Ker R. Marshall

P450 BM3: the very model of a modern flavocytochrome

Flavocytochrome P450 BM3 is a bacterial P450 system in which a fatty acid hydroxylase P450 is fused to a mammalian-like diflavin NADPH-P450 reductase in a single polypeptide. The enzyme is soluble (unlike mammalian P450 redox systems) and its fusion arrangement affords it the highest catalytic activity of any P450 mono-oxygenase. This article discusses the fundamental properties of P450 BM3 and how progress with this model P450 has affected our comprehension of P450 systems in general.

Azole antifungals are potent inhibitors of cytochrome P450 mono-oxygenases and bacterial growth in mycobacteria and streptomycetes

The genome sequence of Mycobacterium tuberculosis has revealed the presence of 20 different cytochrome P450 mono-oxygenases (P450s) within this organism, and subsequent genome sequences of other mycobacteria and of Streptomyces coelicolor have indicated that these actinomycetes also have large complements of P450s, pointing to important physiological roles for these enzymes. The actinomycete P450s include homologues of 14alpha-sterol demethylases, the targets for the azole class of drugs in yeast and fungi. Previously, this type of P450 was considered to be absent from bacteria. When present at low concentrations in growth medium, azole antifungal drugs were shown to be potent inhibitors of the growth of Mycobacterium smegmatis and of Streptomyces strains, indicating that one or more of the P450s in these bacteria were viable drug targets. The drugs econazole and clotrimazole were most effective against M. smegmatis (MIC values of <0.2 and 0.3 micro M, respectively) and were superior inhibitors of mycobacterial growth compared to rifampicin and isoniazid (which had MIC values of 1.2 and 36.5 micro M, respectively). In contrast to their effects on the actinomycetes, the azoles showed minimal effects on the growth of Escherichia coli, which is devoid of P450s. Azole drugs coordinated tightly to the haem iron in M. tuberculosis H37Rv P450s encoded by genes Rv0764c (the sterol demethylase CYP51) and Rv2276 (CYP121). However, the azoles had a higher affinity for M. tuberculosis CYP121, with K(d) values broadly in line with the MIC values for M. smegmatis. This suggested that CYP121 may be a more realistic target enzyme for the azole drugs than CYP51, particularly in light of the fact that an S. coelicolor DeltaCYP51 strain was viable and showed little difference in its sensitivity to azole drugs compared to the wild-type. If the azole drugs prove to inhibit a number of important P450s in M. smegmatis and S. coelicolor, then the likelihood of drug resistance developing in these species should be minimal. This suggests that azole drug therapy may provide a novel antibiotic strategy against strains of M. tuberculosis that have already developed resistance to isoniazid and other front-line drugs.

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.

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme-substrate complex

A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine substrate in the enzyme–substrate complex. However, recent steady‐state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine substrate that binds to the enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady‐state and stopped‐flow techniques with protiated and deuterated substrates. For all substrates used, there is a macroscopic ionization in the enzyme–substrate complex attributed to a deprotonation event required for optimal catalysis with a pKa of 7.4–8.4. In stopped‐flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine substrate. The acid limb of the bell‐shaped pH profile for the rate of flavin reduction over the substrate binding constant (kred/Ks, reporting on ionizations in the free enzyme and/or free substrate) is due to deprotonation of the free substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the enzyme at high pH. The pKa of the free amine is above 9.3 for all substrates, and is greatly perturbed (ΔpKa∼ 2) on binding to the enzyme active site. This perturbation of the substrate amine pKa on binding to the enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the substrate in the enzyme–substrate complex, thus enabling efficient functioning of these enzymes at physiologically relevant pH.

Characterization of Cupriavidus metallidurans CYP116B1 – A thiocarbamate herbicide oxygenating P450–phthalate dioxygenase reductase fusion protein

The novel cytochrome P450/redox partner fusion enzyme CYP116B1 from Cupriavidus metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated low‐spin, cysteinate‐coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe‐2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady‐state reduction of cytochrome c and ferricyanide were supported by both NADPH/NADH, with NADPH used more efficiently (Km[NADPH] = 0.9 ± 0.5 μm and Km[NADH] = 399.1 ± 52.1 μm). Stopped‐flow studies of NAD(P)H‐dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was ‐301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe‐2S and FMN cofactors were more positive than that of the haem iron. Multi‐angle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate‐like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S‐ethyl dipropylthiocarbamate (EPTC) and S‐propyl dipropylthiocarbamate (vernolate), and the subsequent N‐dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate‐oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC.

Cytochromes P450: novel drug targets in the war against multidrug-resistant Mycobacterium tuberculosis

Novel drug strategies are desperately needed to combat the global threat posed by multidrug-resistant strains of Mycobacterium tuberculosis (Mtb). The genome sequence of Mtb has revealed an unprecedented number of cytochrome P450 enzymes in a prokaryote, suggesting fundamental physiological roles for many of these enzymes. Several azole drugs (known inhibitors of cytochromes P450) have been shown to have potent anti-mycobacterial activity, and the most effective azoles have extremely tight binding constants for one of the Mtb P450s (CYP121). The structure of CYP121 has been determined at atomic resolution, revealing novel features of P450 structure, including mixed haem conformations and putative proton-relay pathways from protein surface to haem iron. The structure provides both a platform for investigation of structure/mechanism of cytochrome P450, and for design of inhibitor molecules as novel anti-tubercular agents.

DNA Binding Suppresses Human AIF-M2 Activity and Provides a Connection between Redox Chemistry, Reactive Oxygen Species, and Apoptosis

Human AIF-M2 is an unusual flavoprotein oxidoreductase that binds DNA, nicotinamide coenzyme, and the modified flavin 6-hydroxy-FAD. Using multiple solution methods to investigate the redox chemistry and binding interactions of AIF-M2, we demonstrate that binding of DNA and coenzyme to AIF-M2 is mutually exclusive. We also show that DNA binding does not perturb the redox chemistry of AIF-M2, but it has significant effects on the reduction kinetics of the 6-hydroxy-FAD cofactor by NAD(P)H. Based on quantitative analysis of ligand binding and redox chemistry, we propose a model for the function of AIF-M2. In this model, DNA binding suppresses the redox activity of AIF-M2 by preventing the binding of the reducing coenzyme NAD(P)H. This DNA-mediated suppression of AIF-M2 activity is expected to lower cellular levels of superoxide and peroxide, thereby lessening survival signaling by Ras, NF-κB, or AP-1, as suggested from knock-out studies of the related AIF in human colon cancer cell lines. We show marked differences between AIF-M2 and AIF. DNA and coenzyme binding activity is retained in the C-terminal deletion mutant AIF-M2-(Δ319-613), whereas DNA binds to the C-terminal D3 domain of AIF. Our work provides the first analysis of AIF-M2 ligand interactions and redox chemistry and identifies an important mechanistic connection between coenzyme and DNA binding, redox activity, and the apoptotic function of AIF-M2. Through its DNA binding activity, we suggest that AIF-M2 lessens survival cell signaling in the presence of foreign (e.g. bacterial and (retro)viral) cytosolic DNA, thus contributing to the onset of apoptosis.

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.

Crystal structure and solution characterization of the activation domain of human methionine synthase

Human methionine synthase (hMS) is a multidomain cobalamin‐dependent enzyme that catalyses the conversion of homocysteine to methionine by methyl group transfer. We report here the 1.6 Å crystal structure of the C‐terminal activation domain of hMS. The structure is C‐shaped with the core comprising mixed α and β regions, dominated by a twisted antiparallel β sheet with a β‐meander region. These features, including the positions of the active‐site residues, are similar to the activation domain of Escherichia coli cobalamin‐dependent MS (MetH). Structural and solution studies suggest a small proportion of hMS activation domain exists in a dimeric form, which contrasts with the monomeric form of the E. coli homologue. Fluorescence studies show that human activation domain interacts with the FMN‐binding domain of human methionine synthase reductase (hMSR). This interaction is enhanced in the presence of S‐adenosyl‐methionine. Binding of the D963E/K1071N mutant activation domain to the FMN domain of MSR is weaker than with wild‐type activation domain. This suggests that one or both of the residues D963 and K1071 are important in partner binding. Key differences in the sequences and structures of hMS and MetH activation domains are recognized and include a major reorientation of an extended 310‐containing loop in the human protein. This structural alteration might reflect differences in their respective reactivation complexes and/or potential for dimer formation. The reported structure is a component of the multidomain hMS : MSR complex, and represents an important step in understanding the impact of clinical mutations and polymorphisms in this key electron transfer complex.