Unlike higher organisms, microbes have only a very limited capacity for controlling their (micro)... more Unlike higher organisms, microbes have only a very limited capacity for controlling their (micro)environment. In order for them to survive and compete successfully with other microbes under the heterogeneous and changing conditions that are so characteristic of natural environments, they have evolved the potential to respond to environmental change by changing themselves, both functionally and structurally. This potential for rapid adaptation is uniquely associated with the life style of the microbe and resides in its ...
<p>The composite structures contain all structural features established for the respective ... more <p>The composite structures contain all structural features established for the respective products. Quantities of each structural element fit with the combined data of 1D <sup>1</sup>H NMR integration and methylation analysis, as well as enzymatic degradation studies with α-amylase, dextranase and pullulanase. For comparison the composite structure for the <i>A</i>. <i>chroococcum</i> GtfD polymer from amylose is represented as well. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172622#pone.0172622.ref015" target="_blank">15</a>]</p
<p>(A) (putative) GtfD-like 4,6-α-GTase enzymes, (B) (putative) GtfC-like 4,6-α-GTase enzym... more <p>(A) (putative) GtfD-like 4,6-α-GTase enzymes, (B) (putative) GtfC-like 4,6-α-GTase enzymes, (C) (putative) GtfB-like enzymes, and (D) sucrose-active enzymes. The seven strictly conserved amino acid residues in GH70 enzymes (numbered 1 to 7 above the sequences) are also conserved in GtfD-like proteins. Amino acids that constitute the catalytic triad are highlighted in bold and lightly shaded. Residues forming acceptor subsites -1, +1 and +2 in Gtf180-ΔN [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172622#pone.0172622.ref007" target="_blank">7</a>] are indicated in green, red and blue, respectively. Abbreviations at the bottom: NU = nucleophile, A/B = general acid/base, TS = transition state stabilizer. <sup>a</sup> The protein sequences are annotated by their GenBank Accession number, except for the <i>Burkholderia</i> sp. NFACC38-1 GtfD-like protein sequence that is labeled with its IMG/ER Gene.</p
<p>The crystal structures of the <i>L</i>. <i>reuteri</i> 121 GtfA ... more <p>The crystal structures of the <i>L</i>. <i>reuteri</i> 121 GtfA glucansucrase (left) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172622#pone.0172622.ref010" target="_blank">10</a>] and the <i>B</i>. <i>licheniformis</i> α-amylase (right) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172622#pone.0172622.ref029" target="_blank">29</a>] are included. Domains A, B, C, IV and V are highlighted in blue, green, magenta, yellow and red, respectively. Ig2-like domains are colored in pink. The amino acid residue numbers represent the start of each domain. Conserved regions I-IV are indicated by grey rectangles. Domains A, B, C and IV were assigned in <i>P</i>. <i>beijingensis</i> GtfD by sequence comparison with <i>L</i>. <i>reuteri</i> 121 GtfB.</p
One-carbon compounds (C1) at all oxidation levels between methane and carbon dioxide occur abunda... more One-carbon compounds (C1) at all oxidation levels between methane and carbon dioxide occur abundantly throughout nature. Methane is present in fossil deposits and is formed by methanogenic bacteria. Methanol arises by the hydrolysis of methyl esters and ethers such as pectin and lignin, which are present in plants. Methylated amines occur in plants and animals and are produced by microbial degradation of choline derivatives present in plant membrane material and animal tissue. Formate is a major end-product of mixed-acid fermentation and carbon dioxide is present in the atmosphere and, as carbonates, in natural waters and soil. It is not surprising, therefore, that microorganisms are found in nature which are capable of growth on such compounds as carbon and energy sources.
Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to the... more Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (α), 7 (β) and 8 (γ) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of α-, βand γ-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversifi...
Extensive characterization of the thermostable alpha-amylase of Clostridium thermosulfurogenes EM... more Extensive characterization of the thermostable alpha-amylase of Clostridium thermosulfurogenes EM1, recently reclassified as Thermoanaerobacterium thermosulfurigenes, clearly demonstrated that the enzyme is a cyclodextrin glycosyltransferase (CGTase). Product analysis after incubation of the enzyme with starch revealed formation of alpha-, beta-, and gamma-cyclodextrins, as well as linear sugars. The specific activity for cyclization of this CGTase was similar to those of other CGTases, whereas the specific activity for hydrolysis was relatively high in comparison with other CGTases. Alignment of the amino acid sequence of the T. thermosulfurigenes enzyme with sequences from known bacterial CGTases showed high homology. The four consensus regions of carbohydrate-converting enzymes, as well as a C-terminal raw-starch binding motif, could be identified in the sequence.
An l -aminopeptidase of Pseudomonas putida , used in an industrial process for the hydrolysis of ... more An l -aminopeptidase of Pseudomonas putida , used in an industrial process for the hydrolysis of d,l -amino acid amide racemates, was purified to homogeneity. The highly l -enantioselective enzyme resembled thiol reagent-sensitive alkaline serine proteinases and was strongly activated by divalent cations. It possessed a high substrate specificity for dipeptides and α-H amino acid amides, e.g., l -phenylglycine amide.
Autotrophic growth of Xanthobacter flavus is dependent on the fixation of carbon dioxide via the ... more Autotrophic growth of Xanthobacter flavus is dependent on the fixation of carbon dioxide via the Calvin cycle and on the oxidation of simple organic and inorganic compounds to provide the cell with energy. Maximal induction of the cbb and gap-pgk operons encoding enzymes of the Calvin cycle occurs in the absence of multicarbon substrates and the presence of methanol, formate, hydrogen, or thiosulfate. The LysR-type transcriptional regulator CbbR regulates the expression of the cbb and gap-pgk operons, but it is unknown to what cellular signal CbbR responds. In order to study the effects of low-molecular-weight compounds on the DNA-binding characteristics of CbbR, the protein was expressed in Escherichia coli and subsequently purified to homogeneity. CbbR of X. flavus is a dimer of 36-kDa subunits. DNA-binding assays suggested that two CbbR molecules bind to a 51-bp DNA fragment on which two inverted repeats containing the LysR motif are located. The addition of 200 μM NADPH, but not...
Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are k... more Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are key regulatory enzymes in L-Phe and L-Tyr biosynthesis in Amycolatopsis methanolica. At least two CM proteins, CMIa and CMIb, are required for the single chorismate mutase activity in the wild type. Component CMIa (a homodimeric protein with 16-kDa subunits) was purified to homogeneity (2,717-fold) and kinetically characterized. The partially purified CMIb preparation obtained also contained the single DS (DSI) activity detectable in the wild type. The activities of CMIa and CMIb were inhibited by both L-Phe and L-Tyr. DSI activity was inhibited by L-Trp, L-Phe, and L-Tyr. A leaky L-Phe-requiring auxotroph, mutant strain GH141, grown under L-Phe limitation, possessed additional DS (DSII) and CM (CMII) activities. Synthesis of both CMII and DSII was repressed by L-Phe. An ortho-DL-fluorophenylalanine-resistant mutant of the wild type (strain oFPHE83) that had lost the sensitivity of DSII a...
The ATP-dependent phosphofructokinase (ATP-PFK) of Streptomyces coelicolor A3(2) was purified to ... more The ATP-dependent phosphofructokinase (ATP-PFK) of Streptomyces coelicolor A3(2) was purified to homogeneity (1,600-fold) and characterized (110 kDa, with a single type of subunit of 40 kDa); it is allosterically inhibited by phosphoenolpyruvate. Cloning of the pfk gene of S. coelicolor A3(2) and analysis of the deduced amino acid sequence (343 amino acids; 36,667 Da) revealed high similarities to the PPi-PFK enzyme from Amycolatopsis methanolica (tetramer, nonallosteric; 70%) and to the allosteric ATP-PFK enzymes from other bacteria, e.g., Escherichia coli (tetramer; 37%) and Bacillus stearothermophilus (tetramer, 41%). Further structural and functional analysis of the two actinomycete PFK enzymes should elucidate the features of these proteins that determine substrate specificity (ATP versus PPi) and allosteric (in)sensitivity.
Auxotrophic mutants of the actinomycete Amycolatopsis methanolica requiring l-Phe or l-Tyr were i... more Auxotrophic mutants of the actinomycete Amycolatopsis methanolica requiring l-Phe or l-Tyr were isolated and identified as strains lacking prephenate dehydratase (strain GH71) or arogenate dehydrogenase (strain GH70), respectively. A. methanolica thus employs single pathways only for the biosynthesis of these aromatic amino acids. Anion-exchange chromatography of extracts revealed two peaks with Phe as well as Tyr aminotransferase (AT) activity (Phe/Tyr ATI and Phe/Tyr ATII) and three peaks with prephenate AT activity (Ppa ATI to Ppa ATIII). Phe/Tyr ATI and Ppa ATI coeluted and appear to function as the A. methanolica branched-chain amino acid AT. Ppa ATII probably functions as the aspartate AT. Mutant studies showed that Phe/Tyr ATII is the dominant AT in l-Phe biosynthesis and in l-Tyr catabolism but not in l-Tyr biosynthesis. Biochemical studies showed that Ppa ATIII is highly specific for prephenate and provided evidence that Ppa ATIII is the dominant AT in l-Tyr biosynthesis.
Unlike higher organisms, microbes have only a very limited capacity for controlling their (micro)... more Unlike higher organisms, microbes have only a very limited capacity for controlling their (micro)environment. In order for them to survive and compete successfully with other microbes under the heterogeneous and changing conditions that are so characteristic of natural environments, they have evolved the potential to respond to environmental change by changing themselves, both functionally and structurally. This potential for rapid adaptation is uniquely associated with the life style of the microbe and resides in its ...
<p>The composite structures contain all structural features established for the respective ... more <p>The composite structures contain all structural features established for the respective products. Quantities of each structural element fit with the combined data of 1D <sup>1</sup>H NMR integration and methylation analysis, as well as enzymatic degradation studies with α-amylase, dextranase and pullulanase. For comparison the composite structure for the <i>A</i>. <i>chroococcum</i> GtfD polymer from amylose is represented as well. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172622#pone.0172622.ref015" target="_blank">15</a>]</p
<p>(A) (putative) GtfD-like 4,6-α-GTase enzymes, (B) (putative) GtfC-like 4,6-α-GTase enzym... more <p>(A) (putative) GtfD-like 4,6-α-GTase enzymes, (B) (putative) GtfC-like 4,6-α-GTase enzymes, (C) (putative) GtfB-like enzymes, and (D) sucrose-active enzymes. The seven strictly conserved amino acid residues in GH70 enzymes (numbered 1 to 7 above the sequences) are also conserved in GtfD-like proteins. Amino acids that constitute the catalytic triad are highlighted in bold and lightly shaded. Residues forming acceptor subsites -1, +1 and +2 in Gtf180-ΔN [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172622#pone.0172622.ref007" target="_blank">7</a>] are indicated in green, red and blue, respectively. Abbreviations at the bottom: NU = nucleophile, A/B = general acid/base, TS = transition state stabilizer. <sup>a</sup> The protein sequences are annotated by their GenBank Accession number, except for the <i>Burkholderia</i> sp. NFACC38-1 GtfD-like protein sequence that is labeled with its IMG/ER Gene.</p
<p>The crystal structures of the <i>L</i>. <i>reuteri</i> 121 GtfA ... more <p>The crystal structures of the <i>L</i>. <i>reuteri</i> 121 GtfA glucansucrase (left) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172622#pone.0172622.ref010" target="_blank">10</a>] and the <i>B</i>. <i>licheniformis</i> α-amylase (right) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172622#pone.0172622.ref029" target="_blank">29</a>] are included. Domains A, B, C, IV and V are highlighted in blue, green, magenta, yellow and red, respectively. Ig2-like domains are colored in pink. The amino acid residue numbers represent the start of each domain. Conserved regions I-IV are indicated by grey rectangles. Domains A, B, C and IV were assigned in <i>P</i>. <i>beijingensis</i> GtfD by sequence comparison with <i>L</i>. <i>reuteri</i> 121 GtfB.</p
One-carbon compounds (C1) at all oxidation levels between methane and carbon dioxide occur abunda... more One-carbon compounds (C1) at all oxidation levels between methane and carbon dioxide occur abundantly throughout nature. Methane is present in fossil deposits and is formed by methanogenic bacteria. Methanol arises by the hydrolysis of methyl esters and ethers such as pectin and lignin, which are present in plants. Methylated amines occur in plants and animals and are produced by microbial degradation of choline derivatives present in plant membrane material and animal tissue. Formate is a major end-product of mixed-acid fermentation and carbon dioxide is present in the atmosphere and, as carbonates, in natural waters and soil. It is not surprising, therefore, that microorganisms are found in nature which are capable of growth on such compounds as carbon and energy sources.
Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to the... more Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (α), 7 (β) and 8 (γ) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of α-, βand γ-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversifi...
Extensive characterization of the thermostable alpha-amylase of Clostridium thermosulfurogenes EM... more Extensive characterization of the thermostable alpha-amylase of Clostridium thermosulfurogenes EM1, recently reclassified as Thermoanaerobacterium thermosulfurigenes, clearly demonstrated that the enzyme is a cyclodextrin glycosyltransferase (CGTase). Product analysis after incubation of the enzyme with starch revealed formation of alpha-, beta-, and gamma-cyclodextrins, as well as linear sugars. The specific activity for cyclization of this CGTase was similar to those of other CGTases, whereas the specific activity for hydrolysis was relatively high in comparison with other CGTases. Alignment of the amino acid sequence of the T. thermosulfurigenes enzyme with sequences from known bacterial CGTases showed high homology. The four consensus regions of carbohydrate-converting enzymes, as well as a C-terminal raw-starch binding motif, could be identified in the sequence.
An l -aminopeptidase of Pseudomonas putida , used in an industrial process for the hydrolysis of ... more An l -aminopeptidase of Pseudomonas putida , used in an industrial process for the hydrolysis of d,l -amino acid amide racemates, was purified to homogeneity. The highly l -enantioselective enzyme resembled thiol reagent-sensitive alkaline serine proteinases and was strongly activated by divalent cations. It possessed a high substrate specificity for dipeptides and α-H amino acid amides, e.g., l -phenylglycine amide.
Autotrophic growth of Xanthobacter flavus is dependent on the fixation of carbon dioxide via the ... more Autotrophic growth of Xanthobacter flavus is dependent on the fixation of carbon dioxide via the Calvin cycle and on the oxidation of simple organic and inorganic compounds to provide the cell with energy. Maximal induction of the cbb and gap-pgk operons encoding enzymes of the Calvin cycle occurs in the absence of multicarbon substrates and the presence of methanol, formate, hydrogen, or thiosulfate. The LysR-type transcriptional regulator CbbR regulates the expression of the cbb and gap-pgk operons, but it is unknown to what cellular signal CbbR responds. In order to study the effects of low-molecular-weight compounds on the DNA-binding characteristics of CbbR, the protein was expressed in Escherichia coli and subsequently purified to homogeneity. CbbR of X. flavus is a dimer of 36-kDa subunits. DNA-binding assays suggested that two CbbR molecules bind to a 51-bp DNA fragment on which two inverted repeats containing the LysR motif are located. The addition of 200 μM NADPH, but not...
Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are k... more Chorismate mutase (CM) and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS) are key regulatory enzymes in L-Phe and L-Tyr biosynthesis in Amycolatopsis methanolica. At least two CM proteins, CMIa and CMIb, are required for the single chorismate mutase activity in the wild type. Component CMIa (a homodimeric protein with 16-kDa subunits) was purified to homogeneity (2,717-fold) and kinetically characterized. The partially purified CMIb preparation obtained also contained the single DS (DSI) activity detectable in the wild type. The activities of CMIa and CMIb were inhibited by both L-Phe and L-Tyr. DSI activity was inhibited by L-Trp, L-Phe, and L-Tyr. A leaky L-Phe-requiring auxotroph, mutant strain GH141, grown under L-Phe limitation, possessed additional DS (DSII) and CM (CMII) activities. Synthesis of both CMII and DSII was repressed by L-Phe. An ortho-DL-fluorophenylalanine-resistant mutant of the wild type (strain oFPHE83) that had lost the sensitivity of DSII a...
The ATP-dependent phosphofructokinase (ATP-PFK) of Streptomyces coelicolor A3(2) was purified to ... more The ATP-dependent phosphofructokinase (ATP-PFK) of Streptomyces coelicolor A3(2) was purified to homogeneity (1,600-fold) and characterized (110 kDa, with a single type of subunit of 40 kDa); it is allosterically inhibited by phosphoenolpyruvate. Cloning of the pfk gene of S. coelicolor A3(2) and analysis of the deduced amino acid sequence (343 amino acids; 36,667 Da) revealed high similarities to the PPi-PFK enzyme from Amycolatopsis methanolica (tetramer, nonallosteric; 70%) and to the allosteric ATP-PFK enzymes from other bacteria, e.g., Escherichia coli (tetramer; 37%) and Bacillus stearothermophilus (tetramer, 41%). Further structural and functional analysis of the two actinomycete PFK enzymes should elucidate the features of these proteins that determine substrate specificity (ATP versus PPi) and allosteric (in)sensitivity.
Auxotrophic mutants of the actinomycete Amycolatopsis methanolica requiring l-Phe or l-Tyr were i... more Auxotrophic mutants of the actinomycete Amycolatopsis methanolica requiring l-Phe or l-Tyr were isolated and identified as strains lacking prephenate dehydratase (strain GH71) or arogenate dehydrogenase (strain GH70), respectively. A. methanolica thus employs single pathways only for the biosynthesis of these aromatic amino acids. Anion-exchange chromatography of extracts revealed two peaks with Phe as well as Tyr aminotransferase (AT) activity (Phe/Tyr ATI and Phe/Tyr ATII) and three peaks with prephenate AT activity (Ppa ATI to Ppa ATIII). Phe/Tyr ATI and Ppa ATI coeluted and appear to function as the A. methanolica branched-chain amino acid AT. Ppa ATII probably functions as the aspartate AT. Mutant studies showed that Phe/Tyr ATII is the dominant AT in l-Phe biosynthesis and in l-Tyr catabolism but not in l-Tyr biosynthesis. Biochemical studies showed that Ppa ATIII is highly specific for prephenate and provided evidence that Ppa ATIII is the dominant AT in l-Tyr biosynthesis.
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