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- 2019| MDPIIn dealing with Mycobacterium tuberculosis, the causative agent of the deadliest human disease—tuberculosis (TB)—utilization of cholesterol as a carbon source indicates the possibility of using cholesterol catabolic genes/proteins as novel drug targets. However, studies on cholesterol catabolism in mycobacterial species are scarce, and the number of mycobacterial species utilizing cholesterol as a carbon source is unknown. The availability of a large number of mycobacterial species’ genomic data affords an opportunity to explore and predict mycobacterial species’ ability to utilize cholesterol employing in silico methods. In this study, comprehensive comparative analysis of cholesterol catabolic genes/proteins in 93 mycobacterial species was achieved by deducing a comprehensive cholesterol catabolic pathway, developing a software tool for extracting homologous protein data and using protein structure and functional data. Based on the presence of cholesterol catabolic homologous proteins proven or predicted to be either essential or specifically required for the growth of M. tuberculosis H37Rv on cholesterol, we predict that among 93 mycobacterial species, 51 species will be able to utilize cholesterol as a carbon source. This study’s predictions need further experimental validation and the results should be taken as a source of information on cholesterol catabolism and genes/proteins involved in this process among mycobacterial species.
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- Steroids are found in bacteria and eukaryotes, and genes potentially encoding steroid metabolic enzymes have also been identified in giant viruses. For decades, hydroxylated steroids have been utilized in medicine to treat various human diseases. The hydroxylation of steroids can be achieved using microbial enzymes, especially cytochrome P450 monooxygenases (CYPs/P450s) and is well documented. Understanding the structural determinants that govern the regio- and stereoselectivity of steroid hydroxylation by P450s is essential in order to fully exploit their potential. Herein, we present a comprehensive analysis of the steroid-hydroxylating CYP109 family across the domains of life and delineate the structural determinants that govern steroid hydroxylation. Data mining, annotation, and phylogenetic analysis revealed that CYP109 family members are highly populated in bacteria, and indeed, these members passed from bacteria to archaea by horizontal gene transfer, leading to the evolution of P450s in archaea. Analysis of twelve CYP109 crystal structures revealed large, flexible, and dynamic active site cavities that can accommodate multiple ligands. The correct positioning and orientation of the steroid in the active site cavity and the nature of the C17 substituent on the steroid molecule influence catalysis. In an analogous fashion to the CYP107 family, the amino acid residues within the CYP109 binding pocket involve hydrophilic and hydrophobic interactions, influencing substrate orientations and anchoring and determining the site of hydroxylation and catalytic activity. A handful of amino acids, such as Val84, Val292, and Ser387 in CYP109B4, have been found to play a role in determining the catalytic regiospecificity, and a single amino acid, such as Arg74 in CYP109A2, has been found to be essential for the enzymatic activity. This work serves as a reference for the precise understanding of CYP109 structure–function relationships and for P450 enzymes in general. The findings will guide the genetic engineering of CYP109 enzymes to produce valuable steroid molecules of medicinal and biotechnological importance.
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- 2025| Multidisciplinary Di...Cytochromes P450 are a superfamily of heme-containing monooxygenases involved in a variety of oxidative metabolic reactions, primarily catalyzing the insertion of an oxygen atom into a C-H bond. CYP102 represents the first example of a bacterial P450 that can be classified as a type II (eukaryotic-like) P450 and functions as a catalytically self-sufficient enzyme. These unique features have made CYP102 an attractive system for studying P450 structure and function. However, an overall picture of the specific amino acid residues that are crucial to the functioning of CYP102 and the effect of mutations on the P450 structure and catalysis is yet to be reported. Such an approach will aid protein engineering approaches used to improve this enzyme. To address this research knowledge gap, we have investigated 105 CYP102 crystal structures in this study. We demonstrate that the CYP102 active site is highly dynamic and flexible. Amino acid residues that play critical roles in substrate binding, orientation, and anchoring were identified. Mutational studies highlighted the roles of amino acids and provided possible bioengineering improvement strategies for CYP102. Decoy molecules are a promising agent for deceiving CYP102 and permitting non-native substrates into the active site. Ru(II)-diimine photosensitizers and zinc/cobalt (III) sepulchrate (Co(III)Sep) could be used as alternative electron sources. The present study serves as a reference for understanding the structure–functional analysis of CYP102 family members precisely and of P450 enzymes in general. Significantly, this work contributes to the effort to develop an improved CYP102 enzyme, thereby advancing the field of P450 research and potentially leading to new industrial applications.
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- Tuberculosis (TB) is one of the top infectious diseases causing numerous human deaths in the world. Despite enormous efforts, the physiology of the causative agent, Mycobacterium tuberculosis, is poorly understood. To contribute to better understanding the physiological capacity of these microbes, we have carried out extensive in silico analyses of the 1111 mycobacterial species genomes focusing on revealing the role of the orphan cytochrome P450 monooxygenase (CYP) CYP139 family. We have found that CYP139 members are present in 894 species belonging to three mycobacterial groups: M. tuberculosis complex (850-species), Mycobacterium avium complex (34-species), and non-tuberculosis mycobacteria (10-species), with all CYP139 members belonging to the subfamily “A”. CYP139 members have unique amino acid patterns at the CXG motif. Amino acid conservation analysis placed this family in the 8th among CYP families belonging to different biological domains and kingdoms. Biosynthetic gene cluster analyses have revealed that 92% of CYP139As might be associated with producing different secondary metabolites. Such enhanced secondary metabolic potentials with the involvement of CYP139A members might have provided mycobacterial species with advantageous traits in diverse niches competing with other microbial or viral agents, and might help these microbes infect hosts by interfering with the hosts’ metabolism and immune system.
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- 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.
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- 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.
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- 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.
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- 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.
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- 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.
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- 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.
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