scholarly journals Comparative Analysis, Structural Insights, and Substrate/Drug Interaction of CYP128A1 in Mycobacterium tuberculosis

2020 ◽  
Vol 21 (14) ◽  
pp. 4816
Author(s):  
Nokwanda Samantha Ngcobo ◽  
Zinhle Edith Chiliza ◽  
Wanping Chen ◽  
Jae-Hyuk Yu ◽  
David R. Nelson ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Comparative Analysis ◽  
Biosynthetic Gene Cluster ◽  
Cytochrome P450 Monooxygenases ◽  
Mycobacterial Species ◽  
Mycobacterium Tuberculosis H37rv ◽  
Genome Data ◽  
P450 Monooxygenases ◽  
Substrate Drug ◽  
Structural Insights

Cytochrome P450 monooxygenases (CYPs/P450s) are well known for their role in organisms’ primary and secondary metabolism. Among 20 P450s of the tuberculosis-causing Mycobacterium tuberculosis H37Rv, CYP128A1 is particularly important owing to its involvement in synthesizing electron transport molecules such as menaquinone-9 (MK9). This study employs different in silico approaches to understand CYP128 P450 family’s distribution and structural aspects. Genome data-mining of 4250 mycobacterial species has revealed the presence of 2674 CYP128 P450s in 2646 mycobacterial species belonging to six different categories. Contrast features were observed in the CYP128 gene distribution, subfamily patterns, and characteristics of the secondary metabolite biosynthetic gene cluster (BGCs) between M. tuberculosis complex (MTBC) and other mycobacterial category species. In all MTBC species (except one) CYP128 P450s belong to subfamily A, whereas subfamily B is predominant in another four mycobacterial category species. Of CYP128 P450s, 78% was a part of BGCs with CYP124A1, or together with CYP124A1 and CYP121A1. The CYP128 family ranked fifth in the conservation ranking. Unique amino acid patterns are present at the EXXR and CXG motifs. Molecular dynamic simulation studies indicate that the CYP128A1 bind to MK9 with the highest affinity compared to the azole drugs analyzed. This study provides comprehensive comparative analysis and structural insights of CYP128A1 in M. tuberculosis.

scholarly journals Implementation of homology based and non-homology based computational methods for the identification and annotation of orphan enzymes: using Mycobacterium tuberculosis H37Rv as a case study

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Swati Sinha ◽  
Andrew M. Lynn ◽  
Dhwani K. Desai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Functional Annotation ◽  
Drug Targets ◽  
Sequence Similarity ◽  
Hole Filling ◽  
Mycobacterium Tuberculosis H37rv ◽  
Genome Data ◽  
Candidate Protein ◽  
Computational Procedures

Abstract Background Homology based methods are one of the most important and widely used approaches for functional annotation of high-throughput microbial genome data. A major limitation of these methods is the absence of well-characterized sequences for certain functions. The non-homology methods based on the context and the interactions of a protein are very useful for identifying missing metabolic activities and functional annotation in the absence of significant sequence similarity. In the current work, we employ both homology and context-based methods, incrementally, to identify local holes and chokepoints, whose presence in the Mycobacterium tuberculosis genome is indicated based on its interaction with known proteins in a metabolic network context, but have not been annotated. We have developed two computational procedures using network theory to identify orphan enzymes (‘Hole finding protocol’) coupled with the identification of candidate proteins for the predicted orphan enzyme (‘Hole filling protocol’). We propose an integrated interaction score based on scores from the STRING database to identify candidate protein sequences for the orphan enzymes from M. tuberculosis, as a case study, which are most likely to perform the missing function. Results The application of an automated homology-based enzyme identification protocol, ModEnzA, on M. tuberculosis genome yielded 56 novel enzyme predictions. We further predicted 74 putative local holes, 6 choke points, and 3 high confidence local holes in the genome using ‘Hole finding protocol’. The ‘Hole-filling protocol’ was validated on the E. coli genome using artificial in-silico enzyme knockouts where our method showed 25% increased accuracy, compared to other methods, in assigning the correct sequence for the knocked-out enzyme amongst the top 10 ranks. The method was further validated on 8 additional genomes. Conclusions We have developed methods that can be generalized to augment homology-based annotation to identify missing enzyme coding genes and to predict a candidate protein for them. For pathogens such as M. tuberculosis, this work holds significance in terms of increasing the protein repertoire and thereby, the potential for identifying novel drug targets.

scholarly journals Biochemical and structural insights into the cytochrome P450 reductase from Candida tropicalis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ana C. Ebrecht ◽  
Naadia van der Bergh ◽  
Susan T. L. Harrison ◽  
Martha S. Smit ◽  
B. Trevor Sewell ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Cytochrome P450 Monooxygenases ◽  
Oxygen Species ◽  
Type Ii ◽  
Cytochrome P450 Reductase ◽  
Terminal Part ◽  
X Ray ◽  
P450 Monooxygenases ◽  
Oxygen Complex ◽  
Structural Insights

AbstractCytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs). In addition, it can also reduce other proteins and molecules, including cytochrome c, ferricyanide, and different drugs. Although various CPRs have been functionally and structurally characterized, the overall mechanism and its interaction with different redox acceptors remain elusive. One of the main problems regarding electron transfer between CPRs and CYPs is the so-called “uncoupling”, whereby NAD(P)H derived electrons are lost due to the reduced intermediates’ (FAD and FMN of CPR) interaction with molecular oxygen. Additionally, the decay of the iron-oxygen complex of the CYP can also contribute to loss of reducing equivalents during an unproductive reaction cycle. This phenomenon generates reactive oxygen species (ROS), leading to an inefficient reaction. Here, we present the study of the CPR from Candida tropicalis (CtCPR) lacking the hydrophobic N-terminal part (Δ2–22). The enzyme supports the reduction of cytochrome c and ferricyanide, with an estimated 30% uncoupling during the reactions with cytochrome c. The ROS produced was not influenced by different physicochemical conditions (ionic strength, pH, temperature). The X-ray structures of the enzyme were solved with and without its cofactor, NADPH. Both CtCPR structures exhibited the closed conformation. Comparison with the different solved structures revealed an intricate ionic network responsible for the regulation of the open/closed movement of CtCPR.

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

2020 ◽  
Vol 21 (2) ◽  
pp. 656 ◽  
Author(s):  
Makhosazana Jabulile Khumalo ◽  
Nomfundo Nzuza ◽  
Tiara Padayachee ◽  
Wanping Chen ◽  
Jae-Hyuk Yu ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Secondary Metabolites ◽  
Secondary Metabolite ◽  
Gene Clusters ◽  
Cytochrome P450 Monooxygenases ◽  
Biosynthetic Gene ◽  
Mycobacterial Species ◽  
Biosynthetic Gene Clusters ◽  
P450 Monooxygenases ◽  
Cyanobacterial Species

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.

scholarly journals Ancient Bacterial Class Alphaproteobacteria Cytochrome P450 Monooxygenases Can Be Found in Other Bacterial Species

2021 ◽  
Vol 22 (11) ◽  
pp. 5542
Author(s):  
Nomfundo Nzuza ◽  
Tiara Padayachee ◽  
Puleng Rosinah Syed ◽  
Justyna Dorota Kryś ◽  
Wanping Chen ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Bacterial Species ◽  
Gene Clusters ◽  
Side Chain ◽  
Cytochrome P450 Monooxygenases ◽  
Biosynthetic Gene Clusters ◽  
Genome Data ◽  
P450 Monooxygenases ◽  
Large Numbers ◽  
Cyanobacterial 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.

Mycobactericidal activity of glutaraldehyde solutions

1976 ◽  
Vol 4 (5) ◽  
pp. 408-412
Author(s):  
F M Collins ◽  
V Montalbine
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Aqueous Solutions ◽  
Acidic Solution ◽  
Practical Importance ◽  
Tuberculosis H37rv ◽  
Sodium Bisulfite ◽  
Mycobacterial Species ◽  
Mycobacterium Tuberculosis H37rv ◽  
Acid Ph ◽  
Glutaraldehyde Solution

Aqueous solutions of alkaline glutaraldehyde (buffered at pH 8.5) inactivated a standard suspension of Mycobacterium tuberculosis H37Rv faster than the corresponding acid (pH 3.7 preparation. Quantitative differences in the rate of inactivation of eight other species of Mycobacterium were determined using a 1% solution of alkaline glutaraldehyde and inactivation of residual glutaraldehyde with 1% sodium bisulfite solution. Variations in the rate of kill were observed between the various mycobacterial species tested, but such differences were probably not sufficiently large to be of practical importance. A 2% alkaline glutaraldehyde solution inactivated 10(5) viable M. tuberculosis cells present on the surface of porcelain penicylinders within 5 min at 18 degrees C. This rate of inactivation was faster than in the acidic solution.

scholarly journals Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0142100 ◽  
Author(s):  
Lehlohonolo Benedict Qhanya ◽  
Godfrey Matowane ◽  
Wanping Chen ◽  
Yuxin Sun ◽  
Elizabeth Mpholoseng Letsimo ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Comparative Analysis ◽  
Plant Pathogens ◽  
Cytochrome P450 Monooxygenases ◽  
P450 Monooxygenases ◽  
Genome Wide

scholarly journals Identification and Analysis of the Balhimycin Biosynthetic Gene Cluster and Its Use for Manipulating Glycopeptide Biosynthesis in Amycolatopsis mediterranei DSM5908

1999 ◽  
Vol 43 (7) ◽  
pp. 1565-1573 ◽  
Author(s):  
S. Pelzer ◽  
R. Süßmuth ◽  
D. Heckmann ◽  
J. Recktenwald ◽  
P. Huber ◽  
...  
Keyword(s):  
High Performance ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Cytochrome P450 Monooxygenases ◽  
Amycolatopsis Mediterranei ◽  
Glycosyltransferase Gene ◽  
P450 Monooxygenases ◽  
Dna Fragment ◽  
Sequence Similarities ◽  
Reading Frames

ABSTRACT Seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, Amycolatopsis mediterranei DSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (oxyA to -C) showed significant sequence similarities to cytochrome P450 monooxygenases and one (bhaA) showed similarities to halogenase, and the genesbgtfA to -C showed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography–mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene bgtfB, produced at least four different compounds which were not glycosylated but still antibiotically active.

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