13 results on '"Ying Tao"'
Search Results
2. Surveillance of Bat Coronaviruses in Kenya Identifies Relatives of Human Coronaviruses NL63 and 229E and Their Recombination History
- Author
-
Wanda Markotter, Christina Chommanard, Ying Tao, Mang Shi, Jing Zhang, Edward C. Holmes, Ivan V. Kuzmin, Krista Queen, and Suxiang Tong
- Subjects
0301 basic medicine ,Middle East respiratory syndrome coronavirus ,viruses ,Immunology ,Genome, Viral ,Hipposideros ,medicine.disease_cause ,Microbiology ,Virus ,Evolution, Molecular ,Viral Proteins ,03 medical and health sciences ,Phylogenetics ,Chiroptera ,Virology ,Prevalence ,medicine ,Animals ,Amino Acid Sequence ,Respiratory Tract Infections ,Gene ,Conserved Sequence ,Phylogeny ,Coronavirus ,Recombination, Genetic ,Genetics ,biology ,Phylogenetic tree ,Genetic Variation ,virus diseases ,Sequence Analysis, DNA ,respiratory system ,biology.organism_classification ,Kenya ,respiratory tract diseases ,Triaenops ,Coronavirus NL63, Human ,Phylogeography ,030104 developmental biology ,Genetic Diversity and Evolution ,Insect Science ,Epidemiological Monitoring ,Coronavirus Infections - Abstract
Bats harbor a large diversity of coronaviruses (CoVs), several of which are related to zoonotic pathogens that cause severe disease in humans. Our screening of bat samples collected in Kenya from 2007 to 2010 not only detected RNA from several novel CoVs but, more significantly, identified sequences that were closely related to human CoVs NL63 and 229E, suggesting that these two human viruses originate from bats. We also demonstrated that human CoV NL63 is a recombinant between NL63-like viruses circulating in Triaenops bats and 229E-like viruses circulating in Hipposideros bats, with the breakpoint located near 5′ and 3′ ends of the spike (S) protein gene. In addition, two further interspecies recombination events involving the S gene were identified, suggesting that this region may represent a recombination “hot spot” in CoV genomes. Finally, using a combination of phylogenetic and distance-based approaches, we showed that the genetic diversity of bat CoVs is primarily structured by host species and subsequently by geographic distances. IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs), including ancestors of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and human CoV 229E (HCoV-229E). However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL63-like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that interspecies recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential.
- Published
- 2017
3. The Autographa californica Multiple Nucleopolyhedrovirus ORF78 Is Essential for Budded Virus Production and General Occlusion Body Formation
- Author
-
Seok Hee Lee, Joo Hyun Lee, Yeon Ho Je, Qin Liu, Byung Rae Jin, Woojin Kim, Jae-Young Choi, Xue Ying Tao, Soo Dong Woo, Saes Byeol An, and Song Eun Kim
- Subjects
viruses ,Intranuclear Inclusion Bodies ,Molecular Sequence Data ,Immunology ,Mutant ,Spodoptera ,Biology ,Virus Replication ,Microbiology ,Virus ,Viral Proteins ,Microscopy, Electron, Transmission ,Viral life cycle ,Virology ,Sf9 Cells ,Animals ,Virus Release ,Cell Nucleus ,Genes, Essential ,Gene Expression Profiling ,fungi ,Sequence Analysis, DNA ,Transfection ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Molecular biology ,Nucleopolyhedroviruses ,Genome Replication and Regulation of Viral Gene Expression ,Gene expression profiling ,Autographa californica ,Viral replication ,Insect Science ,DNA, Viral ,Gene Deletion - Abstract
ORF78 ( ac78 ) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a baculovirus core gene of unknown function. To determine the role of ac78 in the baculovirus life cycle, an AcMNPV mutant with ac78 deleted, Ac78KO, was constructed. Quantitative PCR analysis revealed that ac78 is a late gene in the viral life cycle. After transfection into Spodoptera frugiperda cells, Ac78KO produced a single-cell infection phenotype, indicating that no infectious budded viruses (BVs) were produced. The defect in BV production was also confirmed by both viral titration and Western blotting. However, viral DNA replication was unaffected, and occlusion bodies were formed. An analysis of BVs and occlusion-derived viruses (ODVs) revealed that AC78 is associated with both forms of the virions and is an envelope structural protein. Electron microscopy revealed that AC78 also plays an important role in the embedding of ODV into the occlusion body. The results of this study demonstrate that AC78 is a late virion-associated protein and is essential for the viral life cycle.
- Published
- 2013
4. Synthesis and Differential Turnover of the CYS3 Regulatory Protein of Neurospora crassa Are Subject to Sulfur Control
- Author
-
Ying Tao and George A. Marzluf
- Subjects
Saccharomyces cerevisiae Proteins ,Recombinant Fusion Proteins ,Sulfur metabolism ,Gene Expression ,chemistry.chemical_element ,Genetics and Molecular Biology ,Microbiology ,Neurospora crassa ,Fungal Proteins ,chemistry.chemical_compound ,Methionine ,RNA, Messenger ,Molecular Biology ,Derepression ,Fungal protein ,biology ,Sulfates ,Lysine ,Structural gene ,Cystathionine gamma-Lyase ,RNA, Fungal ,biology.organism_classification ,Sulfur ,DNA-Binding Proteins ,Glutamine ,Kinetics ,Biochemistry ,chemistry ,Transcription Factors - Abstract
The transcription factor CYS3 of Neurospora crassa is a positive regulator of the sulfur regulatory circuit which contains many structural genes involved in sulfur metabolism. Expression and degradation of the CYS3 protein are precisely regulated in a sulfur-dependent manner. cys-3 expression was found to be fully repressed by high concentrations of methionine or inorganic sulfate present in the culture medium and to be derepressed when these favored sulfur sources were limited. cys-3 transcripts could be readily detected within 2 h after derepression, whereas the CYS3 protein was not found until after 4 h. CYS3 is stable, with a half-life greater than 4 h under low-sulfur conditions when it is required for cell growth. However, it is degraded relatively quickly when methionine or inorganic sulfate becomes available. Upon sulfur repression, cys-3 transcripts disappeared within 30 min with an estimated half-life of 5 min whereas CYS3 protein almost entirely disappeared in 1 h with a half-life of approximately 10 min. These results suggest that a selective elimination of CYS3 is a highly regulated process. Site-directed mutagenesis showed that Lys-105 of CYS3 is important for its instability. The change of this single residue from lysine to glutamine resulted in a prolonged half life of CYS3 and impaired responsiveness of CYS3 degradation to sulfur level changes.
- Published
- 1998
5. Surveillance of Bat Coronaviruses in Kenya Identifies Relatives of Human Coronaviruses NL63 and 229E and Their Recombination History.
- Author
-
Ying Tao, Mang Shi, Chommanard, Christina, Queen, Krista, Jing Zhang, Markotter, Wanda, Kuzmin, Ivan V., Holmes, Edward C., and Suxiang Tong
- Subjects
- *
CORONAVIRUS diseases , *GENETIC recombination , *ZOONOSES , *PATHOGENIC microorganisms , *SARS disease - Abstract
Bats harbor a large diversity of coronaviruses (CoVs), several of which are related to zoonotic pathogens that cause severe disease in humans. Our screening of bat samples collected in Kenya from 2007 to 2010 not only detected RNA from several novel CoVs but, more significantly, identified sequences that were closely related to human CoVs NL63 and 229E, suggesting that these two human viruses originate from bats. We also demonstrated that human CoV NL63 is a recombinant between NL63-like viruses circulating in Triaenops bats and 229E-like viruses circulating in Hipposideros bats, with the breakpoint located near 5' and 3' ends of the spike (S) protein gene. In addition, two further interspecies recombination events involving the S gene were identified, suggesting that this region may represent a recombination "hot spot" in CoV genomes. Finally, using a combination of phylogenetic and distance-based approaches, we showed that the genetic diversity of bat CoVs is primarily structured by host species and subsequently by geographic distances. IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs), including ancestors of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and human CoV 229E (HCoV-229E). However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL63-like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that interspecies recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
6. Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone
- Author
-
William E. Bentley, Ying Tao, Ayelet Fishman, and Thomas K. Wood
- Subjects
Models, Molecular ,Catechols ,Pseudomonas mendocina ,Quantitative Structure-Activity Relationship ,Pyrogallol ,Microbiology ,Medicinal chemistry ,Substrate Specificity ,chemistry.chemical_compound ,Cresols ,parasitic diseases ,Saturated mutagenesis ,Benzene ,Molecular Biology ,Catechol ,Binding Sites ,biology ,Phenol ,Active site ,Monooxygenase ,biology.organism_classification ,Toluene ,Enzymes and Proteins ,Toluene oxidation ,Hydroquinones ,Protein Subunits ,chemistry ,Biochemistry ,Amino Acid Substitution ,Mutagenesis ,biology.protein ,Oxygenases ,Oxidation-Reduction - Abstract
Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p -cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o -cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m -cresol and p -cresol to 4-methylcatechol (100%), and to oxidize o -methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent V max values of 6.6 ± 0.9 to 10.7 ± 0.1 nmol/min/ mg of protein were obtained for o -, m -, and p -cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ± 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o- monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184 : 344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41 : 3176-3188, 2002). By using o -cresol and o -methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o -cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o -methoxyphenol seven times faster than the wild-type T4MO (1.5 ± 0.2 versus 0.21 ± 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o -methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o -cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o -methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o -cresol. Hence, the regiospecific oxidation of o -methoxyphenol and o -cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o -cresol, so saturation mutagenesis converted T4MO into an ortho -hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p -cresol from toluene; hence, a better para- hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.
- Published
- 2004
7. Oxidation of Benzene to Phenol, Catechol, and 1,2,3-Trihydroxybenzene by Toluene 4-Monooxygenase of Pseudomonas mendocina KR1 and Toluene 3-Monooxygenase of Ralstonia pickettii PKO1
- Author
-
William E. Bentley, Thomas K. Wood, Ayelet Fishman, and Ying Tao
- Subjects
DNA, Bacterial ,Molecular Sequence Data ,Catechols ,Pseudomonas mendocina ,Ralstonia ,Pyrogallol ,Applied Microbiology and Biotechnology ,chemistry.chemical_compound ,Organic chemistry ,Phenol ,Phenols ,Enzymology and Protein Engineering ,Benzene ,Catechol ,Ecology ,biology ,Base Sequence ,Ralstonia pickettii ,biology.organism_classification ,Toluene ,chemistry ,Oxygenases ,Oxidation-Reduction ,Food Science ,Biotechnology ,Nuclear chemistry - Abstract
Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho -monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 μM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 ± 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 ± 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 ± 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 ± 1, 3.1 ± 0.3, and 0.26 ± 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 ± 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 ± 0.07 and 1.5 ± 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 ± 0.8, 4.0 ± 0.6, and 2.4 ± 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 μM).
- Published
- 2004
8. Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para-Hydroxylating Enzyme
- Author
-
Thomas K. Wood, Ayelet Fishman, and Ying Tao
- Subjects
Magnetic Resonance Spectroscopy ,biology ,Stereochemistry ,Ralstonia pickettii ,Nuclear magnetic resonance spectroscopy ,Ralstonia ,Monooxygenase ,biology.organism_classification ,Hydroxylation ,Microbiology ,Toluene ,Enzymes and Proteins ,chemistry.chemical_compound ,Burkholderia ,Biochemistry ,chemistry ,Oxygenases ,Molecular Biology ,Oxidation-Reduction ,Pseudomonas mendocina - Abstract
Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods. Whereas toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 hydroxylates monosubstituted benzenes at the para position and toluene ortho -monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates at the ortho position, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported previously to hydroxylate toluene at the meta position, producing primarily m -cresol (R. H. Olsen, J. J. Kukor, and B. Kaphammer, J. Bacteriol. 176:3749-3756, 1994). Using gas chromatography, we have discovered that T3MO hydroxylates monosubstituted benzenes predominantly at the para position. TG1/pBS(Kan)T3MO cells expressing T3MO oxidized toluene at a maximal rate of 11.5 ± 0.33 nmol/min/mg of protein with an apparent K m value of 250 μM and produced 90% p -cresol and 10% m -cresol. This product mixture was successively transformed to 4-methylcatechol. T4MO, in comparison, produces 97% p -cresol and 3% m -cresol. Pseudomonas aeruginosa PAO1 harboring pRO1966 (the original T3MO-bearing plasmid) also exhibited the same product distribution as that of TG1/pBS(Kan)T3MO. TG1/pBS(Kan)T3MO produced 66% p -nitrophenol and 34% m -nitrophenol from nitrobenzene and 100% p -methoxyphenol from methoxybenzene, as well as 62% 1-naphthol and 38% 2-naphthol from naphthalene; similar results were found with TG1/pBS(Kan)T4MO. Sequencing of the tbu locus from pBS(Kan)T3MO and pRO1966 revealed complete identity between the two, thus eliminating any possible cloning errors. 1 H nuclear magnetic resonance analysis confirmed the structural identity of p -cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO.
- Published
- 2004
9. Autographa californica Multiple Nucleopolyhedrovirus ORF11 Is Essential for Budded-Virus Production and Occlusion-Derived-Virus Envelopment.
- Author
-
Xue Ying Tao, Jae Young Choi, Woo Jin Kim, Saes Byeol An, Qin Liu, Song Eun Kim, Seok Hee Lee, Jong Hoon Kim, Soo Dong Woo, Byung Rae Jin, and Yeon Ho Je
- Subjects
- *
ALFALFA looper , *NUCLEOPOLYHEDROVIRUSES , *BACULOVIRUSES , *ELECTRON microscopy , *DNA replication - Abstract
ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5= rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KOtransfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
10. Sirtuin 1 Regulates Hepatitis B Virus Transcription and Replication by Targeting Transcription Factor AP-1.
- Author
-
Ji-Hua Ren, Ying Tao, Zhen-Zhen Zhang, Wei-Xian Chen, Xue-Fei Cai, Ke Chen, Ko, Ben C. B., Chun-Li Song, Long-Kuan Ran, Wan-Yu Li, Ai-Long Huang, and Juan Chena
- Subjects
- *
SIRTUINS , *HEPATITIS B prevention , *GENETIC transcription , *TRANSCRIPTION factor AP-1 , *MINICHROMOSOME maintenance proteins , *DNA replication , *CIRRHOSIS of the liver , *DISEASE risk factors - Abstract
Chronic hepatitis B virus (HBV) infection is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Nevertheless, the molecular mechanism of HBV replication remains elusive. SIRT1 is a class III histone deacetylase that is a structure component of the HBV cccDNA minichromosome. In this study, we found by using microarray-based gene expression profiling analysis that SIRT1 was upregulated in HBV-expressing cells. Gene silencing of SIRT1 significantly inhibited HBV DNA replicative intermediates, 3.5-kb mRNA, and core protein levels. In contrast, the overexpression of SIRT1 augmented HBV replication. Furthermore, SIRT1 enhanced the activity of HBV core promoter by targeting transcription factor AP-1. The c-Jun subunit of AP-1 was bound to the HBV core promoter region, as demonstrated by using a chromatin immunoprecipitation assay. Mutation of AP-1 binding site or knockdown of AP-1 abolished the effect of SIRT1 on HBV replication. Finally, SIRT1 inhibitor sirtinol also suppressed the HBV DNA replicative intermediate, as well as 3.5-kb mRNA. Our study identified a novel host factor, SIRT1, which may facilitate HBV replication in hepatocytes. These data suggest a rationale for the use of SIRT1 inhibitor in the treatment of HBV infection. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
11. Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone.
- Author
-
Ying Tao, Fishman, Ayelet, Bentley, William E., and Wood, Thomas K.
- Subjects
- *
TOLUENE , *AROMATIC compounds , *MONOOXYGENASES , *OXYGENASES , *PSEUDOMONAS , *BENZENE - Abstract
Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3- methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent Vmax values of 6.6 ± 0.9 to 10.7 ± 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ± 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, Gl03, and Al07 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184:344.349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41:3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 ± 0.2 versus 0.21 ± 0.01 nmol/min/mg of protein). Variant I100L produced 3methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/ A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho.hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
12. Oxidation of Benzene to Phenol, Catechol, and 1,2,3-Trihydroxybenzene by Toluene 4-Monooxygenase of Pseudomonas mendocina KR1 and Toluene 3-Monooxygenase of Ralstonia pickettii PKO1.
- Author
-
Ying Tao, Fishman, Ayelet, Bentley, William E., and Wood, Thomas K.
- Subjects
- *
AROMATIC compounds , *BENZENE , *PHENOL , *CATECHOL , *TOLUENE , *MONOOXYGENASES , *POLYPHENOLS , *HYDROXYLATION , *ESCHERICHIA coli , *ENTEROBACTERIACEAE - Abstract
Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendecina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 µM and under the control of a constitutive/ac promoter, Escherichia coli TGI/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 ± 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 ± 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 ± 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 ± 1, 3.1 ± 0.3, and 0.26 ± 0.09 nmol/min/mg of protein, respectively, and E. coli TGI/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 ± 03 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 ± 0.07 and 1.5 ± 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 ± 0.8, 4.0 ± 0.6, and 2.4 7plusmn; 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 µM). [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
13. NeuroBactrus, a Novel, Highly Effective, and Environmentally Friendly Recombinant Baculovirus Insecticide.
- Author
-
Hee un Shim, Jae Young Choi, Yong Wang, Xue Ying Tao, Qin Liu, Jong Yul Roh, Jae Su Kim, Woo Jin Kim, Soo Dong Woo, Byung Rae Jin, and Yeon Ho Je
- Subjects
- *
RECOMBINANT proteins , *BACULOVIRUS genetics , *TOXICOLOGY of insecticides , *THURINGIENSIN , *ALFALFA looper , *GENE expression in bacteria , *PROMOTERS (Genetics) - Abstract
A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cryl-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrincryl-5-polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf6O3. The polyhedrin-CryI-5-polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an -65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cryl -5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.