of these variants reveal that the introduced substitutions do not cause any significant structural perturbations, and hence these variants resemble the corresponding wild-type domains. The obtained knowledge in understanding the enantioselective styrene epoxidation and the cofactor selectivity in P450 BM3 variants can very likely be generalized to structurally related monooxygenases, substrates, and cofactors.