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

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

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

Construction of world's first Mycobacterium tuberculosis cytochrome P450 monooxygenase library

Published ThesisThe actinomycete Mycobacterium tuberculosis causes Tuberculosis (TB), a chronic lung disease in humans and continues to be one of the greatest threats to mankind. Large number of studies showed that M. tuberculosis cytochrome P450 monooxygenases (P450s) can be used as novel drug target. P450s are mixed function oxidoreductases well known for their role in essential cellular anabolic and catabolic processes. Despite the greater importance of M. tuberculosis P450s as novel drug targets, only four M. tuberculosis P450s (apart from highly conserved CYP51) have been functionally characterized for their in vivo role. The major challenges in M. tuberculosis P450 research is expression of M. tuberculosis P450s and identification of substrate(s). This study is aimed to develop world’s first M. tuberculosis P450 expression library by cloning remaining 14 M. tuberculosis P450s. In order to clone 14 M. tuberculosis P450s a cloning strategy was developed such that all 14 M. tuberculosis P450s was cloned into expression vector. In this study, expression vector pINK_A was modified in its multiple cloning site by incorporating more restriction enzyme sites to accommodate all 14 M. tuberculosis P450s. The modified vector was named as pINK_d. Restriction profiling of 14 M. tuberculosis P450s were carried out and suitable restriction enzymes were selected for directional cloning of M. tuberculosis P450s into pINK_d vector. The pINK_d and 14 M. tuberculosis P450 cDNAs were synthesized and all synthesized P450 cDNA were subsequently cloned into pINK_d. The plasmid DNA clones (or constructs) containing M. tuberculosis P450 cDNA was transformed into E. coli DH5α cells and the recombinant E. coli cells were selected on Luria-Bertani agar plates containing ampicillin antibiotic. Plasmids from the recombinant cells were isolated and subjected to restriction enzyme digestion analysis. Restriction enzyme digestion analysis of plasmids revealed that all 14 M. tuberculosis P450s were successfully cloned as correct size of cDNA corresponding to respective M. tuberculosis P450s was released upon digestion with restriction enzymes. This study will pave the way for expression and characterization of M. tuberculosis P450s. Thus developing M. tuberculosis P450 based novel anti-TB drugs. The M. tuberculosis P450 E. coli library developed in this study will be patented after confirming M. tuberculosis P450s expression. Apart from my Masters study, I also supervised two B. Tech student projects and managed to publish an article with students. Furthermore, I also worked on a few other bioinformatics projects and earned co-authorship. Most of my research articles are published in high impact factor journals. The following is a list of my research articles: 1. IKR Kgosiemang (co-author) (2016) Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s. Scientific Reports 6, Article number: 33099. 2. IKR Kgosiemang (co-author) (2015). Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes. Scientific Reports 5, Article number: 11572. 3. NT Mthakathi, IKR Kgosiemang et al, (2015). Cytochrome P450 monooxygenase analysis in free-living and symbiotic microalgae Coccomyxa sp. C-169 and Chlorella sp. NC64A. Alage 30(3):233-239. In addition to the above credits, I was featured on national TV and in newspapers for discovering a novel drug target. I also presented work at both national and international (Canada) conferences

Green synthesis of Iron oxide and Iron dioxide nanoparticles using Euphorbia tirucalli: characterization and antiproliferative evaluation against three breast cancer cell lines

AbstractResearchers have become increasingly interested in nanoparticles made from plants because of their stability and large surface area. In the current study, iron oxide and iron dioxide nanoparticles were synthesized using aerial parts of the E. tirucalli as a reducing agent. The nanoparticles were analyzed using various techniques, including Ultraviolet-visible spectroscopy, Fourier Transform Infrared spectroscopy, X-ray diffractometer, X-ray photoelectron spectroscopy, X-ray energy dispersive spectroscopy, Scanning electron Microscopy, and Transmission Electron Microscopy. The nanoparticles were then investigated for their antiproliferative effect against MCF-7, SK-BR-3, MDA-MB231, and Vero cell lines. The results confirmed the formation of FeO and FeO2 nanoparticles by color change and a UV absorbance peak between 220–390 nm. EDS analysis showed traces of Fe and O, while TEM confirmed the nanoparticle size of 100 nm. FTIR showed a peak at 514 nm. The FeO-RT NPs demonstrated over 80% antiproliferative activity against the MCF-7 cell line at a concentration of 10 μg/mL. while doxorubicin, FeO-RT NPs, and DCM extract showed similar activity against the MDA-MB231 cell line at 10 and 1 g/mL concentrations. However, Vero and SK-BR-3 cell lines showed decreased antiproliferative activity. This study highlights the environmentally friendly use and safe application of iron oxide NPs in cancer therapy