Comparative analysis of P450 signature motifs EXXR and CXG in the large and diverse kingdom of fungi: identification of evolutionarily conserved amino acid patterns characteristic of P450 family

Published ArticleCytochrome P450 monooxygenases (P450s) are heme-thiolate proteins distributed across the biological kingdoms. P450s are catalytically versatile and play key roles in organisms primary and secondary metabolism. Identification of P450s across the biological kingdoms depends largely on the identification of two P450 signature motifs, EXXR and CXG, in the protein sequence. Once a putative protein has been identified as P450, it will be assigned to a family and subfamily based on the criteria that P450s within a family share more than 40% homology and members of subfamilies share more than 55% homology. However, to date, no evidence has been presented that can distinguish members of a P450 family. Here, for the first time we report the identification of EXXR- and CXG-motifs-based amino acid patterns that are characteristic of the P450 family. Analysis of P450 signature motifs in the under-explored fungal P450s from four different phyla, ascomycota, basidiomycota, zygomycota and chytridiomycota, indicated that the EXXR motif is highly variable and the CXG motif is somewhat variable. The amino acids threonine and leucine are preferred as second and third amino acids in the EXXR motif and proline and glycine are preferred as second and third amino acids in the CXG motif in fungal P450s. Analysis of 67 P450 families from biological kingdoms such as plants, animals, bacteria and fungi showed conservation of a set of amino acid patterns characteristic of a particular P450 family in EXXR and CXG motifs. This suggests that during the divergence of P450 families from a common ancestor these amino acids patterns evolve and are retained in each P450 family as a signature of that family. The role of amino acid patterns characteristic of a P450 family in the structural and/or functional aspects of members of the P450 family is a topic for future research

Medicinal Properties of Selected Asparagus Species: A Review

Asparagus species are naturally distributed along Asia, Africa, and Europe and are known to have numerous biological properties. This review article was aimed to provide an organized summary of current studies on the traditional uses, phytochemistry, and pharmacological and toxicological studies of Asparagus laricinus Burch., Asparagus africanus Lam., Asparagus officinalis L., Asparagus racemosus Willd., and Asparagus densiflorus (Kunth) Jessop to attain and establish new insights for further researches. Information used in this review was obtained from electronic database including PubMed central, Google scholars, Science direct, Scopus, and Sabinet. Based on the present findings, the existing literature still presents some breaches about the mechanism of action of various constituents of these plants, and their relation to other plant compounds in poly-herbal formulations, as well as their long-term use and safety. More in-depth studies are still needed for active compounds and biological activities of Asparagus laricinus, Asparagus africanus, and Asparagus densiflorus. Therefore, innumerable opportunities and possibilities for investigation are still available in novel areas of these plants for future research stud¬ies. It can be concluded that all selected Asparagus species have tremendous potential to improve human health and the pharmacological activities of these plants can be attributed to bioactive phytochemicals they possess

Antimicrobial activity screening of Philenoptera violacea (Klotzsch) schrire and Xanthocercis zambesiaca (Baker) Dumaz-Le-Grand

Published ArticleMicroorganism involvement in cancer has been identified for over a century, and different types of bacteria have been associated with carcinogenesis. Philenoptera violacea (Klotzsch) Schrire and Xanthocercis zambesiaca (Baker) Dumaz-le- Grand plant extracts are traditionally used by South African traditional healers for the treatment of inflammation related disorders; however, their efficacy has not been determined. The aim of this study was to investigate the antimicrobial activity of the mixture of leaves, flower & twig of each plant extract respectively. Antimicrobial activity was determined using p- Iodonitrotetrazolium chloride (INT) assay on gram-positive bacterium: Staphylococcus aureus, S. epidermidis, S. saprohyticus, Bacillus subtilis, and gram-negative bacterium: Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa. Results of antimicrobial activity revealed that both plant extracts had no antimicrobial activity against selected micro-organisms. Thus, we couldn’t support or confirm the antimicrobial activity potential of these plants as reported by traditional healers, however, factors that might have contributed to these results were not excluded

Comparative Genomics and Evolutionary Analysis of Cytochrome P450 Monooxygenases in Fungal Subphylum Saccharomycotina

Published ArticleCytochrome P450 monooxygenases (P450s) are heme-thiolate enzymes and play an important role in the primary and secondary metabolism of living organisms. Genome sequencing analysis of fungal organisms revealed the presence of numerous P450s in their genomes, with few exceptions. P450s in the fungal subphylum Saccharomycotina, which contains biotechnologically important and opportunistic human pathogen yeasts, have been underexplored because there are few P450s in their genomes. In the present study we performed comparative analysis of P450s in 25 yeast species. A hundred and seventy-two P450s were found in 25 yeast species and these are grouped into 13 P450 families and 27 subfamilies. P450s ranged from a minimum of three (Saccharomyces species) to a maximum of 21 (Candida species) in the yeast genomes. Among the P450 families, the CYP52 family showed the highest number of member P450s (71) followed by CYP51 (27), CYP61 (25), CYP56 (20) and CYP501 (11). Pichia pastoris and Dekkera bruxellensis showed a novel P450 family, CYP5489, in their genome. Based on the functional properties of characterized P450s, we conclude that P450s in Saccharomycotina species possibly play a role in organisms’ physiology either in the synthesis of cellular components or in the utilization of simpler organic molecules. The ecological niches of yeast species are highly enriched with simpler organic nutrients and it is well known that yeast species utilize simpler organic nutrients as carbon source efficiently. This might have played a role in compacting yeast genomes and possibly losing a considerable number of P450s during evolution

Ethno-Medical Botany And Some Biological Activities of Ipomoea Oblongata Collected In The Free State Province, South Africa

Published ArticleAbstract: Background and Objective: Literature reporting on the therapeutic uses and biological activities of Ipomoea oblongata (E.Mey.ex Choisy) is very scanty in South Africa. This study was carried out to comprehensively document medicinal use of this species and also determine some of its biological activities namely the antioxidant, total phenolic content and phytochemical properties. Materials and Methods: Ethnobotanical data on the healing applications of I. oblongata was gathered using a semi-structure questionnaire with 12 traditional healers (THs) practicing in the Free State province. The antioxidant properties and the total polyphenol content of I. oblongata extracts (Dichloromethane, DCM, Methanol, MeOH and Water, H2O) were determined by means of the 2, 2-Diphenylpicrylhydrazyl assay and the Folin Ciocalteu method, respectively. Gallic acid and ascorbic acid were used as standards. Phytochemical constituents were determined using standard screening procedures. Results: Roots of I. oblongata were mainly used by THs as remedy for ailments such as cancer, cardiovascular diseases, diarrhoea and nausea, inflammatory diseases, pregnancy and postnatal, respiratory infections, sexually transmitted infections and wounds. The MeOH roots extract of I. oblongata showed remarkable (98% at 0.25mg mL–1 and 99% at 0.5mg mL–1) antioxidant potential. Total phenolic contents of this species showed the Gallic acid equivalence (GAE) of 0.04 for both the MeOH and DCM and 0.03 for the H2O extract. The phytochemical screening exhibited the presence of carbohydrates, glycosides, steroids, terpenoids, alkaloids, flavonoids and tannis. Conclusion: Ipomoea oblongata roots are highly harvested and multi-used by THs against various human ailments, which could be attributed to the present of biologically active pharmaceutical components demonstrated

A Review of South African Traditional Medicinal Plants Used for Treating Fungal Coinfections in COVID-19 Patients with Respiratory Diseases

Fungal infections are still most prevalent in the South African population. Fungal respiratory infections and diseases are the cause of severe clinical challenges and mortality in patients with compromised immune systems. Clinical signs of coronavirus disease of 2019 (COVID-19) such as lung injury, hyperglycemia due to diabetes, host iron and zinc depletion, hypoxia, immunosuppression, steroid therapy, and long-term hospitalization predispose patients to opportunistic fungal infections. Fungal pathogens, including Cryptococcus, Aspergillus, and Candida species, cause coinfections in patients infected with (COVID-19), and this has a negative impact on the patients’ pharmacological management goals. Cryptococcus, Aspergillus, and Candida species cause respiratory infections and illnesses including pneumonia, pulmonary aspergillosis, pulmonary candidiasis, and pulmonary cryptococcosis. South African traditional medicinal plants have been used in the treatment of respiratory symptoms and diseases caused by these fungal pathogens. Medicinal plants contain secondary metabolites possessing antifungal activity against Cryptococcus, Aspergillus, and Candida species. Moreover, medicinal plants are cheaper and easily accessible and are believed to be safe. This review documents the use of South African traditional medicinal plants including Artemisia absinthium, Artemisia afra, Dicoma anomala, Felicia species, Mentha species, Ruta graveolens, and Seasia erosa in the treatment of fungal infections and diseases caused by these pathogens

Genome-wide identification, annotation and characterization of novel thermostable cytochrome P450 monooxygenases from the thermophilic biomass-degrading fungi Thielavia terrestris and Myceliophthora thermophila

Published ArticleCytochrome P450 monooxygenases (P450s) are ubiquitous heme-thiolate proteins that have potential biotechnological application. Thermostable-P450s that can withstand hostile industrial conditions, such as high temperatures, extremes of pH and organic solvents, are needed for biotechnological usage. Here, for the first time, we report a large number of thermostable-P450s from two thermophilic biomass-degrading fungi, Myceliophthora thermophila and Thielavia terrestris. Genome-wide P450 analysis revealed the presence of 79 and 70 P450s (P450ome) in T. terrestris and M. thermophila. Authentic P450s containing both the P450 signature domains (EXXRand CXG) were classified as follows: T. terrestris (50 families and 56 subfamilies) and M. thermophila (49 families and 53 subfamilies). Bioinformatics analysis of P450omes suggested the presence of a large number of thermostable-P450s. Based on aliphatic index cut-off ([90), 14 and 11 P450s were determined to be thermostable in T. terrestris and M. thermophila. Among the thermostable P450s, six P450s from T. terrestris and three from M. thermophila had a melting temperature (Tm) of [65 C, suggesting their hyperthermal tolerance. Analysis of the instability index of two ascomycete P450omes revealed the presence of 12 and 19 in vitro stable P450s in T. terrestris and M. thermophila. Overall, six P450s from T. terrestris and four from M. thermophila showed both thermal tolerance and in vitro stability. Thermophilic ascomycetes P450s are of potential interest from a structural, mechanistic and biotechnological point of view, as five P450s showed higher thermal tolerance and five showed higher in vitro stability compared to the wellcharacterized thermostable-P450s CYP175A1 (bacteria) and CYP119 (archaea)

Cytochrome P450 monooxygenase analysis in free-living and symbiotic microalgae Coccomyxa sp. C-169 and Chlorella sp. NC64A

Published ArticleMicroalgae research is gaining momentum because of their potential biotechnological applications, including the generation of biofuels. Genome sequencing analysis of two model microalgal species, polar free-living Coccomyxa sp. C-169 and symbiotic Chlorella sp. NC64A, revealed insights into the factors responsible for their lifestyle and unravelled biotechnologically valuable proteins. However, genome sequence analysis under-explored cytochrome P450 monooxygenases (P450s), heme-thiolate proteins ubiquitously present in species belonging to different biological kingdoms. In this study we performed genome data-mining, annotation and comparative analysis of P450s in these two model algal species. Sixty-nine P450s were found in two algal species. Coccomyxa sp. showed 40 P450s and Chlorella sp. showed 29 P450s in their genome. Sixty-eight P450s (>100 amino acid in length) were grouped into 32 P450 families and 46 P450 subfamilies. Among the P450 families, 27 P450 families were novel and not found in other biological kingdoms. The new P450 families are CYP745-CYP747, CYP845-CYP863, and CYP904-CYP908. Five P450 families, CYP51, CYP97, CYP710, CYP745, and CYP746, were commonly found between two algal species and 16 and 11 P450 families were unique to Coccomyxa sp. and Chlorella sp. Synteny analysis and gene-structure analysis revealed P450 duplications in both species. Functional analysis based on homolog P450s suggested that CYP51 and CYP710 family members are involved in membrane ergosterol biosynthesis. CYP55 and CYP97 family members are involved in nitric oxide reduction and biosynthesis of carotenoids. This is the first report on comparative analysis of P450s in the microalgal species Coccomyxa sp. C-169 and Chlorella sp. NC64A

Comparative Analyses And Structural Insights Of The Novel Cytochrome P450 Fusion Protein Family CYP5619 In Oomycetes

Published ArticlePhylogenetic and structural analysis of P450 proteins fused to peroxidase/dioxygenase has not been reported yet. We present phylogenetic and in silico structural analysis of the novel P450 fusion family CYP5619 from the deadliest fish pathogenic oomycete, Saprolegnia diclina. Data-mining and annotation of CYP5619 members revealed their unique presence in oomycetes. CYP5619 members have the highest number of conserved amino acids among eukaryotic P450s. The highest number of conserved amino acids (78%) occurred in the peroxidase/dioxygenase domain compared to the P450 domain (22%). In silico structural analysis using a high-quality CYP5619A1 model revealed that CYP5619A1 has characteristic P450 structural motifs including EXXR and CXG. However, the heme-binding domain (CXG) in CYP5619 members was found to be highly degenerated. The in silico substrate binding pattern revealed that CYP5619A1 have a high affinity to medium chain fatty acids. Interestingly, the controlling agent of S. diclina malachite green was predicted to have the highest binding affinity, along with linoleic acid. However, unlike fatty acids, none of the active site amino acids formed hydrogen bonds with malachite green. The study’s results will pave the way for assessing CYP5619A1’s role in S. diclina physiology, including the nature of malachite green binding

Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes

Published ArticleCytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members’ duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes