106 research outputs found
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
Software for molecular docking: a review
Publshed ArticleMolecular docking methodology explores the behavior
of small molecules in the binding site of a target protein.
As more protein structures are determined experimentally
using X-ray crystallography or nuclear magnetic resonance
(NMR) spectroscopy, molecular docking is increasingly used
as a tool in drug discovery. Docking against homologymodeled
targets also becomes possible for proteins whose
structures are not known. With the docking strategies, the
druggability of the compounds and their specificity against a
particular target can be calculated for further lead optimization
processes. Molecular docking programs perform a search algorithm
in which the conformation of the ligand is evaluated
recursively until the convergence to the minimum energy is
reached. Finally, an affinity scoring function, ΞG [U total in
kcal/mol], is employed to rank the candidate poses as the sum
of the electrostatic and van der Waals energies. The driving
forces for these specific interactions in biological systems aim
toward complementarities between the shape and electrostatics
of the binding site surfaces and the ligand or substrate
In silico strategies on prion pathogenic conversion and inhibition from PrPC -PrPSc
Published ArticleTo date, various therapeutic strategies identified numerous anti-prion compounds and antibodies that stabilize PrPC, block the conversion of PrPC-PrPSc and increased effect on PrPSc clearance. However, no suitable drug has been identified clinically so far due to the poor oral absorption, low blood-brain-barrier [BBB] penetration, and high toxicity. Although some of the drugs were proven to be effective in prion-infected cell culture and whole animal models, none of them increased the rate of survival compared to placebo. Areas covered: In this review, the authors highlight the importance of in silico approaches like molecular docking, virtual screening, pharmacophore analysis, molecular dynamics, QSAR, CoMFA and CoMSIA applied to detect molecular mechanisms of prion inhibition and conversion from PrPC-PrPSc. Expert opinion: Several in silico approaches combined with experimental studies have provided many structural and functional clues on the stability and physiological activity of prion mutants. Further, various studies of in silico and in vivo approaches were also shown to identify several new small organic anti-scrapie compounds to decrease the accumulation of PrPres in cell culture, inhibit the aggregation of a PrPC peptide, and possess pharmacokinetic characteristics that confirm the drug-likeness of these compounds
Systematic Identification and Evolutionary Analysis of Catalytically Versatile Cytochrome P450 Monooxygenase Families Enriched in Model Basidiomycete Fungi
Published ArticleGenome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the
presence of numerous cytochrome P450 monooxygenases (P450s) in their genomes, with some exceptions. Considering the
large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and
the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwinβs theory of natural
selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components
degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its
natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450
analysis in the basidiomycete species, Phanerochaete chrysosporium, Phanerochaete carnosa, Agaricus bisporus, Postia
placenta, Ganoderma sp. and Serpula lacrymans, revealed enrichment of 11 P450 families (out of 68 P450 families), CYP63,
CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis
of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of
Phanerochaete chrysosporium and Phanerochaete carnosa, suggesting paralogous evolution of P450s in model
basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns.
P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests
that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in
basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are
catalytically versatile and possibly involved in fungal colonization of plant material. To our knowledge, this is the first report
on the identification and comparative-evolutionary analysis of P450 families enriched in model basidiomycetes
A Fungal P450 (CYP5136A3) Capable of Oxidizing Polycyclic Aromatic Hydrocarbons and Endocrine Disrupting Alkylphenols: Role of Trp129 and Leu324
The model white rot fungus Phanerochaete chrysosporium, which is known for its versatile pollutant-biodegradation ability, possesses an extraordinarily large repertoire of P450 monooxygenases in its genome. However, the majority of these P450s have hitherto unknown function. Our initial studies using a genome-wide gene induction strategy revealed multiple P450s responsive to individual classes of xenobiotics. Here we report functional characterization of a cytochrome P450 monooxygenase, CYP5136A3 that showed common responsiveness and catalytic versatility towards endocrine-disrupting alkylphenols (APs) and mutagenic/carcinogenic polycyclic aromatic hydrocarbons (PAHs). Using recombinant CYP5136A3, we demonstrated its oxidation activity towards APs with varying alkyl side-chain length (C3-C9), in addition to PAHs (3β4 ring size). AP oxidation involves hydroxylation at the terminal carbon of the alkyl side-chain (Ο-oxidation). Structure-activity analysis based on a 3D model indicated a potential role of Trp129 and Leu324 in the oxidation mechanism of CYP5136A3. Replacing Trp129 with Leu (W129L) and Phe (W129F) significantly diminished oxidation of both PAHs and APs. The W129L mutation caused greater reduction in phenanthrene oxidation (80%) as compared to W129F which caused greater reduction in pyrene oxidation (88%). Almost complete loss of oxidation of C3-C8 APs (83β90%) was observed for the W129L mutation as compared to W129F (28β41%). However, the two mutations showed a comparable loss (60β67%) in C9-AP oxidation. Replacement of Leu324 with Gly (L324G) caused 42% and 54% decrease in oxidation activity towards phenanthrene and pyrene, respectively. This mutation also caused loss of activity towards C3-C8 APs (20β58%), and complete loss of activity toward nonylphenol (C9-AP). Collectively, the results suggest that Trp129 and Leu324 are critical in substrate recognition and/or regio-selective oxidation of PAHs and APs. To our knowledge, this is the first report on an AP-oxidizing P450 from fungi and on structure-activity relationship of a eukaryotic P450 for fused-ring PAHs (phenanthrene and pyrene) and AP substrates
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 organismsβ 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
The compound (3-{5-[(2,5-dimethoxyphenyl)amino]-1,3,4-thiadiazolidin-2-yl}-5,8-methoxy-2H-chromen-2-one) inhibits the prion protein conversion from PrPC to PrPSc with lower IC50 in ScN2a cells
Published ArticlePrion diseases are fatal neurodegenerative disorders of the central nervous system characterized by the
accumulation of a protease resistant form (PrPSc) of the cellular prion protein (PrPC) in the brain. Two
types of cellular prion (PrPC) compounds have been identified that appear to affect prion conversion
are known as Effective Binders (EBs) and Accelerators (ACCs). Effective binders shift the balance in favour
of PrPC, whereas Accelerators favour the formation of PrPSc. Molecular docking indicates EBs and ACCs
both bind to pocket-D of the SHaPrPC molecule. However, EBs and ACCs may have opposing effects on
the stability of the salt bridge between Arg156 and Glu196/Glu200. Computational docking data indicate
that the hydrophobic benzamide group of the EB, GFP23 and the 1-(3,3-dimethylcyclohexylidene)piperidinium
group of the ACC, GFP22 play an important role in inhibition and conversion from SHaPrPC to
SHaPrPSc, respectively. Experimentally, NMR confirmed the amide chemical shift perturbations observed
upon the binding of GFP23 to pocket-D of SHaPrPC. Consistent with its role as an ACC, titration of GFP22
resulted in widespread chemical shift changes and signal intensity loss due to protein unfolding. Virtual
screening of a ligand database using the molecular scaffold developed from the set of EBs identified six of
our compounds (previously studied using fluorescence quenching) as being among the top 100 best binders.
Among them, compounds 5 and 6 were found to be particularly potent in decreasing the accumulation
SHaPrPSc in ScN2a cells with an IC50 of 35 mM and 20 mM
The compound (3-{5-[(2,5-dimethoxyphenyl)amino]-1,3,4-thiadiazolidin-2-yl}-5,8-methoxy-2H-chromen-2-one) inhibits the prion protein conversion from PrPC to PrPSc with lower IC50 in ScN2a cells
Published ArticlePrion diseases are fatal neurodegenerative disorders of the central nervous system characterized by the
accumulation of a protease resistant form (PrPSc) of the cellular prion protein (PrPC) in the brain. Two
types of cellular prion (PrPC) compounds have been identified that appear to affect prion conversion
are known as Effective Binders (EBs) and Accelerators (ACCs). Effective binders shift the balance in favour
of PrPC, whereas Accelerators favour the formation of PrPSc. Molecular docking indicates EBs and ACCs
both bind to pocket-D of the SHaPrPC molecule. However, EBs and ACCs may have opposing effects on
the stability of the salt bridge between Arg156 and Glu196/Glu200. Computational docking data indicate
that the hydrophobic benzamide group of the EB, GFP23 and the 1-(3,3-dimethylcyclohexylidene)piperidinium
group of the ACC, GFP22 play an important role in inhibition and conversion from SHaPrPC to
SHaPrPSc, respectively. Experimentally, NMR confirmed the amide chemical shift perturbations observed
upon the binding of GFP23 to pocket-D of SHaPrPC. Consistent with its role as an ACC, titration of GFP22
resulted in widespread chemical shift changes and signal intensity loss due to protein unfolding. Virtual
screening of a ligand database using the molecular scaffold developed from the set of EBs identified six of
our compounds (previously studied using fluorescence quenching) as being among the top 100 best binders.
Among them, compounds 5 and 6 were found to be particularly potent in decreasing the accumulation
SHaPrPSc in ScN2a cells with an IC50 of 35 mM and 20 mM
Isolation and characterisation of endocrine disruptor nonylphenol-using bacteria from South Africa
Published ArticleEndocrine disrupting chemicals (EDCs) are synthetic chemicals that alter the function of endocrine systems in animals including humans. EDCs are considered priority pollutants and worldwide research is ongoing to develop bioremediation strategies to remove EDCs from the environment. An understanding of indigenous microorganisms is important to design efficient bioremediation strategies. However, much of the information available on EDCs has been generated from developed regions. Recent studies have revealed the presence of different EDCs in South African natural resources, but, to date, studies analysing the capabilities of microorganisms to utilise/degrade EDCs have not been reported from South Africa. Here, we report for the first time on the isolation and enrichment of six bacterial strains from six different soil samples collected from the Mpumalanga Province, which are capable of utilising EDC nonylphenol as a carbon source. Furthermore, we performed a preliminary characterisation of isolates concerning their phylogenetic identification and capabilities to degrade nonylphenol. Phylogenetic analysis using 16S rRNA gene sequencing revealed that four isolates belonged to Pseudomonas and the remaining two belonged to Enterobacteria and Stenotrophomonas. All six bacterial species showed degradation of nonylphenol in broth cultures, as HPLC analysis revealed 41β46% degradation of nonylphenol 12 h after addition. The results of this study represent the beginning of identification of microorganisms capable of degrading nonylphenol, and pave the way for further exploration of EDC-degrading microorganisms from South Africa
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)
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