What NMR teaches us about P450 structure, dynamics and function.

Thomas C. Pochapsky

Departments of Chemistry, Biochemistry and Rosenstiel Center for Basic Biomedical Research, Brandeis University, Waltham, Massachusetts, 02454-9110 USA

Solution NMR methods have given us a clearer understanding of the complex conformational and dynamic transitions that occur in the course of the P450 reaction cycle. The ability to characterize P450 structure and dynamics in solution under controlled conditions (with and without substrate, effector and redox partner binding, heme ligation and oxidation states) allow us to parse these states discretely. In particular, using residual dipolar couplings (RDCs) as restraints in solvated molecular dynamics simulations, we can generate solution structural ensembles of particular P450 states that allow direct observation of conformational changes that take place upon changing conditions. H/D exchange, NMR relaxation measurements and line width analyses provide complementary information on dynamics. Examples of these insights from five P450s, the catabolic CYP101A1, macrolide antibiotic biosynthetic MycG, bacterial CYP106A2, CYP119 from T. sulfolobus. and human CYP17A1 will be described, with the theme being how these disparate enzymes show commonality in their behavior.

Human cytochrome P450 7B1 (CYP7B1) 7a-hydroxylates both 25-hydroxycholesterol and 27-hydroxycholesterol during bile acid synthesis. Many inherited mutations of CYP7B1 cause the accumulation of these oxysterol substrates, which in turn result in neurodegeneration. The resulting neuromuscular weakness and loss of control means progressive reduction in mobility causing spastic paraplegia type 5. Additionally, high 27-hydroxycholesterol levels stimulate b-secretase activity, promoting amyloid formation to result in an Alzheimer’s-like pathology correlated with cognitive deficits. Finally, CYP7B1 is one of several sterol P450 enzymes implicated in Parkinson’s Disease. While many CYP7B1 mutations have been described in spastic paraplegia patients in particular, the structural and functional impacts of these mutations are unknown, as is any structure of the wild type CYP7B1.

The first human CYP7B1 structure was determined using X-ray crystallography to a resolution of 2.31 Å. This structure was obtained in the presence of the non-specific azole inhibitor neticonazole, which fits well in the active site but makes few specific interactions with bordering side chains. Residues whose mutations result in spastic paraplegia type 5 were analyzed in the context of their locations and interactions. Some mutations would clearly lead to the loss of significant stabilizing interactions and correlate with little or no folded, heme-containing protein. However, others have unexpected effects on protein folding or stability that are not clear from the structure. A T297A mutation in the central I helix yields folded protein, but with decreased substrate binding and affinity and is also likely affects catalysis. This new molecular understanding of the CYP7B1 structure and the effects of mutations leading to disease could potentially be used to support the development of future therapeutic treatments for spastic paraplegia type 5, Alzheimer’s, and Parkinson’s Disease.

Cytochrome P450 heme containing metalloenzymes (CYPs) are monooxygenases which catalyse a diverse range of oxidation reactions through the activation of dioxygen. However, the high cost of the required nicotinamide cofactors and their need for additional electron transfer proteins limits their use as biocatalysts in larger-scale chemical applications. We have recently identified that a family of these CYP enzymes involved in the oxidation of lignin derived aromatics can function as peroxygenases. We have investigated whether other bacterial CYPs can be converted into efficient peroxygenases through protein engineering of the enzyme’s oxygen activation machinery to more closely resemble those of the natural peroxygenase. We have developed mutants which have significantly higher peroxygenase activity than a single mutant prototype and that function at significantly lower hydrogen peroxide concentrations. The X-ray crystal structures and other spectroscopic techniques (UV-Vis/EPR) revealed significant structural changes at the heme and the oxygen-binding groove providing a rationale for the modified activity. We have extended our mutagenesis strategy to CYP enzymes from a diverse range of bacteria and generated new peroxygenase biocatalysts for the regio- and stereo-selective hydroxylation of steroids, norisoprenoids and drug molecules at relatively low hydrogen peroxide concentrations. When this method is applied to CYP enzymes from thermophilic bacteria these reactions can occur at elevated temperatures enabling enzymatic hydroxylation reactions on a variety of substrates under non-standard biological conditions.

Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, JAPAN

In natural product biosynthesis, monooxygenation and other oxidative modifications diversify the structural complexity of metabolites after the formation of primary carbon scaffolds. Cytochrome P450 enzymes (P450s/CYPs) and non-heme iron- and 2-oxoglutarate-dependent dioxygenases (DOXs) are two major oxygenase families involved in these transformations in plants.

DOX enzymes participate in the initial steps of α-tomatine oxidative metabolism in unripe tomato fruits. Specifically, habrochaitoside synthase (HAS) catalyzes the abstraction of a hydrogen atom from the C20 position of α-tomatine, triggering spontaneous F-ring rearrangement to form habrochaitoside A. Meanwhile, C23 hydroxylase (23DOX) hydroxylates α-tomatine at the C23 position, leading to the biosynthesis of esculeoside A. In potato, the DOX enzyme dioxygenase for potato solanidan synthesis (DPS) hydroxylates solamarine at the 16α position to produce solanine. DPS and its tomato homolog also catalyze 16-hydroxylation of α-tomatine. Despite their high sequence similarity (amino acid sequence identity >50%), these homologous enzymes exhibit distinct metabolic activities toward α-tomatine.

To elucidate the structural basis of substrate recognition and specificity, we determined the crystal structures of the substrate-bound forms of 23DOX, HAS and the DPS homolog. Unexpectedly, co-crystallization with α-tomatine resulted in the capture of filotomatine, an isomer of α-tomatine, in both structures. Notably, the C23 atom of filotomatine was oriented toward the metal center in 23DOX, while the C16 atom faced the metal center in the DPS homolog.

Furthermore, we demonstrated that the substitution of one or two amino acid residues can modulate substrate binding and catalytic activity in HAS and 23DOX. A double mutant of HAS (H82M/N263F) produced 20-hydroxytomatine and a potential overoxidized product. We will also discuss the detailed mechanisms underlying the formation of these mutant-derived products.

A quantum mechanical/molecular mechanical (QM/MM) calculation was also performed on the production mechanism of habrochaitoside A by HAS. The results of this calculation suggest the occurrence of a dehydrogenation reaction at the C20 position of α-tomatine and the subsequent one electron transfer to the Fe and H⁺ abstraction, leading to the F-ring expansion.