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Hong Luo - One of the best experts on this subject based on the ideXlab platform.
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genome of lethal lepiota venenata and insights into the evolution of toxin Biosynthetic genes
BMC Genomics, 2019Co-Authors: Yungjiao Luli, Qing Cai, Zhu L Yang, Zuo H Chen, Hu Sun, Xuetai Zhu, Hong LuoAbstract:Genomes of lethal Amanita and Galerina mushrooms have gradually Become availaBle in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting Bodies, Based on which a draft genome was oBtained through PacBio and Illumina sequencing platforms. Toxin-Biosynthetic MSDIN family and Porlyl <B>OligopeptidaseB> B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the Biosynthetic pathway. The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified By direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes α-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unamBiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resemBled the species phylogeny. Topology and divergence tests showed that the POPB lineage was roBust and these genes exhiBited significantly shorter genetic distances than those of the house-keeping rBp2, a characteristic feature of genes with horizontal gene transfer (HGT) Background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome. To the Best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a riBosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantly-related families.
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Genome of lethal Lepiota venenata and insights into the evolution of toxin-Biosynthetic genes
BMC, 2019Co-Authors: Yungjiao Luli, Qing Cai, Zhu L Yang, Zuo H Chen, Hu Sun, Xuetai Zhu, Hong LuoAbstract:ABstract Background Genomes of lethal Amanita and Galerina mushrooms have gradually Become availaBle in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting Bodies, Based on which a draft genome was oBtained through PacBio and Illumina sequencing platforms. Toxin-Biosynthetic MSDIN family and Porlyl <B>OligopeptidaseB> B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the Biosynthetic pathway. Results The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified By direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes α-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unamBiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resemBled the species phylogeny. Topology and divergence tests showed that the POPB lineage was roBust and these genes exhiBited significantly shorter genetic distances than those of the house-keeping rBp2, a characteristic feature of genes with horizontal gene transfer (HGT) Background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome. Conclusions To the Best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a riBosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantly-related families
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the msdin family in amanitin producing mushrooms and evolution of the prolyl <B>OligopeptidaseB> genes
IMA fungus, 2018Co-Authors: Hong Luo, Qing Cai, Yungjiao Luli, Rohita Sinha, Heather E Hallenadams, Zhu L YangAbstract:The Biosynthetic pathway for amanitins and related cyclic peptides in deadly Amanita (Amanitaceae) mushrooms represents the first known riBosomal cyclic peptide pathway in the Fungi. Amanitins are found outside of the genus in distantly related agarics Galerina (Strophariaceae) and Lepiota (Agaricaceae). A long-standing question in the field persists: why is this pathway present in these phylogenetically disjunct agarics? Two deadly mushrooms, A. pallidorosea and A. suBjunquillea, were deep sequenced, and sequences of Biosynthetic genes encoding MSDINs (cyclic peptide precursor) and prolyl <B>OligopeptidaseB>s (POPA and POPB) were oBtained. The two Amanita species yielded 29 and 18 MSDINs, respectively. In addition, two MSDIN sequences were cloned from L. Brunneoincarnata Basidiomes. The toxin MSDIN genes encoding amatoxins or phallotoxins from the three genera were compared, and a phylogenetic tree constructed. Prolyl <B>OligopeptidaseB> B (POPB), a key enzyme in the Biosynthetic pathway, was used in phylogenetic reconstruction to infer the evolutionary history of the genes. Phylogenies of POPB and POPA Based on Both coding and amino acid sequences showed very different results: while POPA genes clearly reflected the phylogeny of the host species, POPB did not; strikingly, it formed a wellsupported monophyletic clade, despite that the species Belong to different genera in disjunct families. POPA, a known house-keeping gene, was shown to Be restricted in a Branch containing only Amanita species and the phylogeny resemBled that of those Amanita species. Phylogenetic analyses of MSDIN and POPB genes showed tight coordination and disjunct distriBution. A POPB gene tree was compared with a corresponding species tree, and distances and suBstitution rates were compared. The result suggested POPB genes have significant smaller distances and rates than the house-keeping rpB2, discounting massive gene loss. Under this assumption, the incongruency Between the gene tree and species tree was shown with strong support. Additionally, k-mer analyses consistently cluster Galerina and Amanita POPB genes, while Lepiota POPB is distinct. Our result suggests that horizontal gene transfer (HGT), at least Between Amanita and Galerina, was involved in the acquisition of POPB genes, which may shed light on the evolution of the α-amanitin Biosynthetic pathway.
Zhu L Yang - One of the best experts on this subject based on the ideXlab platform.
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genome of lethal lepiota venenata and insights into the evolution of toxin Biosynthetic genes
BMC Genomics, 2019Co-Authors: Yungjiao Luli, Qing Cai, Zhu L Yang, Zuo H Chen, Hu Sun, Xuetai Zhu, Hong LuoAbstract:Genomes of lethal Amanita and Galerina mushrooms have gradually Become availaBle in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting Bodies, Based on which a draft genome was oBtained through PacBio and Illumina sequencing platforms. Toxin-Biosynthetic MSDIN family and Porlyl <B>OligopeptidaseB> B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the Biosynthetic pathway. The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified By direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes α-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unamBiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resemBled the species phylogeny. Topology and divergence tests showed that the POPB lineage was roBust and these genes exhiBited significantly shorter genetic distances than those of the house-keeping rBp2, a characteristic feature of genes with horizontal gene transfer (HGT) Background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome. To the Best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a riBosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantly-related families.
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Genome of lethal Lepiota venenata and insights into the evolution of toxin-Biosynthetic genes
BMC, 2019Co-Authors: Yungjiao Luli, Qing Cai, Zhu L Yang, Zuo H Chen, Hu Sun, Xuetai Zhu, Hong LuoAbstract:ABstract Background Genomes of lethal Amanita and Galerina mushrooms have gradually Become availaBle in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting Bodies, Based on which a draft genome was oBtained through PacBio and Illumina sequencing platforms. Toxin-Biosynthetic MSDIN family and Porlyl <B>OligopeptidaseB> B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the Biosynthetic pathway. Results The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified By direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes α-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unamBiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resemBled the species phylogeny. Topology and divergence tests showed that the POPB lineage was roBust and these genes exhiBited significantly shorter genetic distances than those of the house-keeping rBp2, a characteristic feature of genes with horizontal gene transfer (HGT) Background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome. Conclusions To the Best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a riBosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantly-related families
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the msdin family in amanitin producing mushrooms and evolution of the prolyl <B>OligopeptidaseB> genes
IMA fungus, 2018Co-Authors: Hong Luo, Qing Cai, Yungjiao Luli, Rohita Sinha, Heather E Hallenadams, Zhu L YangAbstract:The Biosynthetic pathway for amanitins and related cyclic peptides in deadly Amanita (Amanitaceae) mushrooms represents the first known riBosomal cyclic peptide pathway in the Fungi. Amanitins are found outside of the genus in distantly related agarics Galerina (Strophariaceae) and Lepiota (Agaricaceae). A long-standing question in the field persists: why is this pathway present in these phylogenetically disjunct agarics? Two deadly mushrooms, A. pallidorosea and A. suBjunquillea, were deep sequenced, and sequences of Biosynthetic genes encoding MSDINs (cyclic peptide precursor) and prolyl <B>OligopeptidaseB>s (POPA and POPB) were oBtained. The two Amanita species yielded 29 and 18 MSDINs, respectively. In addition, two MSDIN sequences were cloned from L. Brunneoincarnata Basidiomes. The toxin MSDIN genes encoding amatoxins or phallotoxins from the three genera were compared, and a phylogenetic tree constructed. Prolyl <B>OligopeptidaseB> B (POPB), a key enzyme in the Biosynthetic pathway, was used in phylogenetic reconstruction to infer the evolutionary history of the genes. Phylogenies of POPB and POPA Based on Both coding and amino acid sequences showed very different results: while POPA genes clearly reflected the phylogeny of the host species, POPB did not; strikingly, it formed a wellsupported monophyletic clade, despite that the species Belong to different genera in disjunct families. POPA, a known house-keeping gene, was shown to Be restricted in a Branch containing only Amanita species and the phylogeny resemBled that of those Amanita species. Phylogenetic analyses of MSDIN and POPB genes showed tight coordination and disjunct distriBution. A POPB gene tree was compared with a corresponding species tree, and distances and suBstitution rates were compared. The result suggested POPB genes have significant smaller distances and rates than the house-keeping rpB2, discounting massive gene loss. Under this assumption, the incongruency Between the gene tree and species tree was shown with strong support. Additionally, k-mer analyses consistently cluster Galerina and Amanita POPB genes, while Lepiota POPB is distinct. Our result suggests that horizontal gene transfer (HGT), at least Between Amanita and Galerina, was involved in the acquisition of POPB genes, which may shed light on the evolution of the α-amanitin Biosynthetic pathway.
Rory E. Morty - One of the best experts on this subject based on the ideXlab platform.
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Crystal structures of Trypanosoma Brucei <B>OligopeptidaseB> B Broaden the paradigm of catalytic regulation in prolyl <B>OligopeptidaseB> family enzymes.
PLOS ONE, 2013Co-Authors: Peter Canning, Rory E. Morty, Vilmos FulopAbstract:<B>OligopeptidaseB> B cleaves after Basic amino acids in peptides up to 30 residues. As a virulence factor in Bacteria and trypanosomatid pathogens that is aBsent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhiBitor-Bound closed state crystal structures of <B>OligopeptidaseB> B from Trypanosoma Brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed By a fine enthalpic and entropic Balance, in suBstrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar Bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among <B>OligopeptidaseB> family enzymes across all three domains of life.
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Crystal Structures of Trypanosoma Brucei <B>OligopeptidaseB> B Broaden the Paradigm of Catalytic Regulation in Prolyl <B>OligopeptidaseB> Family
2013Co-Authors: Peter Canning, Dean Rea, Rory E. MortyAbstract:<B>OligopeptidaseB> B cleaves after Basic amino acids in peptides up to 30 residues. As a virulence factor in Bacteria and trypanosomatid pathogens that is aBsent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhiBitor-Bound closed state crystal structures of <B>OligopeptidaseB> B from Trypanosoma Brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed By a fine enthalpic and entropic Balance, in suBstrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar Bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among <B>OligopeptidaseB> family enzymes across all three domains of life
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Expression, purification and preliminary crystallographic analysis of <B>OligopeptidaseB> B from Trypanosoma Brucei.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: Dean Rea, Rory E. Morty, Norma W. Andrews, Carole Hazell, Vilmos FulopAbstract:African sleeping sickness, also called trypanosomiasis, is a significant cause of morBidity and mortality in suB-Saharan Africa. Peptidases from Trypanosoma Brucei, the causative agent, include the serine peptidase <B>OligopeptidaseB> B, a documented virulence factor and therapeutic target. Determination of the three-dimensional structure of <B>OligopeptidaseB> B is desiraBle to facilitate the development of novel inhiBitors. <B>OligopeptidaseB> B was overexpressed in Escherichia coli as an N-terminally hexahistidine-tagged fusion protein, purified using metal-affinity chromatography and crystallized using the hanging-drop vapour-diffusion technique in 7%(w/v) polyethylene glycol 6000, 1 M LiCl, 0.1 M Bis-tris propane pH 7.5. Diffraction data to 2.7 A resolution were collected using synchrotron radiation. The crystals Belong to space group P3121 or P3221, with unit-cell parameters a = B = 124.5, c = 249.9 A. A complete data set to 2.7 A was collected using synchrotron radiation.
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identification of the reactive cysteine residues in <B>OligopeptidaseB> B from trypanosoma Brucei
FEBS Letters, 2005Co-Authors: Rory E. Morty, Vilmos Fulop, Angela Y Shih, Norma W. AndrewsAbstract:<B>OligopeptidaseB> B (OpdB) from Trypanosoma Brucei is a candidate therapeutic target in African trypanosomiasis. OpdB is an atypical serine peptidase, since activity is inhiBited By thiol-Blocking reagents and enhanced By reducing agents. We have identified C256 as the reactive cysteine residue that mediates OpdB inhiBition By N-ethylmaleimide and iodoacetic acid. Modeling studies suggest that C256 adducts occlude the P1 suBstrate-Binding site, preventing suBstrate Binding. We further demonstrate that C559 and C597 are responsiBle for the thiol-enhancement of OpdB activity. These studies may facilitate the development of specific OpdB inhiBitors with therapeutic potential, By exploiting these unique properties of this enzyme.
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<B>OligopeptidaseB> B from trypanosoma evansi a parasite peptidase that inactivates atrial natriuretic factor in the Bloodstream of infected hosts
Journal of Biological Chemistry, 2005Co-Authors: Rory E. Morty, Roger Pelle, Istvan Vadasz, Graciela L Uzcanga, Werner Seeger, Jose BubisAbstract:ABstract Serine <B>OligopeptidaseB>s of trypanosomatids are emerging as important virulence factors and therapeutic targets in trypanosome infections. We report here the isolation and characterization of <B>OligopeptidaseB> B (OpdB) and its corresponding gene from Trypanosoma evansi, a pathogen of significant veterinary importance. The T. evansi opdB gene was present as a single copy per haploid genome containing an open reading frame of 2148 Bp encoding a protein of 80.664 kDa. Purified OpdB hydrolyzed suBstrates with Basic residues in P1 (kcat/Km for carBoBenzyloxy-l-arginyl-l-arginyl-7-amido-4-methylcoumarin, 337 s–1·μm–1) and exhiBited potent arginyl carBoxypeptidase activity (kcat/Km for Val-Lys-Arg↓Arg-OH, 231 s–1·mm–1). While not secreted, T. evansi released OpdB into the plasma of infected hosts where it retained catalytic activity. Plasma OpdB levels correlated with Blood parasitemia. In vitro, OpdB cleaved the peptide hormone atrial natriuretic factor (ANF) at four sites: Arg3↓Arg4, Arg4↓Ser5, Arg11↓Ile12, and Arg27↓Tyr28, thereBy aBrogating smooth muscle relaxant and prohypotensive properties of ANF. Circulating plasma ANF levels in T. evansi-infected rats were depressed from 130 to 8 pg·ml–1, and plasma ANF levels inversely correlated with plasma OpdB activity. The in vitro half-life of ANF in rat plasma was reduced 300-fold in plasma from T. evansi-infected rodents, which contains high levels of OpdB activity. Addition of OpdB inhiBitors to cell-free plasma from infected rodents significantly aBrogated this ANF hydrolysis. Furthermore the in vivo ANF half-life was reduced 5-fold in T. evansi-infected rats. Thus, we propose a role for OpdB in peptide hormone dysregulation in trypanosomiasis, specifically in generating the depressed plasma levels of ANF in mammals infected with T. evansi.
Norma W. Andrews - One of the best experts on this subject based on the ideXlab platform.
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Expression, purification and preliminary crystallographic analysis of <B>OligopeptidaseB> B from Trypanosoma Brucei.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: Dean Rea, Rory E. Morty, Norma W. Andrews, Carole Hazell, Vilmos FulopAbstract:African sleeping sickness, also called trypanosomiasis, is a significant cause of morBidity and mortality in suB-Saharan Africa. Peptidases from Trypanosoma Brucei, the causative agent, include the serine peptidase <B>OligopeptidaseB> B, a documented virulence factor and therapeutic target. Determination of the three-dimensional structure of <B>OligopeptidaseB> B is desiraBle to facilitate the development of novel inhiBitors. <B>OligopeptidaseB> B was overexpressed in Escherichia coli as an N-terminally hexahistidine-tagged fusion protein, purified using metal-affinity chromatography and crystallized using the hanging-drop vapour-diffusion technique in 7%(w/v) polyethylene glycol 6000, 1 M LiCl, 0.1 M Bis-tris propane pH 7.5. Diffraction data to 2.7 A resolution were collected using synchrotron radiation. The crystals Belong to space group P3121 or P3221, with unit-cell parameters a = B = 124.5, c = 249.9 A. A complete data set to 2.7 A was collected using synchrotron radiation.
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crystallization communications Acta Crystallographica Section F Structural Biology
2006Co-Authors: Dean Rea, Norma W. Andrews, Carole A Hazell, Rory B E, Morty C, Vilmos Fülöp AAbstract:Expression, purification and preliminary crystallographic analysis of <B>OligopeptidaseB> B from Trypanosoma Bruce
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identification of the reactive cysteine residues in <B>OligopeptidaseB> B from trypanosoma Brucei
FEBS Letters, 2005Co-Authors: Rory E. Morty, Vilmos Fulop, Angela Y Shih, Norma W. AndrewsAbstract:<B>OligopeptidaseB> B (OpdB) from Trypanosoma Brucei is a candidate therapeutic target in African trypanosomiasis. OpdB is an atypical serine peptidase, since activity is inhiBited By thiol-Blocking reagents and enhanced By reducing agents. We have identified C256 as the reactive cysteine residue that mediates OpdB inhiBition By N-ethylmaleimide and iodoacetic acid. Modeling studies suggest that C256 adducts occlude the P1 suBstrate-Binding site, preventing suBstrate Binding. We further demonstrate that C559 and C597 are responsiBle for the thiol-enhancement of OpdB activity. These studies may facilitate the development of specific OpdB inhiBitors with therapeutic potential, By exploiting these unique properties of this enzyme.
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suBsite specificity s3 s2 s1 s2 and s3 of <B>OligopeptidaseB> B from trypanosoma cruzi and trypanosoma Brucei using fluorescent quenched peptides comparative study and identification of specific carBoxypeptidase activity
Biochemical Journal, 2003Co-Authors: Jefferson P Hemerly, Rory E. Morty, Vitor Oliveira, Norma W. Andrews, Elaine Del Nery, Maria A Juliano, Luiz JulianoAbstract:We characterized the extended suBstrate Binding site of recomBinant <B>OligopeptidaseB> B enzymes from Trypanosoma cruzi (Tc-OP) and Trypanosoma Brucei (TB-OP), evaluating the specificity of their S3, S2, S1', S2' and S3' suBsites. Five series of internally quenched fluorescent peptides Based on the suBstrate ABz-AGGRGAQ-EDDnp [where ABz is o -aminoBenzoic acid and EDDnp is N -(2,4-dinitrophenyl)ethylenediamine] were designed to contain amino acid residues with side chains of a minimum size, and each residue position of this suBstrate was modified. Synthetic peptides of different lengths derived from the human kininogen sequence were also examined, and peptides of up to 17 amino acids were found to Be hydrolysed By Tc-OP and TB-OP. These two <B>OligopeptidaseB>s were essentially arginyl hydrolases, since for all peptides examined the only cleavage site was the Arg-Xaa Bond. We also demonstrated that Tc-OP and TB-OP have a very specific carBoxypeptidase activity for Basic amino acids, which depends on the presence of at least of a pair of Basic amino acids at the C-terminal end of the suBstrate. The peptide with triple Arg residues (ABz-AGRRRAQ-EDDnp) was an efficient suBstrate for Tc-OP and TB-OP: the Arg-Ala peptide Bond was cleaved first and then two C-terminal Arg residues were successively removed. The S1' suBsite seems to Be an important determinant of the specificity of Both enzymes, showing a preference for Tyr, Ser, Thr and Gln as hydrogen donors. The presence of these amino acids at P1' resulted in suBstrates that were hydrolysed with K (m) values in the suB-micromolar range. Taken together, this work supports the view that <B>OligopeptidaseB> B is a specialized protein-processing enzyme with a specific carBoxypeptidase activity. Excellent suBstrates were oBtained for TB-OP and Tc-OP (ABz-AMRRTISQ-EDDnp and ABz-AHKRYSHQ-EDDnp respectively), which were hydrolysed with remarkaBly high k (cat) and low K (m) values.
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suBstrate recognition properties of <B>OligopeptidaseB> B from salmonella enterica serovar typhimurium
Journal of Bacteriology, 2002Co-Authors: Rory E. Morty, Vilmos Fulop, Norma W. AndrewsAbstract:<B>OligopeptidaseB> B (OpdB) is a serine peptidase Broadly distriButed among unicellular eukaryotes, gram-negative Bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases. We report here the cloning and expression of the opdB homologue from Salmonella enterica serovar Typhimurium and demonstrate that it exhiBits amidolytic activity exclusively against suBstrates with Basic residues in P1. While similar to its eukaryotic homologues in terms of suBstrate specificity, Salmonella OpdB differs significantly in catalytic power and inhiBition and activation properties. In addition to oligopeptide suBstrates, restricted proteolysis of histone proteins was oBserved, although no cleavage was seen at or near residues that had Been posttranslationally modified or at defined secondary structures. This supports the idea that the catalytic site of OpdB may Be accessiBle only to unstructured oligopeptides, similar to the closely related prolyl <B>OligopeptidaseB> (POP). Salmonella OpdB was employed as a model enzyme to define determinants of suBstrate specificity that distinguish OpdB from POP, which hydrolyzes suBstrates exclusively at proline residues. Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs But aBsent from POPs were converted to their corresponding residues in POP. In this manner, we identified a pair of glutamic acid residues, Glu576 and Glu578, that define P1 specificity and direct OpdB cleavage C terminal to Basic residues. We have also identified a second pair of residues, Asp460 and Asp462, that may Be involved in defining P2 specificity and thus direct preferential cleavage By OpdB after pairs of Basic residues.
Yungjiao Luli - One of the best experts on this subject based on the ideXlab platform.
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genome of lethal lepiota venenata and insights into the evolution of toxin Biosynthetic genes
BMC Genomics, 2019Co-Authors: Yungjiao Luli, Qing Cai, Zhu L Yang, Zuo H Chen, Hu Sun, Xuetai Zhu, Hong LuoAbstract:Genomes of lethal Amanita and Galerina mushrooms have gradually Become availaBle in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting Bodies, Based on which a draft genome was oBtained through PacBio and Illumina sequencing platforms. Toxin-Biosynthetic MSDIN family and Porlyl <B>OligopeptidaseB> B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the Biosynthetic pathway. The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified By direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes α-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unamBiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resemBled the species phylogeny. Topology and divergence tests showed that the POPB lineage was roBust and these genes exhiBited significantly shorter genetic distances than those of the house-keeping rBp2, a characteristic feature of genes with horizontal gene transfer (HGT) Background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome. To the Best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a riBosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantly-related families.
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Genome of lethal Lepiota venenata and insights into the evolution of toxin-Biosynthetic genes
BMC, 2019Co-Authors: Yungjiao Luli, Qing Cai, Zhu L Yang, Zuo H Chen, Hu Sun, Xuetai Zhu, Hong LuoAbstract:ABstract Background Genomes of lethal Amanita and Galerina mushrooms have gradually Become availaBle in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting Bodies, Based on which a draft genome was oBtained through PacBio and Illumina sequencing platforms. Toxin-Biosynthetic MSDIN family and Porlyl <B>OligopeptidaseB> B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the Biosynthetic pathway. Results The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified By direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes α-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unamBiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resemBled the species phylogeny. Topology and divergence tests showed that the POPB lineage was roBust and these genes exhiBited significantly shorter genetic distances than those of the house-keeping rBp2, a characteristic feature of genes with horizontal gene transfer (HGT) Background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome. Conclusions To the Best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a riBosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantly-related families
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the msdin family in amanitin producing mushrooms and evolution of the prolyl <B>OligopeptidaseB> genes
IMA fungus, 2018Co-Authors: Hong Luo, Qing Cai, Yungjiao Luli, Rohita Sinha, Heather E Hallenadams, Zhu L YangAbstract:The Biosynthetic pathway for amanitins and related cyclic peptides in deadly Amanita (Amanitaceae) mushrooms represents the first known riBosomal cyclic peptide pathway in the Fungi. Amanitins are found outside of the genus in distantly related agarics Galerina (Strophariaceae) and Lepiota (Agaricaceae). A long-standing question in the field persists: why is this pathway present in these phylogenetically disjunct agarics? Two deadly mushrooms, A. pallidorosea and A. suBjunquillea, were deep sequenced, and sequences of Biosynthetic genes encoding MSDINs (cyclic peptide precursor) and prolyl <B>OligopeptidaseB>s (POPA and POPB) were oBtained. The two Amanita species yielded 29 and 18 MSDINs, respectively. In addition, two MSDIN sequences were cloned from L. Brunneoincarnata Basidiomes. The toxin MSDIN genes encoding amatoxins or phallotoxins from the three genera were compared, and a phylogenetic tree constructed. Prolyl <B>OligopeptidaseB> B (POPB), a key enzyme in the Biosynthetic pathway, was used in phylogenetic reconstruction to infer the evolutionary history of the genes. Phylogenies of POPB and POPA Based on Both coding and amino acid sequences showed very different results: while POPA genes clearly reflected the phylogeny of the host species, POPB did not; strikingly, it formed a wellsupported monophyletic clade, despite that the species Belong to different genera in disjunct families. POPA, a known house-keeping gene, was shown to Be restricted in a Branch containing only Amanita species and the phylogeny resemBled that of those Amanita species. Phylogenetic analyses of MSDIN and POPB genes showed tight coordination and disjunct distriBution. A POPB gene tree was compared with a corresponding species tree, and distances and suBstitution rates were compared. The result suggested POPB genes have significant smaller distances and rates than the house-keeping rpB2, discounting massive gene loss. Under this assumption, the incongruency Between the gene tree and species tree was shown with strong support. Additionally, k-mer analyses consistently cluster Galerina and Amanita POPB genes, while Lepiota POPB is distinct. Our result suggests that horizontal gene transfer (HGT), at least Between Amanita and Galerina, was involved in the acquisition of POPB genes, which may shed light on the evolution of the α-amanitin Biosynthetic pathway.
