Adrian J. Dunford

What makes a P450 tick

The cytochromes P450 (P450s) are probably natures most versatile enzymes in terms of both their vast substrate range and the diverse types of molecular transformations performed across the P450 enzyme superfamily. The P450s exquisitely perform highly specific oxidative chemistry, utilizing a sophisticated catalytic reaction mechanism. Recent studies have provided the first definitive characterization of the transient reaction cycle intermediate (compound I) responsible for the majority of P450 oxidative reactions. This major advance comes at a time when P450 engineering has facilitated the elucidation of several mammalian P450 structures and generated P450 variants with novel substrate specificity and reactivity. This review describes recent advances in P450 research and the ramifications for biotechnological and biomedical exploitation of these enzymes.

Biodiversity of cytochrome P450 redox systems

P450s (cytochrome P450 mono-oxygenases) are a superfamily of haem-containing mono-oxygenase enzymes that participate in a wide range of biochemical pathways in different organisms from all of the domains of life. To facilitate their activity, P450s require sequential delivery of two electrons passed from one or more redox partner enzymes. Although the P450 enzymes themselves show remarkable similarity in overall structure, it is increasingly apparent that there is enormous diversity in the redox partner systems that drive the P450 enzymes. This paper examines some of the recent advances in our understanding of the biodiversity of the P450 redox apparatus, with a particular emphasis on the redox systems in the pathogen Mycobacterium tuberculosis.

Characterization of active site structure in CYP121. A cytochrome P450 essential for viability of Mycobacterium tuberculosis H37Rv.

Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.

Rapid P450 Heme Iron Reduction by Laser Photoexcitation of Mycobacterium tuberculosis CYP121 and CYP51B1 ANALYSIS OF CO COMPLEXATION REACTIONS AND REVERSIBILITY OF THE P450/P420 EQUILIBRIUM

We demonstrate that photoexcitation of NAD(P)H reduces heme iron of Mycobacterium tuberculosis P450s CYP121 and CYP51B1 on the microsecond time scale. Rates of formation for the ferrous-carbonmonoxy (FeII-CO) complex were determined across a range of coenzyme/CO concentrations. CYP121 reaction transients were biphasic. A hyperbolic dependence on CO concentration was observed, consistent with the presence of a CO binding site in ferric CYP121. CYP51B1 absorption transients for FeII-CO complex formation were monophasic. The reaction rate was second order with respect to [CO], suggesting the absence of a CO-binding site in ferric CYP51B1. In the absence of CO, heme iron reduction by photoexcited NAD(P)H is fast (∼10,000–11,000 s–1) with both P450s. For CYP121, transients revealed initial production of the thiolate-coordinated (P450) complex (absorbance maximum at 448 nm), followed by a slower phase reporting partial conversion to the thiol-coordinated P420 species (at 420 nm). The slow phase amplitude increased at lower pH values, consistent with heme cysteinate protonation underlying the transition. Thus, CO binding occurs to the thiolate-coordinated ferrous form prior to cysteinate protonation. For CYP51B1, slow conversions of both the ferrous/FeII-CO forms to species with spectral maxima at 423/421.5 nm occurred following photoexcitation in the absence/presence of CO. This reflected conversion from ferrous thiolate- to thiol-coordinated forms in both cases, indicating instability of the thiolate-coordinated ferrous CYP51B1. CYP121 FeII-CO complex pH titrations revealed reversible spectral transitions between P450 and P420 forms. Our data provide strong evidence for P420 formation linked to reversible heme thiolate protonation, and demonstrate key differences in heme chemistry and CO binding for CYP121 and CYP51B1.

The crystal structure of the FAD/NADPH binding domain of flavocytochrome P450 BM3

We report the crystal structure of the FAD/NADPH‐binding domain (FAD domain) of the biotechnologically important Bacillus megaterium flavocytochrome P450 BM3, the last domain of the enzyme to be structurally resolved. The structure was solved in both the absence and presence of the ligand NADP+, identifying important protein interactions with the NADPH 2′‐phosphate that helps to dictate specificity for NADPH over NADH, and involving residues Tyr974, Arg966, Lys972 and Ser965. The Trp1046 side chain shields the FAD isoalloxazine ring from NADPH, and motion of this residue is required to enable NADPH‐dependent FAD reduction. Multiple binding interactions stabilize the FAD cofactor, including aromatic stacking with the adenine group from the side chains of Tyr860 and Trp854, and several interactions with FAD pyrophosphate oxygens, including bonding to tyrosines 828, 829 and 860. Mutagenesis of C773 and C999 to alanine was required for successful crystallization, with C773A predicted to disfavour intramolecular and intermolecular disulfide bonding. Multiangle laser light scattering analysis showed wild‐type FAD domain to be near‐exclusively dimeric, with dimer disruption achieved on treatment with the reducing agent dithiothreitol. By contrast, light scattering showed that the C773A/C999A FAD domain was monomeric. The C773A/C999A FAD domain structure confirms that Ala773 is surface exposed and in close proximity to Cys810, with this region of the enzyme’s connecting domain (that links the FAD domain to the FMN‐binding domain in P450 BM3) located at a crystal contact interface between FAD domains. The FAD domain crystal structure enables molecular modelling of its interactions with its cognate FMN (flavodoxin‐like) domain within the BM3 reductase module.

CYP121, CYP51 and associated redox systems in Mycobacterium tuberculosis: Towards deconvoluting enzymology of P450 systems in a human pathogen

An extraordinary array of P450 (cytochrome P450) enzymes are encoded on the genome of the human pathogen Mycobacterium tuberculosis (Mtb) and in related mycobacteria and actinobacteria. These include the first characterized sterol 14alpha-demethylase P450 (CYP51), a known target for azole and triazole drugs in yeasts and fungi. To date, only two Mtb P450s have been characterized in detail: CYP51 and CYP121. The CYP121 P450 shows structural relationships with P450 enzymes involved in synthesis of polyketide antibiotics. Both P450s exhibit tight binding to a range of azole drugs (e.g. clotrimazole and fluconazole) and the same drugs also have potent effects on growth of mycobacteria (but not of e.g. Escherichia coli). Atomic structures are available for both Mtb CYP51 and CYP121, revealing modes of azole binding and intriguing mechanistic and structural aspects. This paper reviews our current knowledge of these and the other P450 systems in Mtb including recent data relating to the reversible conversion of the CYP51 enzyme between P450 (thiolate-co-ordinated) and P420 (thiol-co-ordinated) species on reduction of the haem iron in the absence of a P450 substrate. The accessory flavoprotein and iron-sulfur proteins required to drive P450 catalysis are also discussed, providing an overview of the current state of knowledge of Mtb P450 redox systems.

Analysis of the oxidation of short chain alkynes by flavocytochrome P450 BM3

Bacillus megaterium flavocytochrome P450 BM3 (BM3) is a high activity fatty acid hydroxylase, formed by the fusion of soluble cytochrome P450 and cytochrome P450 reductase modules. Short chain (C6, C8) alkynes were shown to be substrates for BM3, with productive outcomes (i.e. alkyne hydroxylation) dependent on position of the carbon-carbon triple bond in the molecule. Wild-type P450 BM3 catalyses ω-3 hydroxylation of both 1-hexyne and 1-octyne, but is suicidally inactivated in NADPH-dependent turnover with non-terminal alkynes. A F87G mutant of P450 BM3 also undergoes turnover-dependent heme destruction with the terminal alkynes, pointing to a key role for Phe87 in controlling regioselectivity of alkyne oxidation. The terminal alkynes access the BM3 heme active site led by the acetylene functional group, since hydroxylated products are not observed near the opposite end of the molecules. For both 1-hexyne and 1-octyne, the predominant enantiomeric product formed (up to ∼90%) is the (S)-(-)-1-alkyn-3-ol form. Wild-type P450 BM3 is shown to be an effective oxidase catalyst of terminal alkynes, with strict regioselectivity of oxidation and potential biotechnological applications. The absence of measurable octanoic or hexanoic acid products from oxidation of the relevant 1-alkynes is also consistent with previous studies suggesting that removal of the phenyl group in the F87G mutant does not lead to significant levels of ω-oxidation of alkyl chain substrates.

Chapter 8:Structure, Mechanism and Function of Cytochrome P450 Enzymes

The cytochromes P450 constitute a superfamily of heme b-containing monoxygenase enzymes found in organisms from all of the domains of life. They catalyse the two-electron reduction of molecular oxygen bound to their ferrous heme iron, resulting in its scission and the introduction of a single atom of oxygen into a substrate molecule, with the remaining oxygen atom used in production of a molecule of water. The cytochromes P450 have numerous functions critical to human physiology — including key roles in steroid synthesis and in the metabolism of drugs and xenobiotics. In microbes they are increasingly recognized to be important enzymes for biotechnologically relevant transformations — for instance in the synthesis of polyketides and the oxidation of cholesterol. This chapter will focus on the structural and mechanistic properties of the cytochromes P450, including recent studies that have led to major breakthroughs in the spectroscopic characterization of the reactive iron-oxo species responsible for the substrate oxidation events. It will also consider wider aspects of cytochrome P450 catalysis, including the diverse redox partner systems used by these hemoprotein enzymes to facilitate catalysis. In addition, the biomedical relevance of various cytochromes P450 will be highlighted, and selected biotechnological applications of these enzymes reviewed. The cytochromes P450 are an important class of enzymes capable of highly regio- and stereoselective substrate oxidations that are often impossible to achieve using synthetic chemistry approaches. Their applicability for industrially relevant transformations is now well recognized and they are increasingly valued as tools in industrial biotechnology.