Structure-Function Analysis of the Essential Mycobacterium tuberculosis P450 Drug Target, CYP121A1.

Cytochrome P450 CYP121A1 is a well-known drug target against Mycobacterium tuberculosis, the human pathogen that causes the deadly disease tuberculosis (TB). CYP121A1 is a unique P450 enzyme because it uses classical and non-classical P450 catalytic processes and has distinct structural features among P450s. However, a detailed investigation of CYP121A1 protein structures in terms of active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 53 CYP121A1 crystal structures were investigated in this study. Critical amino acids required for CYP121A1's overall activity were identified and highlighted this enzyme's rigid architecture and substrate selectivity. The CYP121A1-fluconazole crystal structure revealed a novel azole drug-P450 binding mode in which azole heme coordination was facilitated by a water molecule. Fragment-based inhibitor approaches revealed that CYP121A1 can be inhibited by molecules that block the substrate channel or by directly interacting with the P450 heme. This study serves as a reference for the precise understanding of CYP121A1 interactions with different ligands and the structure-function analysis of P450 enzymes in general. Our findings provide critical information for the synthesis of more specific CYP121A1 inhibitors and their development as novel anti-TB drugs.

Structure-Function Analysis of the Biotechnologically Important Cytochrome P450 107 (CYP107) Enzyme Family.

Cytochrome P450 monooxygenases (CYPs; P450s) are a superfamily of heme-containing enzymes that are recognized for their vast substrate range and oxidative multifunctionality. CYP107 family members perform hydroxylation and epoxidation processes, producing a variety of biotechnologically useful secondary metabolites. Despite their biotechnological importance, a thorough examination of CYP107 protein structures regarding active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 44 CYP107 crystal structures were investigated in this study. We demonstrate that the CYP107 active site cavity is very flexible, with ligand binding reducing the volume of the active site in some situations and increasing volume size in other instances. Polar interactions between the substrate and active site residues result in crucial salt bridges and the formation of proton shuttling pathways. Hydrophobic interactions, however, anchor the substrate within the active site. The amino acid residues within the binding pocket influence substrate orientation and anchoring, determining the position of the hydroxylation site and hence direct CYP107's catalytic activity. Additionally, the amino acid dynamics within and around the binding pocket determine CYP107's multifunctionality. This study serves as a reference for understanding the structure-function analysis of CYP107 family members precisely and the structure-function analysis of P450 enzymes in general. Finally, this work will aid in the genetic engineering of CYP107 enzymes to produce novel molecules of biotechnological interest.

Genome-Wide Analysis of the Cytochrome P450 Monooxygenases in the Lichenized Fungi of the Class Lecanoromycetes.

Lichens are unique organisms that exhibit a permanent symbiosis between fungi and algae or fungi and photosynthetic bacteria. Lichens have been found to produce biotechnologically valuable secondary metabolites. A handful of studies showed that tailoring enzymes such as cytochrome P450 monooxygenases (CYPs/P450s) play a key role in synthesizing these metabolites. Despite the critical role of P450s in the biosynthesis of secondary metabolites, the systematic analysis of P450s in lichens has yet to be reported. This study is aimed to address this research gap. A genome-wide analysis of P450s in five lichens from the fungal class Lecanoromycetes revealed the presence of 434 P450s that are grouped into 178 P450 families and 345 P450 subfamilies. The study indicated that none of the P450 families bloomed, and 15 P450 families were conserved in all five Lecanoromycetes. Lecanoromycetes have more P450s and higher P450 family diversity compared to Pezizomycetes. A total of 73 P450s were found to be part of secondary metabolite gene clusters, indicating their potential involvement in the biosynthesis of secondary metabolites. Annotation of P450s revealed that CYP682BG1 and CYP682BG2 from Cladonia grayi and Pseudevernia furfuracea (physodic acid chemotype) are involved in the synthesis of grayanic acid and physodic acid, CYP65FQ2 from Stereocaulon alpinum is involved in the synthesis of atranorin, and CYP6309A2 from Cladonia uncialis is involved in the synthesis of usnic acid. This study serves as a reference for future annotation of P450s in lichens.

Pezizomycetes Genomes Reveal Diverse P450 Complements Characteristic of Saprotrophic and Ectomycorrhizal Lifestyles.

Cytochrome P450 monooxygenases (CYPs/P450s) are heme proteins that play a role in organisms' primary and secondary metabolism. P450s play an important role in organism adaptation since lifestyle influences P450 composition in their genome. This phenomenon is well-documented in bacteria but less so in fungi. This study observed this phenomenon where diverse P450 complements were identified in saprophytic and ectomycorrhizal Pezizomycetes. Genome-wide data mining, annotation, and phylogenetic analysis of P450s in 19 Pezizomycetes revealed 668 P450s that can be grouped into 153 P450 families and 245 P450 subfamilies. Only four P450 families, namely, CYP51, CYP61, CYP5093, and CYP6001, are conserved across 19 Pezizomycetes, indicating their important role in these species. A total of 5 saprophyte Pezizomycetes have 103 P450 families, whereas 14 ectomycorrhizal Pezizomycetes have 89 P450 families. Only 39 P450 families were common, and 50 and 64 P450 families, respectively, were unique to ectomycorrhizal and saprophytic Pezizomycetes. These findings suggest that the switch from a saprophytic to an ectomycorrhizal lifestyle led to both the development of diverse P450 families as well as the loss of P450s, which led to the lowest P450 family diversity, despite the emergence of novel P450 families in ectomycorrhizal Pezizomycetes.

An Unprecedented Number of Cytochrome P450s Are Involved in Secondary Metabolism in Salinispora Species.

Cytochrome P450 monooxygenases (CYPs/P450s) are heme thiolate proteins present in species across the biological kingdoms. By virtue of their broad substrate promiscuity and regio- and stereo-selectivity, these enzymes enhance or attribute diversity to secondary metabolites. Actinomycetes species are well-known producers of secondary metabolites, especially Salinispora species. Despite the importance of P450s, a comprehensive comparative analysis of P450s and their role in secondary metabolism in Salinispora species is not reported. We therefore analyzed P450s in 126 strains from three different species Salinispora arenicola, S. pacifica, and S. tropica. The study revealed the presence of 2643 P450s that can be grouped into 45 families and 103 subfamilies. CYP107 and CYP125 families are conserved, and CYP105 and CYP107 families are bloomed (a P450 family with many members) across Salinispora species. Analysis of P450s that are part of secondary metabolite biosynthetic gene clusters (smBGCs) revealed Salinispora species have an unprecedented number of P450s (1236 P450s-47%) part of smBGCs compared to other bacterial species belonging to the genera Streptomyces (23%) and Mycobacterium (11%), phyla Cyanobacteria (8%) and Firmicutes (18%) and the classes Alphaproteobacteria (2%) and Gammaproteobacteria (18%). A peculiar characteristic of up to six P450s in smBGCs was observed in Salinispora species. Future characterization Salinispora species P450s and their smBGCs have the potential for discovering novel secondary metabolites.

Contrasting Health Effects of Bacteroidetes and Firmicutes Lies in Their Genomes: Analysis of P450s, Ferredoxins, and Secondary Metabolite Clusters.

Species belonging to the bacterial phyla Bacteroidetes and Firmicutes represent over 90% of the gastrointestinal microbiota. Changes in the ratio of these two bacterial groups were found to have contrasting health effects, including obesity and inflammatory diseases. Despite the availability of many bacterial genomes, comparative genomic studies on the gene pools of these two bacterial groups concerning cytochrome P450 monooxygenases (P450s), ferredoxins, and secondary metabolite biosynthetic gene clusters (smBGCs) are not reported. This study is aimed to address this research gap. The study revealed the presence of diverse sets of P450s, ferredoxins, and smBGCs in their genomes. Bacteroidetes species have the highest number of P450 families, ferredoxin cluster-types, and smBGCs compared to Firmicutes species. Only four P450 families, three ferredoxin cluster types, and five smBGCs are commonly shared between these two bacterial groups. Considering the above facts, we propose that the contrasting effects of these two bacterial groups on the host are partly due to the distinct nature of secondary metabolites produced by these organisms. Thus, the cause of the contrasting health effects of these two bacterial groups lies in their gene pools.

Saprophytic to Pathogenic Mycobacteria: Loss of Cytochrome P450s Vis a Vis Their Prominent Involvement in Natural Metabolite Biosynthesis.

Cytochrome P450 monooxygenases (P450s/CYPs) are ubiquitous enzymes with unique regio- and stereo-selective oxidation activities. Due to these properties, P450s play a key role in the biosynthesis of natural metabolites. Mycobacterial species are well-known producers of complex metabolites that help them survive in diverse ecological niches, including in the host. In this study, a comprehensive analysis of P450s and their role in natural metabolite synthesis in 2666 mycobacterial species was carried out. The study revealed the presence of 62,815 P450s that can be grouped into 182 P450 families and 345 subfamilies. Blooming (the presence of more than one copy of the same gene) and expansion (presence of the same gene in many species) were observed at the family and subfamily levels. CYP135 was the dominant family in mycobacterial species. The mycobacterial species have distinct P450 profiles, indicating that lifestyle impacts P450 content in their genome vis a vis P450s, playing a key role in organisms' adaptation. Analysis of the P450 profile revealed a gradual loss of P450s from non-pathogenic to pathogenic mycobacteria. Pathogenic mycobacteria have more P450s in biosynthetic gene clusters that produce natural metabolites. This indicates that P450s are recruited for the biosynthesis of unique metabolites, thus helping these pathogens survive in their niches. This study is the first to analyze P450s and their role in natural metabolite synthesis in many mycobacterial species.

Diversification of Ferredoxins across Living Organisms.

Ferredoxins, iron-sulfur (Fe-S) cluster proteins, play a key role in oxidoreduction reactions. To date, evolutionary analysis of these proteins across the domains of life have been confined to observing the abundance of Fe-S cluster types (2Fe-2S, 3Fe-4S, 4Fe-4S, 7Fe-8S (3Fe-4s and 4Fe-4S) and 2[4Fe-4S]) and the diversity of ferredoxins within these cluster types was not studied. To address this research gap, here we propose a subtype classification and nomenclature for ferredoxins based on the characteristic spacing between the cysteine amino acids of the Fe-S binding motif as a subtype signature to assess the diversity of ferredoxins across the living organisms. To test this hypothesis, comparative analysis of ferredoxins between bacterial groups, Alphaproteobacteria and Firmicutes and ferredoxins collected from species of different domains of life that are reported in the literature has been carried out. Ferredoxins were found to be highly diverse within their types. Large numbers of alphaproteobacterial species ferredoxin subtypes were found in Firmicutes species and the same ferredoxin subtypes across the species of Bacteria, Archaea, and Eukarya, suggesting shared common ancestral origin of ferredoxins between Archaea and Bacteria and lateral gene transfer of ferredoxins from prokaryotes (Archaea/Bacteria) to eukaryotes. This study opened new vistas for further analysis of diversity of ferredoxins in living organisms.

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.

In Silico Analysis of P450s and Their Role in Secondary Metabolism in the Bacterial Class Gammaproteobacteria.

The impact of lifestyle on shaping the genome content of an organism is a well-known phenomenon and cytochrome P450 enzymes (CYPs/P450s), heme-thiolate proteins that are ubiquitously present in organisms, are no exception. Recent studies focusing on a few bacterial species such as Streptomyces, Mycobacterium, Cyanobacteria and Firmicutes revealed that the impact of lifestyle affected the P450 repertoire in these species. However, this phenomenon needs to be understood in other bacterial species. We therefore performed genome data mining, annotation, phylogenetic analysis of P450s and their role in secondary metabolism in the bacterial class Gammaproteobacteria. Genome-wide data mining for P450s in 1261 Gammaproteobacterial species belonging to 161 genera revealed that only 169 species belonging to 41 genera have P450s. A total of 277 P450s found in 169 species grouped into 84 P450 families and 105 P450 subfamilies, where 38 new P450 families were found. Only 18% of P450s were found to be involved in secondary metabolism in Gammaproteobacterial species, as observed in Firmicutes as well. The pathogenic or commensal lifestyle of Gammaproteobacterial species influences them to such an extent that they have the lowest number of P450s compared to other bacterial species, indicating the impact of lifestyle on shaping the P450 repertoire. This study is the first report on comprehensive analysis of P450s in Gammaproteobacteria.

Impact of lifestyle on cytochrome P450 monooxygenase repertoire is clearly evident in the bacterial phylum Firmicutes.

Cytochrome P450 monooxygenases (CYPs/P450s), heme thiolate proteins, are well known for their role in organisms' primary and secondary metabolism. Research on eukaryotes such as animals, plants, oomycetes and fungi has shown that P450s profiles in these organisms are affected by their lifestyle. However, the impact of lifestyle on P450 profiling in bacteria is scarcely reported. This study is such an example where the impact of lifestyle seems to profoundly affect the P450 profiles in the bacterial species belonging to the phylum Firmicutes. Genome-wide analysis of P450s in 972 Firmicutes species belonging to 158 genera revealed that only 229 species belonging to 37 genera have P450s; 38% of Bacilli species, followed by 14% of Clostridia and 2.7% of other Firmicutes species, have P450s. The pathogenic or commensal lifestyle influences P450 content to such an extent that species belonging to the genera Streptococcus, Listeria, Staphylococcus, Lactobacillus, Lactococcus and Leuconostoc do not have P450s, with the exception of a handful of Staphylococcus species that have a single P450. Only 18% of P450s are found to be involved in secondary metabolism and 89 P450s that function in the synthesis of specific secondary metabolites are predicted. This study is the first report on comprehensive analysis of P450s in Firmicutes.

More P450s Are Involved in Secondary Metabolite Biosynthesis in Streptomyces Compared to Bacillus, Cyanobacteria, and Mycobacterium.

Unraveling the role of cytochrome P450 monooxygenases (CYPs/P450s), heme-thiolate proteins present in living and non-living entities, in secondary metabolite synthesis is gaining momentum. In this direction, in this study, we analyzed the genomes of 203 Streptomyces species for P450s and unraveled their association with secondary metabolism. Our analyses revealed the presence of 5460 P450s, grouped into 253 families and 698 subfamilies. The CYP107 family was found to be conserved and highly populated in Streptomyces and Bacillus species, indicating its key role in the synthesis of secondary metabolites. Streptomyces species had a higher number of P450s than Bacillus and cyanobacterial species. The average number of secondary metabolite biosynthetic gene clusters (BGCs) and the number of P450s located in BGCs were higher in Streptomyces species than in Bacillus, mycobacterial, and cyanobacterial species, corroborating the superior capacity of Streptomyces species for generating diverse secondary metabolites. Functional analysis via data mining confirmed that many Streptomyces P450s are involved in the biosynthesis of secondary metabolites. This study was the first of its kind to conduct a comparative analysis of P450s in such a large number (203) of Streptomyces species, revealing the P450s' association with secondary metabolite synthesis in Streptomyces species. Future studies should include the selection of Streptomyces species with a higher number of P450s and BGCs and explore the biotechnological value of secondary metabolites they produce.

Comprehensive Analyses of Cytochrome P450 Monooxygenases and Secondary Metabolite Biosynthetic Gene Clusters in Cyanobacteria.

The prokaryotic phylum Cyanobacteria are some of the oldest known photosynthetic organisms responsible for the oxygenation of the earth. Cyanobacterial species have been recognised as a prosperous source of bioactive secondary metabolites with antibacterial, antiviral, antifungal and/or anticancer activities. Cytochrome P450 monooxygenases (CYPs/P450s) contribute to the production and diversity of various secondary metabolites. To better understand the metabolic potential of cyanobacterial species, we have carried out comprehensive analyses of P450s, predicted secondary metabolite biosynthetic gene clusters (BGCs), and P450s located in secondary metabolite BGCs. Analysis of the genomes of 114 cyanobacterial species identified 341 P450s in 88 species, belonging to 36 families and 79 subfamilies. In total, 770 secondary metabolite BGCs were found in 103 cyanobacterial species. Only 8% of P450s were found to be part of BGCs. Comparative analyses with other bacteria Bacillus, Streptomyces and mycobacterial species have revealed a lower number of P450s and BGCs and a percentage of P450s forming part of BGCs in cyanobacterial species. A mathematical formula presented in this study revealed that cyanobacterial species have the highest gene-cluster diversity percentage compared to Bacillus and mycobacterial species, indicating that these diverse gene clusters are destined to produce different types of secondary metabolites. The study provides fundamental knowledge of P450s and those associated with secondary metabolism in cyanobacterial species, which may illuminate their value for the pharmaceutical and cosmetics industries.

Distribution and Diversity of Cytochrome P450 Monooxygenases in the Fungal Class Tremellomycetes.

Tremellomycetes, a fungal class in the subphylum Agaricomycotina, contain well-known opportunistic and emerging human pathogens. The azole drug fluconazole, used in the treatment of diseases caused by some species of Tremellomycetes, inhibits cytochrome P450 monooxygenase CYP51, an enzyme that converts lanosterol into an essential component of the fungal cell membrane ergosterol. Studies indicate that mutations and over-expression of CYP51 in species of Tremellomycetes are one of the reasons for fluconazole resistance. Moreover, the novel drug, VT-1129, that is in the pipeline is reported to exert its effect by binding and inhibiting CYP51. Despite the importance of CYPs, the CYP repertoire in species of Tremellomycetes has not been reported to date. This study intends to address this research gap. Comprehensive genome-wide CYP analysis revealed the presence of 203 CYPs (excluding 16 pseudo-CYPs) in 23 species of Tremellomycetes that can be grouped into 38 CYP families and 72 CYP subfamilies. Twenty-three CYP families are new and three CYP families (CYP5139, CYP51 and CYP61) were conserved across 23 species of Tremellomycetes. Pathogenic cryptococcal species have 50% fewer CYP genes than non-pathogenic species. The results of this study will serve as reference for future annotation and characterization of CYPs in species of Tremellomycetes.