Evolution Of P450s Research Articles

A cytochrome P450 insecticide detoxification mechanism is not conserved across the Megachilidae family of bees.

Recent work has demonstrated that many bee species have specific cytochrome P450 enzymes (P450s) that can efficiently detoxify certain insecticides. The presence of these P450s, belonging or closely related to the CYP9Q subfamily (CYP9Q-related), is generally well conserved across the diversity of bees. However, the alfalfa leafcutter bee, Megachile rotundata, lacks CYP9Q-related P450s and is 170-2500 times more sensitive to certain insecticides than bee pollinators with these P450s. The extent to which these findings apply to other Megachilidae bee species remains uncertain. To address this knowledge gap, we sequenced the transcriptomes of four Megachile species and leveraged the data obtained, in combination with publicly available genomic data, to investigate the evolution and function of P450s in the Megachilidae. Our analyses reveal that several Megachilidae species, belonging to the Lithurgini, Megachilini and Anthidini tribes, including all species of the Megachile genus investigated, lack CYP9Q-related genes. In place of these genes Megachile species have evolved phylogenetically distinct CYP9 genes, the CYP9DM lineage. Functional expression of these P450s from M. rotundata reveal they lack the capacity to metabolize the neonicotinoid insecticides thiacloprid and imidacloprid. In contrast, species from the Osmiini and Dioxyini tribes of Megachilidae have CYP9Q-related P450s belonging to the CYP9BU subfamily that are able to detoxify thiacloprid. These findings provide new insight into the evolution of P450s that act as key determinants of insecticide sensitivity in bees and have important applied implications for pesticide risk assessment.

A conserved hymenopteran-specific family of cytochrome P450s protects bee pollinators from toxic nectar alkaloids

Many plants produce chemical defense compounds as protection against antagonistic herbivores. However, how beneficial insects such as pollinators deal with the presence of these potentially toxic chemicals in nectar and pollen is poorly understood. Here, we characterize a conserved mechanism of plant secondary metabolite detoxification in the Hymenoptera, an order that contains numerous highly beneficial insects. Using phylogenetic and functional approaches, we show that the CYP336 family of cytochrome P450 enzymes detoxifies alkaloids, a group of potent natural insecticides, in honeybees and other hymenopteran species that diverged over 281 million years. We linked this function to an aspartic acid residue within the main access channel of CYP336 enzymes that is highly conserved within this P450 family. Together, these results provide detailed insights into the evolution of P450s as a key component of detoxification systems in hymenopteran species and reveal the molecular basis of adaptations arising from interactions between plants and beneficial insects.

Lifestyles Shape the Cytochrome P450 Repertoire of the Bacterial Phylum Proteobacteria.

For the last six decades, cytochrome P450 monooxygenases (CYPs/P450s), heme thiolate proteins, have been under the spotlight due to their regio- and stereo-selective oxidation activities, which has led to the exploration of their applications in almost all known areas of biology. The availability of many genome sequences allows us to understand the evolution of P450s in different organisms, especially in the Bacteria domain. The phenomenon that “P450s play a key role in organisms’ adaptation vis a vis lifestyle of organisms impacts P450 content in their genome” was proposed based on studies on a handful of individual bacterial groups. To have conclusive evidence, one must analyze P450s and their role in secondary metabolism in species with diverse lifestyles but that belong to the same category. We selected species of the phylum Proteobacteria classes, Alpha, Beta, Gamma, Delta, and Epsilon, to address this research gap due to their diverse lifestyle and ancient nature. The study identified that the lifestyle of alpha-, beta-, gamma-, delta-, and epsilon-proteobacterial species profoundly affected P450 profiles in their genomes. The study determined that irrespective of the species associated with different proteobacterial classes, pathogenic species or species adapted to a simple lifestyle lost or had few P450s in their genomes. On the contrary, species with saprophytic or complex lifestyles had many P450s and secondary metabolite biosynthetic gene clusters. The study findings prove that the phenomenon mentioned above is factual, and there is no link between the number and diversity of P450s and the age of the bacteria.

Ancient Bacterial Class Alphaproteobacteria Cytochrome P450 Monooxygenases Can Be Found in Other Bacterial Species.

Cytochrome P450 monooxygenases (CYPs/P450s), heme-thiolate proteins, are well-known players in the generation of chemicals valuable to humans and as a drug target against pathogens. Understanding the evolution of P450s in a bacterial population is gaining momentum. In this study, we report comprehensive analysis of P450s in the ancient group of the bacterial class Alphaproteobacteria. Genome data mining and annotation of P450s in 599 alphaproteobacterial species belonging to 164 genera revealed the presence of P450s in only 241 species belonging to 82 genera that are grouped into 143 P450 families and 214 P450 subfamilies, including 77 new P450 families. Alphaproteobacterial species have the highest average number of P450s compared to Firmicutes species and cyanobacterial species. The lowest percentage of alphaproteobacterial species P450s (2.4%) was found to be part of secondary metabolite biosynthetic gene clusters (BGCs), compared other bacterial species, indicating that during evolution large numbers of P450s became part of BGCs in other bacterial species. Our study identified that some of the P450 families found in alphaproteobacterial species were passed to other bacterial species. This is the first study to report on the identification of CYP125 P450, cholesterol and cholest-4-en-3-one hydroxylase in alphaproteobacterial species (Phenylobacterium zucineum) and to predict cholesterol side-chain oxidation capability (based on homolog proteins) by P. zucineum.

Similarities, variations, and evolution of cytochrome P450s in Streptomyces versus Mycobacterium

Cytochrome P450 monooxygenases (P450s) found in all domains of life are known for their catalytic versatility and stereo- and regio-specific activity. While the impact of lifestyle on P450 evolution was reported in many eukaryotes, this remains to be addressed in bacteria. In this report, Streptomyces and Mycobacterium, belonging to the phylum Actinobacteria, were studied owing to their contrasting lifestyles and impacts on human. Analyses of all P450s and those predicted to be associated with secondary metabolism have revealed that different lifestyles have affected the evolution of P450s in these bacterial genera. We have found that while species in both genera have essentially the same number of P450s in the genome, Streptomyces P450s are much more diverse than those of Mycobacterium. Moreover, despite both belonging to Actinobacteria, only 21 P450 families were common, and 123 and 56 families were found to be unique to Streptomyces and Mycobacterium, respectively. The presence of a large and diverse number of P450s in Streptomyces secondary metabolism contributes to antibiotic diversity, helping to secure the niche. Conversely, based on the currently available functional data, types of secondary metabolic pathways and associated P450s, mycobacterial P450s seem to play a role in utilization or synthesis of lipids.

Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s.

Since the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria −42; fungi −19; plant −28; animal −22; plant and animal −1 and common P450 family −1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study’s results offer new understanding of the dynamic structural nature of P450s.

Systematic Identification and Evolutionary Analysis of Catalytically Versatile Cytochrome P450 Monooxygenase Families Enriched in Model Basidiomycete Fungi

Genome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s) in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin’s theory of natural selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450 analysis in the basidiomycete species, Phanerochaete chrysosporium, Phanerochaete carnosa, Agaricus bisporus, Postia placenta, Ganoderma sp. and Serpula lacrymans, revealed enrichment of 11 P450 families (out of 68 P450 families), CYP63, CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of Phanerochaete chrysosporium and Phanerochaete carnosa, suggesting paralogous evolution of P450s in model basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns. P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are catalytically versatile and possibly involved in fungal colonization of plant material. To our knowledge, this is the first report on the identification and comparative-evolutionary analysis of P450 families enriched in model basidiomycetes.

Unusual properties of the cytochrome P450 superfamily

During the early years of cytochrome P450 research, a picture of conserved properties arose from studies of mammalian forms of these monooxygenases. They included the protohaem prosthetic group, the cysteine residue that coordinates to the haem iron and the reduced CO difference spectrum. Alternatively, the most variable feature of P450s was the enzymatic activities, which led to the conclusion that there are a large number of these enzymes, most of which have yet to be discovered. More recently, studies of these enzymes in other eukaryotes and in prokaryotes have led to the discovery of unexpected P450 properties. Many are variations of the original properties, whereas others are difficult to explain because of their unique nature relative to the rest of the known members of the superfamily. These novel properties expand our appreciation of the broad view of P450 structure and function, and generate curiosity concerning the evolution of P450s. In some cases, structural properties, previously not found in P450s, can lead to enzymatic activities impacting the biological function of organisms containing these enzymes; whereas, in other cases, the biological reason for the variations are not easily understood. Herein, we present particularly interesting examples in detail rather than cataloguing them all.

Insights into the function and evolution of P450s in plant steroid metabolism

Numerous cytochrome P450 monooxygenases (P450s) have been known to be involved in the biosynthesis and metabolism of triterpenoids and steroids. This review will survey the oxidative reactions by such P450s and provide insights into the evolution of the steroid-biosynthetic P450 genes in the plant kingdom. Special emphasis is placed on brassinosteroids (BRs), plant steroid hormones, that play essential roles in the regulation of plant growth and development. Several P450s involved in BR biosynthesis and catabolism have recently been characterized by recombinant protein experiments, revealing a new route of the BR biosynthetic pathway.

The microsomal mixed function oxidase system of amphibians and reptiles: components, activities and induction

This article reviews current research in amphibian and reptilian cytochromes P450, important to the overall understanding of xenobiotic metabolism in the ecosystem and the evolution of P450s. Amphibians and reptilians contain the normal mixed function oxidase system (MFO). In general the MFO content and activities are less than those found in mammals, but only a few of the known activities have been examined in these vertebrate classes. Research to date has focused on two families of cytochromes P450, CYP1 and 2. The isoforms examined catalyze the classic activities but there have been notable absences. The total number of isoforms present and the breadth of substrates metabolized are yet unknown. Induction by foreign compounds (xenobiotics) is lengthier and yields lower levels of induced activity than is typically found in mammals. When these animals are pretreated with 3-methylcholanthrene (3MC) and β-naphthaflavone (BNF), which are known to induce the same isoform in mammals, multiple isoforms are induced with different activities. Phenobarbital-pretreatment in turtles and alligators induces cytochromes P450 and suggestive data indicates induction in the lizard Agama lizard and the newt Pleurodeles waltl. In amphibians and reptiles a CYP2B protein does appear to be present along with constitutive activities associated with the 2 family of cytochromes P450. The markedly different response to classic inducers combined with lower or absent activities alters the view of how amphibians and reptilians respond to xenobiotic challenges.

MOLECULAR-GENETIC ANALYSIS OF PLANT CYTOCHROME P450-DEPENDENT MONOOXYGENASES.

Cytochrome P450-dependent monooxygenases are a large group of heme-containing enzymes, most of which catalyze NADPH- and O2-dependent hydroxylation reactions. The cloning of plant P450s has been hampered because these membrane-localized proteins are typically present in low abundance and are often unstable to purification. Since the cloning of the first plant P450 gene in 1990, there has been an explosion in the rate at which genes encoding plant P450s have been identified. These successes have largely been the result of advances in purification techniques, as well as the application of alternative methods such as mutant- and PCR-based cloning strategies. The availability of these cloned genes has made possible the analysis of P450 gene regulation and may soon reveal aspects of the evolution of P450s in plants. This new knowledge will significantly improve our understanding of many metabolic pathways and may permit their manipulation in the near future.