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Paul J Luzio - One of the best experts on this subject based on the ideXlab platform.
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endolysosomes are the principal intracellular sites of acid Hydrolase activity
Current Biology, 2016Co-Authors: Nicholas A Bright, Luther J. Davis, Paul J LuzioAbstract:The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid Hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid Hydrolase active. We found that endolysosomes are the principal organelles in which acid Hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid Hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid Hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-Hydrolase-active endolysosomes and acid-Hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-Hydrolase-active, acidic endolysosomes and acid Hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle.
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endolysosomes are the principal intracellular sites of acid Hydrolase activity
Current Biology, 2016Co-Authors: Nicholas A Bright, Luther J. Davis, Paul J LuzioAbstract:The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid Hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid Hydrolase active. We found that endolysosomes are the principal organelles in which acid Hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid Hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid Hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-Hydrolase-active endolysosomes and acid-Hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-Hydrolase-active, acidic endolysosomes and acid Hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle.
Linda C. Deveaux - One of the best experts on this subject based on the ideXlab platform.
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Glycoside Hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism.
Biotechnology for Biofuels, 2018Co-Authors: William A. Apel, Peter P. Sheridan, Linda C. DeveauxAbstract:Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside Hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside Hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Experiments were performed to understand the effect of monosaccharides on gene expression during growth on the polysaccharide, WAX. Molecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside Hydrolases were downregulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, showing a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside Hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside Hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell. Glycoside Hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside Hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside Hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside Hydrolases were not tested as part of the research discussed, many of the glycoside Hydrolases found in the A. acidocaldarius Type Strain appear to have a broader substrate range than that represented by the glycoside Hydrolase family in which the enzymes were categorized.
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Glycoside Hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism
BMC, 2018Co-Authors: Brady D. Lee, Peter P. Sheridan, William A. Apel, Linda C. DeveauxAbstract:Abstract Background Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside Hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside Hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Experiments were performed to understand the effect of monosaccharides on gene expression during growth on the polysaccharide, WAX. Results Molecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside Hydrolases were downregulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, showing a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside Hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside Hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell. Conclusions Glycoside Hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside Hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside Hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside Hydrolases were not tested as part of the research discussed, many of the glycoside Hydrolases found in the A. acidocaldarius Type Strain appear to have a broader substrate range than that represented by the glycoside Hydrolase family in which the enzymes were categorized
Jurgen Pleiss - One of the best experts on this subject based on the ideXlab platform.
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the pha depolymerase engineering database a systematic analysis tool for the diverse family of polyhydroxyalkanoate pha depolymerases
BMC Bioinformatics, 2009Co-Authors: Michael Knoll, Virginia Martinez, Thomas M Hamm, Florian Wagner, Jurgen PleissAbstract:Polyhydroxyalkanoates (PHAs) can be degraded by many microorganisms using intra- or extracellular PHA depolymerases. PHA depolymerases are very diverse in sequence and substrate specificity, but share a common α/β-Hydrolase fold and a catalytic triad, which is also found in other α/β-Hydrolases.
Alexander G. Mclennan - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a Nudix Hydrolase from Deinococcus radiodurans with a marked specificity for (deoxy)ribonucleoside 5'-diphosphates
BMC Biochemistry, 2004Co-Authors: David I Fisher, Jared L Cartwright, Hideyoshi Harashima, Hiroyuki Kamiya, Alexander G. MclennanAbstract:Background Nudix Hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix Hydrolases, an unexpectedly large number. These may contribute to radioresistance by removing mutagenic oxidised and otherwise damaged nucleotides. Characterisation of these Hydrolases is necessary to understand the reason for their presence. Here, we report the cloning and characterisation of the DR0975 gene product, a nudix Hydrolase that appears to be unique to this organism. Results The DR0975 gene was cloned and expressed as a 20 kDa histidine-tagged recombinant product in Escherichia coli. Substrate analysis of the purified enzyme showed it to act primarily as a phosphatase with a marked preference for (deoxy)nucleoside 5'-diphosphates (dGDP > ADP > dADP > GDP > dTDP > UDP > dCDP > CDP). K_m for dGDP was 110 μM and k_cat was 0.18 s^-1 under optimal assay conditions (pH 9.4, 7.5 mM Mg^2+). 8-Hydroxy-2'-deoxyguanosine 5'-diphosphate (8-OH-dGDP) was also a substrate with a K_m of 170 μM and k_cat of 0.13 s^-1. Thus, DR0975 showed no preference for 8-OH-dGDP over dGDP. Limited pyrophosphatase activity was also observed with NADH and some (di)adenosine polyphosphates but no other substrates. Expression of the DR0975 gene was undetectable in logarithmic phase cells but was induced at least 30-fold in stationary phase. Superoxide, but not peroxide, stress and slow, but not rapid, dehydration both caused a slight induction of the DR0975 gene. Conclusion Nucleotide substrates for nudix Hydrolases conform to the structure NDP-X, where X can be one of several moieties. Thus, a preference for (d)NDPs themselves is most unusual. The lack of preference for 8-OH-dGDP over dGDP as a substrate combined with the induction in stationary phase, but not by peroxide or superoxide, suggests that the function of DR09075 may be to assist in the recycling of nucleotides under the very different metabolic requirements of stationary phase. Thus, if DR0975 does contribute to radiation resistance, this contribution may be indirect.
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nudix Hydrolases that degrade dinucleoside and diphosphoinositol polyphosphates also have 5 phosphoribosyl 1 pyrophosphate prpp pyrophosphatase activity that generates the glycolytic activator ribose 1 5 bisphosphate
Journal of Biological Chemistry, 2002Co-Authors: D Fisher, Alexander G. Mclennan, Stephen T Safrany, Peter Strike, Jared L CartwrightAbstract:Abstract A total of 17 Nudix Hydrolases were tested for their ability to hydrolyze 5-phosphoribosyl 1-pyrophosphate (PRPP). All 11 enzymes that were active toward dinucleoside polyphosphates with 4 or more phosphate groups as substrates were also able to hydrolyze PRPP, whereas the 6 that could not and that have coenzyme A, NDP-sugars, or pyridine nucleotides as preferred substrates did not degrade PRPP. The products of hydrolysis were ribose 1,5-bisphosphate and Pi. Active PRPP pyrophosphatases included the diphosphoinositol polyphosphate phosphoHydrolase (DIPP) subfamily of Nudix Hydrolases, which also degrade the non-nucleotide diphosphoinositol polyphosphates. K m andk cat values for PRPP hydrolysis for theDeinococcus radiodurans DR2356 (di)nucleoside polyphosphate Hydrolase, the human diadenosine tetraphosphate Hydrolase, and human DIPP-1 (diadenosine hexaphosphate and diphosphoinositol polyphosphate Hydrolase) were 1 mm and 1.5 s−1, 0.13 mm and 0.057 s−1, and 0.38 mm and 1.0 s−1, respectively. Active site mutants of theCaenorhabditis elegans diadenosine tetraphosphate Hydrolase had no activity, confirming that the same active site is responsible for nucleotide and PRPP hydrolysis. Comparison of the specificity constants for nucleotide, diphosphoinositol polyphosphate, and PRPP hydrolysis suggests that PRPP is a significant substrate for theD. radiodurans DR2356 enzyme and for the DIPP subfamily. In the latter case, generation of the glycolytic activator ribose 1,5-bisphosphate may be a new function for these enzymes.
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molecular cloning of diadenosine tetraphosphatase from pig small intestinal mucosa and identification of sequence blocks common to diadenosine polyphosphate Hydrolases and phosphorylases
The International Journal of Biochemistry & Cell Biology, 1997Co-Authors: S Hankin, Katrine A Wintero, Alexander G. MclennanAbstract:Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) pyrophosphoHydrolase is the enzyme responsible for reducing intracellular levels of the stress-responsive nucleotide diadenosine 5',5"'-P1,P4-tetraphosphate. In order to gain more information on the relationships between the enzymes hydrolysing diadenosine polyphosphates in different eukaryotes, the Ap4A Hydrolase and a corresponding cDNA have been isolated from pig small intestinal mucosa by standard procedures. The enzyme is a typical mammalian Ap4A Hydrolase (Km = 0.8 microM) being sensitive to inhibition by fluoride (Ki = 24 microM) and adenosine 5'-tetraphosphate (Ki = 10 nM) and yielding ATP and AMP as products. A low Km Ap4A Hydrolase (Km = 0.3 microM) was also isolated from rabbit small intestinal mucosa. These enzymes differ from the rat intestinal mucosal Hydrolase, which has much higher values of Km for Ap4A and Ki for adenosine 5'-tetraphosphate. A cDNA encoding the pig enzyme was isolated from a pig ileum cDNA library. The derived amino acid sequence of the 16.8 kDa gene product shows 88% identity and 96% similarity to that of the human enzyme. The sequence has the same modification of the MutT motif found in the human enzyme in which a threonine residue replaces a hydrophobic amino acid. Sequences comparisons among eukaryotic diadenosine polyphosphate Hydrolases and phosphorylases reveal two blocks of amino acid similarity, including a motif, Z[AD]Gx[ED]AGQ, which may be involved in polyphosphate binding by the Hydrolases, and an invariant histidine residue that may be involved in catalysis. These sequence similarities may have arisen by convergent evolution.
Streit, Wolfgang R. - One of the best experts on this subject based on the ideXlab platform.
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New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes
'American Society for Microbiology', 2018Co-Authors: Danso Dominik, Schmeisser Christel, Chow Jennifer, Zimmermann Wolfgang, Wei Ren, Leggewie Christian, Li Xiangzhen, Hazen Terry, Streit, Wolfgang R.Abstract:Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly alpha- and beta-Hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET Hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET Hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET Hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes. Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET Hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET Hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil. IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET Hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET Hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET Hydrolases. Finally, we describe the biochemical traits of four novel PET Hydrolases
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New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes.
eScholarship University of California, 2018Co-Authors: Danso Dominik, Schmeisser Christel, Chow Jennifer, Zimmermann Wolfgang, Wei Ren, Leggewie Christian, Li Xiangzhen, Hazen Terry, Streit, Wolfgang R.Abstract:Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and β-Hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET Hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET Hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET Hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET Hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET Hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil.IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET Hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET Hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET Hydrolases. Finally, we describe the biochemical traits of four novel PET Hydrolases
