The Experts below are selected from a list of 2043 Experts worldwide ranked by ideXlab platform
Rong Xiao - One of the best experts on this subject based on the ideXlab platform.
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coupled r carbonyl reductase and glucose dehydrogenase catalyzes r 1 phenyl 1 2 ethanediol biosynthesis with excellent stereochemical selectivity
Process Biochemistry, 2015Co-Authors: Xiaotian Zhou, Rongzhen Zhang, Hongbo Liang, Jiawei Jiang, Rong XiaoAbstract:Abstract The biotransformation of 2-Hydroxyacetophenone to (R)-1-phenyl-1, 2-ethanediol (PED) by NADH-dependent (R)-carbonyl reductase (RCR) from Candida parapsilosis is slow and gives low yields, probably as a result of insufficient cofactors. To improve the biotransformation efficiency of (R)-PED from 2-hydroxyacetophenon, an enzyme-coupling system containing RCR and glucose dehydrogenase (GDH) was constructed to strengthen NADH-recycling pathway in Escherichia coli, in which the Shine-Dalgarno sequence and the aligned spacing sequence were used as linkers between them. The introduction of glucose dehydrogenase had little affects on the cell-growth. The co-expression conditions of RCR and glucose dehydrogenase was optimized to rebalance their catalytic functions. The ratio of kcat/KM for enzyme-coupling system catalyzing 2-HAP and glucose was about 1.0, suggesting the good balance between the functions of RCR and GDH. The rebalanced system gave excellent performance in (R)-PED biotransformation: an optical purity of 99.9% and a yield of 99.9% at optimal conditions: 35 °C and pH 7.0. The introduction of glucose dehydrogenase stimulated increases of 23.8% and 63.8%, in optical purity and yield of (R)-PED, and simultaneously reduced the reaction time two-fold. This work provided a valuable method for efficient chiral alcohol production through protein-expression and biotransformation optimization to rebalance cofactor pathways.
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carbonyl reductase scrii from candida parapsilosis catalyzes anti prelog reaction to s 1 phenyl 1 2 ethanediol with absolute stereochemical selectivity
Bioresource Technology, 2011Co-Authors: Rongzhen Zhang, Yawei Geng, Wenchi Zhang, Shanshan Wang, Rong XiaoAbstract:An (S)-specific carbonyl reductase (SCRII) was purified to homogeneity from Candida parapsilosis by following an anti-Prelog reducing activity of 2-Hydroxyacetophenone. Peptide mass fingerprinting analysis shows SCRII belongs to short-chain dehydrogenase/reductase family. Its coding gene was cloned and overexpressed in Escherichia coli. The recombinant SCRII displays the similar enzymatic characterization and catalytic properties to SCR. It catalyzes the enantioselective reduction of 2-Hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with excellent optical purity of 100% in higher yield than SCR. Based on the sequence-structure alignment, several single-point mutations inside or adjacent to the substrate-binding loop or active site were designed. With respect to recombinant native SCRII, the A220 and E228 mutations almost lost enantioselectivity towards 2-Hydroxyacetophenone reduction. The catalytic efficiencies (kcat/Km) for the A220 or E228 variants are <7% that of the unmutated enzyme. This work provides an excellent catalyst for enantiopure alcohol preparation and the lethal mutations of A220 and E228 suggest their importance in substrate-binding and/or catalysis.
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complementary selectivity to s 1 phenyl 1 2 ethanediol forming candida parapsilosis by expressing its carbonyl reductase in escherichia coli for r specific reduction of 2 Hydroxyacetophenone
Biocatalysis and Biotransformation, 2008Co-Authors: Yao Nie, Rong Xiao, Hai Yan Wang, Zhi Hao SunAbstract:An (R)-specific carbonyl reductase from Candida parapsilosis CCTCCM203011 (CprCR) was shown to catalyze the asymmetric reduction of 2-Hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol (PED), which is a critical chiral building block in organic synthesis. The gene (rcr) encoding CprCR was cloned based on the amino acid sequences of tryptic fragments of the enzyme. Sequence analysis revealed that rcr is comprised of 1008 nucleotides encoding a 35 977 Da polypeptide, and shares similarity to proteins of the medium-chain dehydrogenase/reductase (MDR) superfamily. Recombinant rcr expressed in Escherichia coli showed a specific 2-Hydroxyacetophenone-reducing activity. Using rcr expressing cells, (R)-PED was obtained by asymmetric reduction, which is complementary in enantiomeric configuration to (S)-PED obtained by using whole cells of C. parapsilosis. After optimization of reaction conditions, (R)-PED was produced at 95.5% enantiomeric excess with a yield of 92.6% when isopropanol was used for cofactor regene...
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purification characterization gene cloning and expression of a novel alcohol dehydrogenase with anti prelog stereospecificity from candida parapsilosis
Applied and Environmental Microbiology, 2007Co-Authors: Yao Nie, Hai Yan Wang, Ming Yang, Rong XiaoAbstract:An alcohol dehydrogenase from Candida parapsilosis CCTCC M203011 was characterized along with its biochemical activity and structural gene. The amino acid sequence shows similarity to those of the short-chain dehydrogenase/reductases but no overall identity to known proteins. This enzyme with unusual stereospecificity catalyzes an anti-Prelog reduction of 2-Hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol.
Marco W. Fraaije - One of the best experts on this subject based on the ideXlab platform.
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bvmo catalysed dynamic kinetic resolution of racemic benzyl ketones in the presence of anion exchange resins
Organic and Biomolecular Chemistry, 2010Co-Authors: Cristina Rodriguez, Marco W. Fraaije, Gonzalo De Gonzalo, Ana Riozmartinez, Daniel Torres E Pazmino, Vicente GotorAbstract:4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB was employed in the presence of a weak anion exchange resin to perform dynamic kinetic resolutions of racemic benzyl ketones with high conversions and good optical purities. Different parameters that affect to the efficiency of the enzymatic Baeyer–Villiger oxidation and racemisation were analyzed in order to optimize the activity and selectivity of the biocatalytic system.
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hydroquinone dioxygenase from pseudomonas fluorescens acb a novel member of the family of nonheme iron ii dependent dioxygenases
Journal of Bacteriology, 2008Co-Authors: Marielle J H Moonen, Marco W. Fraaije, Silvia A Synowsky, Willy A M Van Den Berg, Adrie H Westphal, Albert J R Heck, Robert H H Van Den Heuvel, Willem J H Van BerkelAbstract:Hydroquinone 1,2-dioxygenase (HQDO), an enzyme involved in the catabolism of 4-Hydroxyacetophenone in Pseudomonas fluorescens ACB, was purified to apparent homogeneity. Ligandation with 4-hydroxybenzoate prevented the enzyme from irreversible inactivation. HQDO was activated by iron(II) ions and catalyzed the ringfission of a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes. HQDO was inactivated by 2,2-dipyridyl, o-phenanthroline, and hydrogen peroxide and inhibited by phenolic compounds. The inhibition with 4-hydroxybenzoate (Ki 14 M) was competitive with hydroquinone. Online size-exclusion chromatographymass spectrometry revealed that HQDO is an 22 heterotetramer of 112.4 kDa, which is composed of an -subunit of 17.8 kDa and a -subunit of 38.3 kDa. Each -subunit binds one molecule of 4-hydroxybenzoate and one iron(II) ion. N-terminal sequencing and peptide mapping and sequencing based on matrix-assisted laser desorption ionization—two-stage time of flight analysis established that the HQDO subunits are encoded by neighboring open reading frames (hapC and hapD) of a gene cluster, implicated to be involved in 4-Hydroxyacetophenone degradation. HQDO is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The enzyme shows insignificant sequence identity with known dioxygenases.
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4 Hydroxyacetophenone monooxygenase from pseudomonas fluorescens acb a novel flavoprotein catalyzing baeyer villiger oxidation of aromatic compounds
FEBS Journal, 2001Co-Authors: Nanne M. Kamerbeek, Willem J. H. Van Berkel, Marco W. Fraaije, Maria Lle J H Moonen, Dick B JanssenAbstract:A novel flavoprotein that catalyses the NADPH-dependent oxidation of 4-Hydroxyacetophenone to 4-hydroxyphenyl acetate, was purified to homogeneity from Pseudomonas fluorescens ACB. Characterization of the purified enzyme showed that 4-Hydroxyacetophenone monooxygenase (HAPMO) is a homodimer of approximate to 140 kDa with each subunit containing a noncovalently bound FAD molecule. HAPMO displays a tight coupling between NADPH oxidation and substrate oxygenation. Besides 4-Hydroxyacetophenone a wide range of other acetophenones are readily converted via a Baeyer-Villiger rearrangement reaction into the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 amino-acid protein with a deduced mass of 71 884 Da. Except for an N-terminal extension of approximate to 135 residues, the sequence of HAPMO shares significant similarity with two known types of Baeyer-Villiger monooxygenases: cyclohexanone monooxygenase (27-33% sequence identity) and steroid monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functional role of a recently identified flavoprotein sequence motif (ATG) was explored by site-directed mutagenesis. Replacement of the strictly conserved glycine (G490) resulted in a dramatic effect on catalysis. From a kinetic analysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding.
Dick B Janssen - One of the best experts on this subject based on the ideXlab platform.
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4 Hydroxyacetophenone monooxygenase from pseudomonas fluorescens acb a novel flavoprotein catalyzing baeyer villiger oxidation of aromatic compounds
FEBS Journal, 2001Co-Authors: Nanne M. Kamerbeek, Willem J. H. Van Berkel, Marco W. Fraaije, Maria Lle J H Moonen, Dick B JanssenAbstract:A novel flavoprotein that catalyses the NADPH-dependent oxidation of 4-Hydroxyacetophenone to 4-hydroxyphenyl acetate, was purified to homogeneity from Pseudomonas fluorescens ACB. Characterization of the purified enzyme showed that 4-Hydroxyacetophenone monooxygenase (HAPMO) is a homodimer of approximate to 140 kDa with each subunit containing a noncovalently bound FAD molecule. HAPMO displays a tight coupling between NADPH oxidation and substrate oxygenation. Besides 4-Hydroxyacetophenone a wide range of other acetophenones are readily converted via a Baeyer-Villiger rearrangement reaction into the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 amino-acid protein with a deduced mass of 71 884 Da. Except for an N-terminal extension of approximate to 135 residues, the sequence of HAPMO shares significant similarity with two known types of Baeyer-Villiger monooxygenases: cyclohexanone monooxygenase (27-33% sequence identity) and steroid monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functional role of a recently identified flavoprotein sequence motif (ATG) was explored by site-directed mutagenesis. Replacement of the strictly conserved glycine (G490) resulted in a dramatic effect on catalysis. From a kinetic analysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding.
Nanne M. Kamerbeek - One of the best experts on this subject based on the ideXlab platform.
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4 Hydroxyacetophenone monooxygenase from pseudomonas fluorescens acb a novel flavoprotein catalyzing baeyer villiger oxidation of aromatic compounds
FEBS Journal, 2001Co-Authors: Nanne M. Kamerbeek, Willem J. H. Van Berkel, Marco W. Fraaije, Maria Lle J H Moonen, Dick B JanssenAbstract:A novel flavoprotein that catalyses the NADPH-dependent oxidation of 4-Hydroxyacetophenone to 4-hydroxyphenyl acetate, was purified to homogeneity from Pseudomonas fluorescens ACB. Characterization of the purified enzyme showed that 4-Hydroxyacetophenone monooxygenase (HAPMO) is a homodimer of approximate to 140 kDa with each subunit containing a noncovalently bound FAD molecule. HAPMO displays a tight coupling between NADPH oxidation and substrate oxygenation. Besides 4-Hydroxyacetophenone a wide range of other acetophenones are readily converted via a Baeyer-Villiger rearrangement reaction into the corresponding phenyl acetates. The P. fluorescens HAPMO gene (hapE) was characterized. It encoded a 640 amino-acid protein with a deduced mass of 71 884 Da. Except for an N-terminal extension of approximate to 135 residues, the sequence of HAPMO shares significant similarity with two known types of Baeyer-Villiger monooxygenases: cyclohexanone monooxygenase (27-33% sequence identity) and steroid monooxygenase (33% sequence identity). The HAPMO sequence contains several sequence motifs indicative for the presence of two Rossman fold domains involved in FAD and NADPH binding. The functional role of a recently identified flavoprotein sequence motif (ATG) was explored by site-directed mutagenesis. Replacement of the strictly conserved glycine (G490) resulted in a dramatic effect on catalysis. From a kinetic analysis of the G490A mutant it is concluded that the observed sequence motif serves a structural function which is of importance for NADPH binding.
Rabus R. - One of the best experts on this subject based on the ideXlab platform.
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The predicted σ(54)-dependent regulator EtpR is essential for expression of genes for anaerobic p-ethylphenol and p-Hydroxyacetophenone degradation in "Aromatoleum aromaticum" EbN1
2015Co-Authors: Kant M., Dörries M., Wöhlbrand L., Rabus R.Abstract:BACKGROUND: The denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 anaerobically utilizes a multitude of aromatic compounds via specific peripheral degradation routes. Compound-specific formation of these catabolic modules is assumed to be mediated by specific transcriptional activators. In case of the recently elucidated p-ethylphenol/p-Hydroxyacetophenone pathway, the highly substrate-specific regulation was implicated to involve the predicted σ(54)-dependent, NtrC-type regulator EbA324. The latter was suggested to control the expression of the two neighboring gene clusters encoding the catabolic enzymes as well as a corresponding putative solvent efflux system. In the present study, a molecular genetic approach was used to study the predicted function of EbA324. RESULTS: An unmarked in frame ΔebA324 (here renamed as ΔetpR; p-ethylphenol regulator) deletion mutation was generated. The ΔetpR mutant was unable to grow anaerobically with either p-ethylphenol or p-Hydroxyacetophenone. Growth similar to the wild type was restored in the ΔetpR mutant background by in trans expression of plasmid-born etpR. Furthermore, expression of the "p-ethylphenol" gene clusters as well as corresponding protein formation was shown to depend on the presence of both, EtpR and either p-ethylphenol or p-Hydroxyacetophenone. In the wild type, the etpR gene appears to be constitutively expressed and its expression level not to be modulated upon effector presence. Comparison with the regulatory domains of known phenol- and alkylbenzene-responsive NtrC-type regulators of Pseudomonas spp. and Thauera aromatica allowed identifying >60 amino acid residues in the regulatory domain (in particular positions 149 to 192 of EtpR) that may contribute to the effector specificity viz. presumptively restricted effector spectrum of EtpR. CONCLUSIONS: This study provides experimental evidence for the genome predicted σ(54)-dependent regulator EtpR (formerly EbA324) of "A. aromaticum" EbN1 to be responsive to p-ethylphenol, as well as its degradation intermediate p-Hydroxyacetophenone, and to control the expression of genes involved in the anaerobic degradation of these two aromatic growth substrates. Overall, the presented results advance our understanding on the regulation of anaerobic aromatic compound catabolism, foremost based on the sensory discrimination of structurally similar substrates
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Molecular genetic and crystal structural analysis of 1-(4-hydroxyphenyl)-ethanol dehydrogenase from 'Aromatoleum aromaticum' EbN1.
2015Co-Authors: Höffken H., Wöhlbrand L., Breuer M., Hauer B., Rabus R.Abstract:he dehydrogenation of 1-(4-hydroxyphenyl)-ethanol to 4-Hydroxyacetophenone represents the second reaction step during anaerobic degradation of p-ethylphenol in the denitrifying bacterium ‘Aromatoleum aromaticum' EbN1. Previous proteogenomic studies identified two different proteins (ChnA and EbA309) as possible candidates for catalyzing this reaction [Wöhlbrand et al: J Bacteriol 2008;190:5699-5709]. Physiological-molecular characterization of newly generated unmarked in-frame deletion and complementation mutants allowed defining ChnA (renamed here as Hped) as the enzyme responsible for 1-(4-hydroxyphenyl)-ethanol oxidation. Hped [1-(4-hydroxyphenyl)-ethanol dehydrogenase] belongs to the ‘classical' family within the short-chain alcohol dehydrogenase/reductase (SDR) superfamily. Hped was overproduced in Escherichia coli, purified and crystallized. The X-ray structures of the apo- and NAD+-soaked form were resolved at 1.5 and 1.1 Å, respectively, and revealed Hped as a typical homotetrameric SDR. Modeling of the substrate 4-Hydroxyacetophenone (reductive direction of Hped) into the active site revealed the structural determinants of the strict (R)-specificity of Hped (Phe187), contrasting the (S)-specificity of previously reported 1-phenylethanol dehydrogenase (Ped; Tyr93) from strain EbN1 [Höffken et al: Biochemistry 2006;45:82-93]
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Supplementary Material for: Molecular Genetic and Crystal Structural Analysis of 1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase from ‘Aromatoleum aromaticum' EbN1
2015Co-Authors: Höffken H.w., Wöhlbrand L., Breuer M., Hauer B., Rabus R.Abstract:The dehydrogenation of 1-(4-hydroxyphenyl)-ethanol to 4-Hydroxyacetophenone represents the second reaction step during anaerobic degradation of p-ethylphenol in the denitrifying bacterium ‘Aromatoleum aromaticum' EbN1. Previous proteogenomic studies identified two different proteins (ChnA and EbA309) as possible candidates for catalyzing this reaction [Wöhlbrand et al: J Bacteriol 2008;190:5699-5709]. Physiological-molecular characterization of newly generated unmarked in-frame deletion and complementation mutants allowed defining ChnA (renamed here as Hped) as the enzyme responsible for 1-(4-hydroxyphenyl)-ethanol oxidation. Hped [1-(4-hydroxyphenyl)-ethanol dehydrogenase] belongs to the ‘classical' family within the short-chain alcohol dehydrogenase/reductase (SDR) superfamily. Hped was overproduced in Escherichia coli, purified and crystallized. The X-ray structures of the apo- and NAD+-soaked form were resolved at 1.5 and 1.1 Å, respectively, and revealed Hped as a typical homotetrameric SDR. Modeling of the substrate 4-Hydroxyacetophenone (reductive direction of Hped) into the active site revealed the structural determinants of the strict (R)-specificity of Hped (Phe187), contrasting the (S)-specificity of previously reported 1-phenylethanol dehydrogenase (Ped; Tyr93) from strain EbN1 [Höffken et al: Biochemistry 2006;45:82-93]
