Abstract:Sterol 14 ␣ -demethylase (14DM) is a cytochrome P-450 involved in sterol biosynthesis in eukaryotes. It was reported that Mycobacterium smegmatis also makes cholesterol and that cholesterol is essential to Mycobacterium tuberculosis (MT) infection, although the origin of the cholesterol is unknown. A protein product from MT having about 30% sequence identity with eukaryotic 14 ␣ -demethylases has been found to convert sterols to their 14-demethyl products indicating that a sterol pathway might exist in MT. To … Show more
“…Increase in the turnover number for A197G is by a factor of 7. The calculated rate of LS demethylation of the WT protein (0.14 min -1 ) is within the range of values measured previously toward DHL (30) or obtusifoliol (42). Taking into consideration the low efficiency of enzymatic reduction of MT-CYP51 and destabilization of the reduced state observed for several mutants in an attempt to detect residual activity, we varied the experimental conditions for the mutants with no detectable LS demethylation.…”
Section: Resultssupporting
confidence: 79%
“…The 10 conserved amino acids throughout the whole CYP51 family in the B‘ helix/BC loop and helices F and G are seen in the alignment in Figure . 1 Sequence alignment of 44 CYP51 family members from different biological kingdoms [bacteria ( − ), mycetozoa (), plants ( − ), fungi ( − ), and animals ( − )] in the regions of the B‘ helix/BC loop and helices F and G. The sequences were taken from NCBA, SWISS-PROT, TrEMBL, and TIGR databases. The alignment was performed using Clustal W1.81 and prepared in ESPript 2.0 programs.…”
Section: Resultsmentioning
confidence: 99%
“…In the region of SRS1 (B′ helix/BC loop), V87A is not expressed in the P450 form while the expression level and activity of the V87I mutant are comparable to WT. Substitu- 5), plants (6)(7)(8)(9)(10)(11)(12)(13)(14)(15), fungi , and animals (39)(40)(41)(42)(43)(44)] in the regions of the B′ helix/BC loop and helices F and G. The sequences were taken from NCBA, SWISS-PROT, TrEMBL, and TIGR databases. The alignment was performed using Clustal W1.81 and prepared in ESPript 2.0 programs.…”
Section: Resultsmentioning
confidence: 99%
“…Together with the unchanged affinity (apparent K d ), it points out that neither of these residues participates in substrate binding directly but their substitutions affect orientation or the amount of the bound substrate. Because of low solubility of the sterols the fraction of the P450 molecules, which change spin state upon titration with the substrates in vitro in the CYP51 family usually, does not exceed 10% (42,43,49). Using HPCD to dissolve the sterols allowed us to reach 18% spin state transition for WT.…”
CYP51 (sterol 14 alpha-demethylase) is an essential enzyme in sterol biosynthetic pathways and the only P450 gene family having catalytically identical orthologues in different biological kingdoms. The proteins have low sequence similarity across phyla, and the whole family contains about 40 completely conserved amino acid residues. Fifteen of these residues lie in the secondary structural elements predicted to form potential substrate recognition sites within the P450 structural fold. The role of 10 of these residues, in the B' helix/BC loop, helices F and G, has been studied by site-directed mutagenesis using as a template the soluble sterol 14 alpha-demethylase of known structure, CYP51 from Mycobacterium tuberculosis (MT) and the human orthologue. Single amino acid substitutions of seven residues (Y76, F83, G84, D90, L172, G175, and R194) result in loss of the ability of the mutant MTCYP51 to metabolize lanosterol. Residual activity of D195A is very low, V87A is not expressed as a P450, and A197G has almost 1 order of magnitude increased activity. After purification, all of the mutants show normal spectral properties, heme incorporation, and the ability to be reduced enzymatically and to interact with azole inhibitors. Profound influence on the catalytic activity correlates well with the spectral response to substrate binding, effect of substrate stabilization on the reduced state of the P450, and substrate-enhanced efficiency of enzymatic reduction. Mutagenesis of corresponding residues in human CYP51 implies that the conserved amino acids might be essential for the evolutionary conservation of sterol 14 alpha-demethylation from bacteria to mammals.
“…Increase in the turnover number for A197G is by a factor of 7. The calculated rate of LS demethylation of the WT protein (0.14 min -1 ) is within the range of values measured previously toward DHL (30) or obtusifoliol (42). Taking into consideration the low efficiency of enzymatic reduction of MT-CYP51 and destabilization of the reduced state observed for several mutants in an attempt to detect residual activity, we varied the experimental conditions for the mutants with no detectable LS demethylation.…”
Section: Resultssupporting
confidence: 79%
“…The 10 conserved amino acids throughout the whole CYP51 family in the B‘ helix/BC loop and helices F and G are seen in the alignment in Figure . 1 Sequence alignment of 44 CYP51 family members from different biological kingdoms [bacteria ( − ), mycetozoa (), plants ( − ), fungi ( − ), and animals ( − )] in the regions of the B‘ helix/BC loop and helices F and G. The sequences were taken from NCBA, SWISS-PROT, TrEMBL, and TIGR databases. The alignment was performed using Clustal W1.81 and prepared in ESPript 2.0 programs.…”
Section: Resultsmentioning
confidence: 99%
“…In the region of SRS1 (B′ helix/BC loop), V87A is not expressed in the P450 form while the expression level and activity of the V87I mutant are comparable to WT. Substitu- 5), plants (6)(7)(8)(9)(10)(11)(12)(13)(14)(15), fungi , and animals (39)(40)(41)(42)(43)(44)] in the regions of the B′ helix/BC loop and helices F and G. The sequences were taken from NCBA, SWISS-PROT, TrEMBL, and TIGR databases. The alignment was performed using Clustal W1.81 and prepared in ESPript 2.0 programs.…”
Section: Resultsmentioning
confidence: 99%
“…Together with the unchanged affinity (apparent K d ), it points out that neither of these residues participates in substrate binding directly but their substitutions affect orientation or the amount of the bound substrate. Because of low solubility of the sterols the fraction of the P450 molecules, which change spin state upon titration with the substrates in vitro in the CYP51 family usually, does not exceed 10% (42,43,49). Using HPCD to dissolve the sterols allowed us to reach 18% spin state transition for WT.…”
CYP51 (sterol 14 alpha-demethylase) is an essential enzyme in sterol biosynthetic pathways and the only P450 gene family having catalytically identical orthologues in different biological kingdoms. The proteins have low sequence similarity across phyla, and the whole family contains about 40 completely conserved amino acid residues. Fifteen of these residues lie in the secondary structural elements predicted to form potential substrate recognition sites within the P450 structural fold. The role of 10 of these residues, in the B' helix/BC loop, helices F and G, has been studied by site-directed mutagenesis using as a template the soluble sterol 14 alpha-demethylase of known structure, CYP51 from Mycobacterium tuberculosis (MT) and the human orthologue. Single amino acid substitutions of seven residues (Y76, F83, G84, D90, L172, G175, and R194) result in loss of the ability of the mutant MTCYP51 to metabolize lanosterol. Residual activity of D195A is very low, V87A is not expressed as a P450, and A197G has almost 1 order of magnitude increased activity. After purification, all of the mutants show normal spectral properties, heme incorporation, and the ability to be reduced enzymatically and to interact with azole inhibitors. Profound influence on the catalytic activity correlates well with the spectral response to substrate binding, effect of substrate stabilization on the reduced state of the P450, and substrate-enhanced efficiency of enzymatic reduction. Mutagenesis of corresponding residues in human CYP51 implies that the conserved amino acids might be essential for the evolutionary conservation of sterol 14 alpha-demethylation from bacteria to mammals.
“…The CYP51 family is a substrate-specific enzyme that catalyses the 14α-demethylation of sterols (Ortiz de Montellano, 2005). In Mtb, CYP51B1 catalyses the conversion of lanosterol and closely related 14αmethyl sterols to 14α-desmethyl derivatives (Bellamine et al, 1999;Bellamine et al, 2001;. Whether CYP51B1 is a suitable target for drug development or not is still questionable because Mtb does not have a functional sterol biosynthetic pathway as the genes coding for squalene epoxidase and oxidosqualene cyclase, two key enzymes required for construction of the sterol skeleton, are absent in Mtb, in addition to the fact that CYP51B1 is not required for mycobacterial growth (Sassetti et al, 2003;Sassetti et al, 2001;McLean et al, 2010).…”
Tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent, ranking above HIV/AIDS with 6.3 million new cases of TB reported in 2016. TB is an air-borne disease associated with the aerobic bacterium Mycobacterium tuberculosis (Mtb), which mainly infects the lungs. Aerosolization of diseased pulmonary secretions, by coughing, sneezing and speaking, discharge the Mtb bacilli into the atmosphere. Infected aerosol droplet nuclei sized 1-μm are largely trapped in the upper nasal passages or are expelled into the pharynx by the mucociliary mechanism of the lower respiratory tract and are harmlessly swallowed and digested. Infected persons may overcome the initial TB infection, resulting in the development of asymptomatic latent TB. About 10% of individuals may develop the active disease after infection; where the bacteria undergo more rapid growth and overcome the host immune system. In cases of multi-drug resistant (MDR) strains, and extreme drug-resistant (XDR) strains, treatment fails, and the bacteria propagate and attack the host, leading to death from systemic infection. Due to the increased spread of TB worldwide, both the academic and industrial communities have initiated intensive research to develop new therapeutics targeting new enzymes such as cytochrome P450s in Mtb.
The primary diversification of eukaryotes involved protozoa, especially zooflagellates-flagellate protozoa without plastids. Understanding the origins of the higher eukaryotic kingdoms (two purely heterotrophic, Animalia and Fungi, and two primarily photosynthetic, Plantae and Chromista) depends on clarifying evolutionary relationships among the phyla of the ancestral kingdom Protozoa. We therefore sequenced 18S rRNA genes from 10 strains from the protozoan phyla Choanozoa and Apusozoa. Eukaryote diversity is encompassed by three early-radiating, arguably monophyletic groups: Amoebozoa, opisthokonts, and bikonts. Our taxon-rich rRNA phylogeny for eukaryotes allowing for intersite rate variation strongly supports the opisthokont clade (animals, Choanozoa, Fungi). It agrees with the view that Choanozoa are sisters of or ancestral to animals and reveals a novel nonflagellate choanozoan lineage, Ministeriida, sister either to choanoflagellates, traditionally considered animal ancestors, or to animals. Maximum likelihood trees suggest that within animals Placozoa are derived from medusozoan Cnidaria (we therefore place Placozoa as a class within subphylum Medusozoa of the Cnidaria) and hexactinellid sponges evolved from demosponges. The bikont and amoebozoan radiations are both very ill resolved. Bikonts comprise the kingdoms Plantae and Chromista and three major protozoan groups: alveolates, excavates, and Rhizaria. Our analysis weakly suggests that Apusozoa, represented by Ancyromonas and the apusomonads ( Apusomonas and the highly diverse and much more ancient genus Amastigomonas, from which it evolved), are not closely related to other Rhizaria and may be the most divergent bikont lineages. Although Ancyromonas and apusomonads appear deeply divergent in 18S rRNA trees, the trees neither refute nor support the monophyly of Apusozoa. The bikont phylum Cercozoa weakly but consistently appears as sister to Retaria (Foraminifera; Radiolaria), together forming a hitherto largely unrecognized major protozoan assemblage (core Rhizaria) in the eukaryote tree. Both 18S rRNA sequence trees and a rare deletion show that nonciliate haplosporidian and paramyxid parasites of shellfish (together comprising the Ascetosporea) are not two separate phyla, as often thought, but part of the Cercozoa, and may be related to the plant-parasitic plasmodiophorids and phagomyxids, which were originally the only parasites included in the Cercozoa. We discuss rRNA trees in relation to other evidence concerning the basal diversification and root of the eukaryotic tree and argue that bikonts and opisthokonts, at least, are holophyletic. Amoebozoa and bikonts may be sisters-jointly called anterokonts, as they ancestrally had an anterior cilium, not a posterior one like opisthokonts; this contrasting ciliary orientation may reflect a primary divergence in feeding mode of the first eukaryotes. Anterokonts also differ from opisthokonts in sterol biosynthesis (cycloartenol versus lanosterol pathway), major exoskeletal polymers (cellulose versus chiti...
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