Monooxygenases

All known P450-containing monooxygenase systems share common structural and functional domain architecture. Apart from P450 itself, these systems can comprise several fundamentally different protein components or domains, all of which are shared by other multicomponent/multidomain enzyme systems with various functions: FAD flavoprotein or domain, FMN domain, Fe2S2 ferredoxin, Fe3S4 ferredoxin, and cytochrome b5. Either FMN domain, ferredoxins or cytochrome bs serve as the electron transport intermediate between the FAD domain and P450. The molecular evolution of both P450-containing systems and of each particular component does not follow phylogeny in general. Gene fusion and horizontal gene transfer events can lead to the appearance of novel redox chains in the same manner that artificial chimeric proteins can be constructed by humans. Recent studies using genetic and protein engineering techniques to investigate the separate domains and their interaction are described.

To facilitate access to electronic resources for all researchers working in the field of P450 proteins and P450-containing systems, a WorldWide Web server has been established called The Directory of P450-containing Systems at <http://www.icgeb.trieste.it/p450/>. Currently it contains the most up-to-date list of sequences of both the P450 superfamily and proteins mediating electron transfer to P450, i.e. NADPH:P450 reductases, specific NAD(P)H:ferredoxin reductases, cytochrome b5 reductases, ferredoxins and cytochromes b5, and their homologues from different enzyme systems. All the referenced sequences are provided with accession numbers and cross-links to major sequence databanks: PIR, SWISS-PROT, EMBL/GenBank and PRF.

Twenty five derivatives of the drugs terfenadine and ebastine have been designed, synthesized and evaluated as inhibitors of recombinant human CYP2J2. Compound 14, which has an imidazole substituent, is a good non-competitive inhibitor of CYP2J2 (IC(50)=400nM). It is not selective towards CYP2J2 as it also efficiently inhibits the other main vascular CYPs, such as CYP2B6, 2C8, 2C9 and 3A4; however, it could be an interesting tool to inhibit all these vascular CYPs. Compounds 4, 5 and 13, which have a propyl, allyl and benzo-1,3-dioxole terminal group, respectively, are selective CYP2J2 inhibitors. Compound 4 is a high-affinity, competitive inhibitor and alternative substrate of CYP2J2 (K(i)=160+/-50nM). Compounds 5 and 13 are efficient mechanism-based inhibitors of CYP2J2 (k(inact)/K(i) values approximately 3000Lmol(-1)s(-1)). Inactivation of CYP2J2 by 13 is due to the formation of a stable iron-carbene bond which occurs upon CYP2J2-catalyzed oxidation of 13 with a partition ratio o...

This paper reviews the classification of the P450 superfamily which is mainly based on sequence homology. The widely accepted classification by Nebert et al. [(1991) DNA Cell Biol. 10, 1-14] as well as the results of a ‘two-step’ multiple sequence alignment technique show that the molecular evolution of P450s, in contrast to that of many protein families, does not follow phylogeny. The data suggest that during the evolution of P450s, gene duplications and gene fusions, horizontal gene transfer and intron loss events have occurred. ‘Weak’ and ‘strong’ hierarchies in the clustering of P450 sequences were revealed. A novel evolutionary tree of the P450 superfamily has been constructed using a multiple alignment of consensus sequences. The simple classification of known P450-containing monooxygenase systems into three-, two- and one-component systems is further discussed. Particularly, the multidomain enzyme, nitric oxide synthase (NOS), should be classified as an example of a eukaryotic one-component P450 system since its N-terminal (haem) domain exhibits similarity with microsomal P450s.

The Directory of P450-containing Systems on World-Wide Web has been designed to facilitate access to electronic resources for all researchers working in the field of P450-containing and related enzyme systems. Currently, it contains the most up-to-date list of sequences of both the P450 superfamily and proteins mediating electron transfer to P450, i.e. NADPH:P450 reductases, specific NAD(P)H:ferredoxin reductases, cytochrome b5 reductases, ferredoxins and cytochromes b5, and their homologues. All the referenced sequences are provided with accession numbers and links to major sequence databanks: PIR, SWISS-PROT, EMBL/GenBank and PRF. An associated database of steroid substrates and products of P450-dependent reactions has also been developed.

The choice of bioinorganic motifs by Nature results in a spectacular variety of active-site structures even within the same protein family. Here, we use the concept of the bioinorganic motif to discuss the function and evolution of P450-containing and other related systems. Apart from P450, these systems include a FAD flavoprotein or domain, a FMN domain, ferredoxins and cytochrome b5. Analysis of available complete genomes can shed light on what an ancestral P450-containing system could be.

The psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125, isolated from Antarctic seawater, was used as recipient for a biodegradative gene of the mesophilic Pseudomonas stutzeri OX1. tou cluster, coding for Toluene o-Xylene Monooxygenase (ToMO), was successfully ...

The novel protein p33MONOX (p33Monooxygenase) was over-expressed in neuroblastoma cells demonstrating its inhibitory effect on the phosphorylation of the App (amyloid precursor protein) and Bcl2 (B-cell lymphoma 2) proteins but mediating higher activation of Mapk1/3 (mitogen-activated protein kinase 1/3). We employed a variety of cell biology techniques to show the localization of p33MONOX to the cytoplasm of pyramidal neurons in the mouse brain hippocampus. We also carried out a yeast-two-hybrid screening plus co-immunoprecipitation and bio-informatics to determine COBRA1 (cofactor of BRCA1 (breast cancer type 1)), NOL12 (nucleolar protein 12), and PRNP (prion protein) as p33MONOX-interacting proteins. Bio-computational analyses revealed a flavine-containing monooxygenase (FMO)-1 motif, thus linking p33MONOX to a group of previously characterized proteins, the MICALs (molecule interacting with CasL). Concluding, p33MONOX might regulate pre- and post-transcriptional control of dynamic processes related to growth cone guidance.

The cytochrome P-450-dependent monooxygenase system was examined in microsomal fractions prepared from 42 post mortem human livers and 9 lungs and kidneys. Electron microscopy studies indicated that the human liver samples were relatively free of mitochondrial and plasma membrane contamination, but samples of kidney and lung were less pure. The microsomal fractions from all organs were judged to be relatively free of haemoglobin and methaemoglobin. The specific enzyme activities for several drug substrates for the monooxygenase, NADPH-cytochrome c reductase activity and the content of the microsomal cytochromes were measured. The values of the biochemical parameters studied were found to be quite variable and the values for the human liver were appreciably lower than those obtained with liver microsomes from laboratory rodents. The enzyme activities of the human kidney and lung microsomal fractions were 1-10% of those seen for human liver samples, except for NADPH-cytochrome c(P-450...

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b5 content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23-2.1 nmol/min/mg and 0.5-11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individ...

Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated as playing key roles in climate control and in the biogeochemical cycling of sulfur. Metabolism of DMS by Bacteria has been previously identified as an important sink of DMS in soils and in the marine environment; however, relatively little is known about the physiology or biochemistry of Bacteria that metabolism DMS. The key enzyme of DMS oxidation in Hyphomicrobium spp. – DMS monooxygenase - has been purified and characterised from H. sulfonivorans. It has been shown to be a two-componant monooxygenase, related to bacterial luciferase, comprising two subunits – an FMNH2-dependent DMS monooxygenase (DmoA) and an NADHdependent FMN oxidoreductase (DmoB). For DMS, DMS monooxygenase from H. sulfonivorans has a Vmax of 1250 nmol DMS oxidised min-1 (mg protein)-1 and a kM of 16.5μM, corresponding to a kCAT of 5.2s-1. DMS oxidation in terms of acting as a sole-carbon source and as a supplementary energy source has been demonstrated in methylotrophic and heterotrophic bacteria. Chemolithoheterotrophic growth in which DMS carbon is assimilated to biomass whilst DMS sulfur is oxidised to tetrathionate with a net energy gain has been demonstrated in “M. thiooxidans”. Both “internal” and “external” chemolithoheterotrophy has been observed in “M. thiooxidans”, with endogenous and exogenous thiosulfate being oxidised to tetrathionate with a net energy gain. As far as can be found from the literature, this is the first recorded production of a polythionate from an organosulfur compound, as such, representing a potential new step in the biogeochemical sulfur cycle. Stable-isotope probing with [13C2]-DMS has been performed for the first time and has confirmed Methylophaga spp. as dominant DMS-oxidising Bacteria in the marine environment. The oxidation of marine thiosulfate to tetrathionate has been demonstrating during a phytoplankton bloom, indicating that chemolithoheterotrophic Bacteria are active during the bloom. Preliminary analyses have been carried out on the genome sequence of “Methylophaga thiooxidans” and the genes encoding the major enzymes of formaldehyde assimilation via the KDPG aldolase variant RuMP pathway have been identified. Genes encoding key enzymes involved in the dissimilation of methanol and methylated amines have been indentified, in addition to those involved in nitrogen uptake from ammonia, nitrate, nitrite and urea. Chemoorganoheterotrophic growth, coupling the oxidation of DMS to DMSO with ATP formation, has been demonstrated in Sagittula stellata E-37T, though the enzyme(s) responsible for this oxidation remain unclear.