**Preface to "Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 2.0"**

Nearly 70 years ago, R.T. Williams and B.B. Brodie developed the concept of drug metabolism and described the types of reactions and mechanisms by which the body facilitates drug excretion. A decade later, a protein that absorbs at 450 nm in the presence of carbon monoxide was independently discovered by Klingenberg and Garfinkel. Five years later, Omura and Sato identified this protein as cytochrome P450 (P450 or CYP). Numerous P450 investigations in over 100,000 papers have established the enzyme's prominent role in drug metabolism and endobiotic biosynthesis. They have also identified different types of P450s with diverse ranges of substrate specificity and functions. These enzymes are essential for life and are widely distributed among archaea, prokaryotes, and eukaryotes.

The success of and interest in the first Special Issue, titled "Cytochromes P450: Drug Metabolism and Bioactivation", spawned a second issue to include additional topics, titled "Cytochrome P450: Drug Metabolism and Bioactivation and Biodiversity". This book comprises 12 original papers and 3 reviews that were published in this Special Issue and recommended by the *IJMS* Editor. As Guest Editors, we present the work described below.

The first five papers examine human P450s and the human P450 reductase.

The first paper investigates access to the substrate of CYP3A4, the major human liver P450. The authors used *in silico* modeling to analyze access channels for the substrate and product of CYP3A4. Calculations were performed with version 2 of the CCCPP software, which was developed for this project. The article provides a detailed description of these channels, together with associated quantitative data.

The second paper reports the use of X-ray crystallography to explain the mechanism-based inactivation of CYP3A4 by three suicide substrates: mibefradil, an antihypertensive drug that was quickly withdrawn from the market; a semi-synthetic antibiotic azamulin; and a natural furanocoumarin, 6,7-dihydroxy-bergamottin. These findings can increase our understanding of suicide substrate binding and inhibitory mechanisms and can be used to improve the predictions of binding, metabolic sites, and inhibitory/inactivation potential of newly developed drugs.

The third paper investigates the membrane interaction of two P450 enzymes of the 2C family, CYP2C9 and CYP2C19, the structures of which have been solved in their truncated forms (without the N-terminal transmembrane helix) by X-ray crystallography. The aim of this work was to model the missing transmembrane helix and to study its interaction with the phospholipid layer. The authors provided a mechanistic interpretation of experimentally observed effects of mutagenesis on substrate selectivity.

The fourth paper examines electron transfer between NADPH–cytochrome P450 reductase (CPR) and CYP2C8. The study used *in vitro* and fast kinetic methods to study electron transfer and hydrogen peroxide production for three polymorphic forms of CYP2C8, namely, \*1, \*2, and \*3. Anaerobic stopped-flow measurements revealed that the kinetics of the first electron transfer in these genetic variants were altered compared with those of CYP2C8\*1 (wildtype), suggesting that electron transfer from CPR is disfavored.

The fifth paper presents the interactions between CPR and cytochrome P450. CPR is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a dynamic conformational equilibrium between open and closed conformations during electron transfer (ET). This study used *in silico* and *in vitro* approaches to probe the effects of a specific mutation in the hinge segment of CPR on electron transfer with three different human P450 isoforms. The investigation showed that CPR has a highly flexible hinge that results in a conformational distribution of open CPR conformers that can accommodate ET interactions with a variety of redox partners.

The next three papers review the clinical implications of cytochrome P450s as possible clinical biomarkers.

The first paper in this series reviews the role of cytochrome P450 (CYP450) enzymes in the field of cardio-oncology. The paper highlights the importance of cardiac medications in preventive cardio-oncology for high-risk patients or in the management of cardiotoxicities during or following cancer treatment. Common interactions between anticancer and cardiovascular drugs are reviewed. This work emphasizes that metabolic differences between drugs can lead to unpredictable bioavailability, which can drive inter-individual variability in drug disposition and cardiovascular toxicity.

The clinical implications of cytochrome P450 family 4 are reviewed in the next paper. This P450 family is responsible for the metabolism of fatty acids, xenobiotics, therapeutic drugs, and signaling molecules, including eicosanoids, leukotrienes, and prostanoids. Genetic polymorphisms within this P450 family have been associated with a range of human diseases; for example, genetic variants of the CYP4A11 and 4F2 genes have been associated with cardiovascular diseases. The risk of cancer is increased with mutations in CYP4B1, CYP4Z1, and other CYP4 genes that generate 20-hydroxyeicosatetraenoic acid (20-HETE). CYP4V2 gene variants are associated with ocular disease, while those of CYP4F22 are linked to skin disease, and CYP4F3B is associated with dysfunctions in the inflammatory response.

The focus of the final paper in this series is on CYPs within circulating extracellular vesicles (EVs). Recent studies have revealed an abundance of several CYPs in plasma EVs and other cell-derived EVs. This review covers the abundance of CYPs in plasma EVs and EVs derived from CYP-expressing cells, as well as the potential role of EV CYPs in cell–cell communication and their application with respect to novel biomarkers and therapeutic interventions. Moreover, studies have demonstrated that CYP-containing EVs can cause xenobiotic-induced toxicity via cell–cell interactions. Ultimately, mitigating CYP-mediated toxicity will require an understanding of the mechanism by which CYPs are loaded in EVs, as well as their circulation via plasma and their role in extrahepatic cells.

The next six papers are studies from K. Syed's group, who analyzed microorganism genomes to identify cytochrome P450s, collectively known as the CYPome.

The first paper from K. Syed's group describes the CYPome of bacterial species in the *Bacillus* genus that produce secondary metabolites. Genetic studies on these bacteria recently revealed the presence of secondary metabolite biosynthetic gene clusters (BGCs). *In silico* analysis of 128 species within the *Bacillus* genus identified 507 P450s divided into 13 families and 28 subfamilies. A large number of P450 genes were found within secondary metabolite BGCs and are associated with specific P450 families.

The CYPomes of the *Mycobacteria* (*M.*) genus, which includes species that are involved in tuberculosis (*M. tuberculosis*) and leprosy (*M. leprae*), are described in the next paper from K. Syed's group. This paper shows that the cytochrome P450 139 A (CYP139A) subfamily is present in 894 species in three mycobacterial groups: *M. tuberculosis* complex (850 species), *Mycobacterium avium* complex (34 species), and non-tuberculosis mycobacteria (10 species). Biosynthetic gene cluster analyses suggest that 92% of CYP139A members produce different secondary metabolites

K. Syed's group examined CYPs of the fungal world in their third paper. The paper analyzes the subphylum Agaricomycotina within the dimorphic fungus class Tremellomycetes. Analysis of the CYPome revealed 203 CYPs (excluding 16 pseudo-CYPs) in 23 species of Tremellomycetes; the identified proteins are grouped into 38 CYP families and 72 subfamilies. Of the identified CYPs, 23 CYP families are new, and 3 CYP families (CYP5139, CYP51, and CYP61) are conserved across 23 species within the fungal class Tremellomycetes. This paper focuses on the CYP51 family in particular because mutations lead to resistance to fungicides such as fluconazole.

The focus of the fourth paper from K. Syed's group is on the cyanobacterial CYPome. Cyanobacteria are the oldest known photosynthetic organisms and responsible for the oxygenation of the Earth's atmosphere. Analysis of the genomes of 114 cyanobacterial species revealed 341 P450s from 88 species within 36 families and 79 subfamilies. In total, 770 secondary metabolite BGCs were found in 103 cyanobacterial species. Comparative analyses were performed with other bacterial species in the *Bacillus*, *Streptomyces*, and *Mycobacteria* genera. Compared with cyanobacteria, the species in these genera had fewer P450s and BGCs and smaller P450 fractions within a given BGC.

The CYPomes of *Streptomyces* and secondary metabolites of bacteria from *Bacillus*, *Cyanobacterium*, and *Mycobacterium* are compared in their fifth paper. Bacteria in the *Streptomyces* genus have a higher number of P450s than species from the *Bacillus* genus and Cyanobacteria. The average number of secondary metabolite BGCs and the number of P450s within BGCs were found to be higher in bacteria from the *Streptomyces* genus than the other examined bacterial species. This result corroborates the superior capacity of *Streptomyces* bacteria to generate diverse secondary metabolites. The CYP107 family was found consistently in bacterial species of the *Streptomyces* and *Bacillus* genera, implying a central role in secondary metabolite synthesis.

Using *in silico* approaches, the penultimate paper provided by K. Syed's group investigates CYP128 in bacterial species from the *Mycobacterium* genus, which is best known for the tuberculosis-causing *Mycobacterium* (*M.*) *tuberculosis*. Genomic analysis of the bacteria revealed a large number of CYP128s that fall into six categories. The paper also examines the interrelationships and patterns of CYP128 genes and biosynthetic gene clusters (BGCs) in different mycobacterial species. The authors report different features in the CYP128 gene distribution, subfamily patterns, and characteristics of the secondary metabolite biosynthetic gene clusters (BGCs) between the *M. tuberculosis* complex (MTBC) and other mycobacterial species. In all MTBC species (except one), CYP128 P450s belong to subfamily A, whereas subfamily B is predominant in four other mycobacterial species. Of CYP128 P450s, 78% are in BGCs with CYP124A1 or with both CYP124A1 and CYP121A1. The CYP128 family ranks fifth in conservation among species. Unique amino acid patterns are present in the EXXR and CXG motifs. Molecular dynamic simulation studies indicate that CYP128A1 binds to MK9 with a higher affinity compared with the azole drugs analyzed. This study provides a comprehensive comparative analysis and structural insights into CYP128A1 in *M. tuberculosis*.

Finally, the last paper explores cytochrome P450 in the white-back planthopper, *Sogatellas* (*S.*) *furcifera*, an insect and major rice pest in China and in several other rice-growing countries of Asia. The aim of the study was to explore key genes related to the development of resistance to the insecticide sulfoxaflor in *S. furcifera* and to verify their functions. The paper reports the predicted interactions between CYP6FD1 and CYP4FD2 and sulfoxaflor. It also predicts that CYP6FD1 will have higher metabolic activity with respect to sulfoxaflor.
