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Adrian Goldman - One of the best experts on this subject based on the ideXlab platform.
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Structure and function of the 3-carboxy-cis,cis-Muconate lactonizing enzyme from the protocatechuate degradative pathway of Agrobacterium radiobacter S2.
The FEBS journal, 2006Co-Authors: Sad Halak, Lari Lehtio, Tamara Basta, Sibylle Bürger, Matthias Contzen, Andreas Stolz, Adrian GoldmanAbstract:3-carboxy-cis,cis-Muconate lactonizing enzymes participate in the protocatechuate branch of the 3-oxoadipate pathway of various aerobic bacteria. The gene encoding a 3-carboxy-cis,cis-Muconate lactonizing enzyme (pcaB1S2) was cloned from a gene cluster involved in protocatechuate degradation by Agrobacterium radiobacter strain S2. This gene encoded for a 3-carboxy-cis,cis-Muconate lactonizing enzyme of 353 amino acids − significantly smaller than all previously studied 3-carboxy-cis,cis-Muconate lactonizing enzymes. This enzyme, ArCMLE1, was produced in Escherichia coli and shown to convert not only 3-carboxy-cis,cis-Muconate but also 3-sulfoMuconate. ArCMLE1 was purified as a His-tagged enzyme variant, and the basic catalytic constants for the conversion of 3-carboxy-cis,cis-Muconate and 3-sulfoMuconate were determined. In contrast, Agrobacterium tumefaciens 3-carboxy-cis,cis-Muconate lactonizing enzyme 1 could not, despite 87% sequence identity to ArCMLE1, use 3-sulfoMuconate as substrate. The crystal structure of ArCMLE1 was determined at 2.2 A resolution. Consistent with the sequence, it showed that the C-terminal domain, present in all other members of the fumarase II family, is missing in ArCMLE1. Nonetheless, both the tertiary and quaternary structures, and the structure of the active site, are similar to those of Pseudomonas putida 3-carboxy-cis,cis-Muconate lactonizing enzyme. One principal difference is that ArCMLE1 contains an Arg, as opposed to a Trp, in the active site. This indicates that activation of the carboxylic nucleophile by a hydrophobic environment is not required for lactonization, unlike earlier proposals [Yang J, Wang Y, Woolridge EM, Arora V, Petsko GA, Kozarich JW & Ringe D (2004) Biochemistry43, 10424–10434]. We identified citrate and isocitrate as noncompetitive inhibitors of ArCMLE1, and found a potential binding pocket for them on the enzyme outside the active site.
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the structure of pseudomonas p51 cl Muconate lactonizing enzyme co evolution of structure and dynamics with the dehalogenation function
Protein Science, 2003Co-Authors: Tommi Kajander, Michael Schlömann, Lari Lehtio, Adrian GoldmanAbstract:Bacterial Muconate lactonizing enzymes (MLEs) catalyze the conversion of cis,cis-Muconate as a part of the β-ketoadipate pathway, and some MLEs are also able to dehalogenate chlorinated Muconates (Cl-MLEs). The basis for the Cl-MLEs dehalogenating activity is still unclear. To further elucidate the differences between MLEs and Cl-MLEs, we have solved the structure of Pseudomonas P51 Cl-MLE at 1.95 A resolution. Comparison of Pseudomonas MLE and Cl-MLE structures reveals the presence of a large cavity in the Cl-MLEs. The cavity may be related to conformational changes on substrate binding in Cl-MLEs, at Gly52. Site-directed mutagenesis on Pseudomonas MLE core positions to the equivalent Cl-MLE residues showed that the variant Thr52Gly was rather inactive, whereas the Thr52Gly-Phe103Ser variant had regained part of the activity. These residues form a hydrogen bond in the Cl-MLEs. The Cl-MLE structure, as a result of the Thr-to-Gly change, is more flexible than MLE: As a mobile loop closes over the active site, a conformational change at Gly52 is observed in Cl-MLEs. The loose packing and structural motions in Cl-MLE may be required for the rotation of the lactone ring in the active site necessary for the dehalogenating activity of Cl-MLEs. Furthermore, we also suggest that differences in the active site mobile loop sequence between MLEs and Cl-MLEs result in lower active site polarity in Cl-MLEs, possibly affecting catalysis. These changes could result in slower product release from Cl-MLEs and make it a better enzyme for dehalogenation of substrate.
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3 carboxy cis cis Muconate lactonizing enzyme from neurospora crassa mad phasing with 80 selenomethionines
Acta Crystallographica Section D-biological Crystallography, 2002Co-Authors: Michael C Merckel, Tommi Kajander, A Thompson, Ashley M Deacon, Gunter J Grossmann, Nisse Kalkkinen, Adrian GoldmanAbstract:The structure of 3-carboxy-cis,cis-Muconate lactonizing enzyme from Neurospora crassa was determined at 3.0 A resolution. Phase information was derived from a multiwavelength anomalous dispersion (MAD) experiment conducted at three wavelengths using crystals of fully substituted selenomethionine protein. However, the structure determination was not routine owing to the relatively poor quality of the diffraction data and the large number of twofolds in the unit cell. Eventually, 80 selenium sites were identified by the combined use of direct methods and real-space map interpretation. This represents one of the largest selenium substructures solved and used for phasing. Some of the difficulties in the structure determination and the methods used to address them are discussed.
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the structure of neurospora crassa 3 carboxy cis cis Muconate lactonizing enzyme a β propeller cycloisomerase
Structure, 2002Co-Authors: Tommi Kajander, John W Kozarich, Michael C Merckel, A Thompson, Ashley M Deacon, Paul Mazur, Adrian GoldmanAbstract:Abstract Muconate lactonizing enzymes (MLEs) convert cis,cis -Muconates to muconolactones in microbes as part of the β-ketoadipate pathway; some also dehalogenate Muconate derivatives of xenobiotic haloaromatics. There are three different MLE classes unrelated by evolution. We present the X-ray structure of a eukaryotic MLE, Neurospora crassa 3-carboxy- cis,cis -Muconate lactonizing enzyme ( Nc CMLE) at 2.5 A resolution, with a seven-bladed β propeller fold. It is related neither to bacterial MLEs nor to other β propeller enzymes, but is structurally similar to the G protein β subunit. It reveals a novel metal-independent cycloisomerase motif unlike the bacterial metal cofactor MLEs. Together, the bacterial MLEs and Nc CMLE structures comprise a striking structural example of functional convergence in enzymes for 1,2-addition-elimination of carboxylic acids. Nc CMLE and bacterial MLEs may enhance the reaction rate differently: the former by electrophilic catalysis and the latter by electrostatic stabilization of the enolate.
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structural basis for the activity of two Muconate cycloisomerase variants toward substituted Muconates
Proteins, 1999Co-Authors: Ursula Schell, Michael Schlömann, Adrian Goldman, Tommi Kajander, Sari HelinAbstract:We have refined to 2.3 A resolution two Muconate cycloisomerase (MCIase) variant structures, F329I and I54V, that differ from each other and from wild-type in their activity toward cis,cis-Muconate (CCM) and substituted CCMs. The working and free R-factors for F329I are 17.4/21.6% and for I54V, 17.6/22.3% with good stereochemistry. Except for the mutated residue, there are no significant changes in structure. To understand the differences in enzymatic properties we docked substituted CCMs and CCM into the active sites of the variants and wild type. The extra space the mutations create appears to account for most of the enzymatic differences. The lack of other structural changes explains why, although structurally equivalent changes occur in chloroMuconate cycloisomerase (CMCIase), the changes in themselves do not convert a MCIase into a dehalogenating CMCIase. Reanalysis of the CMCIase structure revealed only one general acid/base, K169. The structural implication is that, in 2-chloro-CCM conversion by CMCIase, the lactone ring of 5-chloromuconolactone rotates before dehalogenation to bring the acidic C4 proton next to K169. Therefore, K169 alone performs both required protonation and deprotonation steps, the first at C5 as in MCIase, and the second, after ring rotation, at C4. This distinguishes CMCIase from α/β barrel isomerases and racemases, which use two different bases. Proteins 1999;34:125–136. © 1999 Wiley-Liss, Inc.
Michael Schlömann - One of the best experts on this subject based on the ideXlab platform.
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the structure of pseudomonas p51 cl Muconate lactonizing enzyme co evolution of structure and dynamics with the dehalogenation function
Protein Science, 2003Co-Authors: Tommi Kajander, Michael Schlömann, Lari Lehtio, Adrian GoldmanAbstract:Bacterial Muconate lactonizing enzymes (MLEs) catalyze the conversion of cis,cis-Muconate as a part of the β-ketoadipate pathway, and some MLEs are also able to dehalogenate chlorinated Muconates (Cl-MLEs). The basis for the Cl-MLEs dehalogenating activity is still unclear. To further elucidate the differences between MLEs and Cl-MLEs, we have solved the structure of Pseudomonas P51 Cl-MLE at 1.95 A resolution. Comparison of Pseudomonas MLE and Cl-MLE structures reveals the presence of a large cavity in the Cl-MLEs. The cavity may be related to conformational changes on substrate binding in Cl-MLEs, at Gly52. Site-directed mutagenesis on Pseudomonas MLE core positions to the equivalent Cl-MLE residues showed that the variant Thr52Gly was rather inactive, whereas the Thr52Gly-Phe103Ser variant had regained part of the activity. These residues form a hydrogen bond in the Cl-MLEs. The Cl-MLE structure, as a result of the Thr-to-Gly change, is more flexible than MLE: As a mobile loop closes over the active site, a conformational change at Gly52 is observed in Cl-MLEs. The loose packing and structural motions in Cl-MLE may be required for the rotation of the lactone ring in the active site necessary for the dehalogenating activity of Cl-MLEs. Furthermore, we also suggest that differences in the active site mobile loop sequence between MLEs and Cl-MLEs result in lower active site polarity in Cl-MLEs, possibly affecting catalysis. These changes could result in slower product release from Cl-MLEs and make it a better enzyme for dehalogenation of substrate.
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mechanism of chloride elimination from 3 chloro and 2 4 dichloro cis cis Muconate new insight obtained from analysis of Muconate cycloisomerase variant catb k169a
Journal of Bacteriology, 2001Co-Authors: Ursula Kaulmann, Stefan R Kaschabek, Michael SchlömannAbstract:ChloroMuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-cis,cis-Muconate to cis-dienelactone (cis-4-carboxymethylenebut-2-en-4-olide), while usual Muconate cycloisomerases transform the same substrate to the bacteriotoxic protoanemonin. Formation of protoanemonin requires that the cycloisomerization of 3-chloro-cis,cis-Muconate to 4-chloromuconolactone is completed by protonation of the exocyclic carbon of the presumed enol/enolate intermediate before chloride elimination and decarboxylation take place to yield the final product. The formation of cis-dienelactone, in contrast, could occur either by dehydrohalogenation of 4-chloromuconolactone or, more directly, by chloride elimination from the enol/enolate intermediate. To reach a better understanding of the mechanisms of chloride elimination, the proton-donating Lys169 of Pseudomonas putida Muconate cycloisomerase was changed to alanine. As expected, substrates requiring protonation, such as cis,cis-Muconate as well as 2- and 3-methyl-, 3-fluoro-, and 2-chloro-cis,cis-Muconate, were not converted at a significant rate by the K169A variant. However, the variant was still active with 3-chloro- and 2,4-dichloro-cis,cis-Muconate. Interestingly, cis-dienelactone and 2-chloro-cis-dienelactone were formed as products, whereas the wild-type enzyme forms protoanemonin and the not previously isolated 2-chloroprotoanemonin, respectively. Thus, the chloroMuconate cycloisomerases may avoid (chloro-)protoanemonin formation by increasing the rate of chloride abstraction from the enol/enolate intermediate compared to that of proton addition to it.
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A new type of Muconate cycloisomerase from Rhodococcus rhodochrous strain 89.
Biochemistry. Biokhimiia, 2001Co-Authors: Inna P. Solyanikova, Michael Schlömann, L. A. GolovlevaAbstract:Muconate cycloisomerase (MCI) was purified from Rhodococcus rhodochrous 89 grown on phenol. The enzyme appears to contain two different type subunits with molecular masses 35.5 and 37 kD. The N-terminal amino acid sequence of both subunits showed more similarity to corresponding enzymes from gram-negative bacteria than to one from Rhodococcus opacus 1CP. MCI from R. rhodochrous 89, like analogous enzymes from gram-negative bacteria, can convert 2-chloroMuconate (2-CM) with the formation of both, 2- and 5-chloromuconolactones (CML) as intermediates. Nevertheless, its unique ability to convert 5-CML to cis- but not to trans-dienelactone sets it apart from all known chloroMuconate cycloisomerases from gram-negative and gram-positive bacteria.
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structural basis for the activity of two Muconate cycloisomerase variants toward substituted Muconates
Proteins, 1999Co-Authors: Ursula Schell, Michael Schlömann, Adrian Goldman, Tommi Kajander, Sari HelinAbstract:We have refined to 2.3 A resolution two Muconate cycloisomerase (MCIase) variant structures, F329I and I54V, that differ from each other and from wild-type in their activity toward cis,cis-Muconate (CCM) and substituted CCMs. The working and free R-factors for F329I are 17.4/21.6% and for I54V, 17.6/22.3% with good stereochemistry. Except for the mutated residue, there are no significant changes in structure. To understand the differences in enzymatic properties we docked substituted CCMs and CCM into the active sites of the variants and wild type. The extra space the mutations create appears to account for most of the enzymatic differences. The lack of other structural changes explains why, although structurally equivalent changes occur in chloroMuconate cycloisomerase (CMCIase), the changes in themselves do not convert a MCIase into a dehalogenating CMCIase. Reanalysis of the CMCIase structure revealed only one general acid/base, K169. The structural implication is that, in 2-chloro-CCM conversion by CMCIase, the lactone ring of 5-chloromuconolactone rotates before dehalogenation to bring the acidic C4 proton next to K169. Therefore, K169 alone performs both required protonation and deprotonation steps, the first at C5 as in MCIase, and the second, after ring rotation, at C4. This distinguishes CMCIase from α/β barrel isomerases and racemases, which use two different bases. Proteins 1999;34:125–136. © 1999 Wiley-Liss, Inc.
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substrate specificity of and product formation by Muconate cycloisomerases an analysis of wild type enzymes and engineered variants
Applied and Environmental Microbiology, 1998Co-Authors: Martin Dominik Vollmer, Adrian Goldman, Ursula Schell, Helga Hoier, Hansjurgen Hecht, Janosch A D Groning, Michael SchlömannAbstract:Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-Muconate, the product of catechol ring cleavage, to (4S)-muconolactone. ChloroMuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the Muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter "calcoaceticus" ADP1. We show that they transform, in addition to cis,cis-Muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis, cis-Muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-Muconate. Based on known three-dimensional structures, variants of P. putida Muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloroMuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloroMuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-Muconate conversion. The rate of 2-chloro-cis,cis-Muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-Muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of Muconate and chloroMuconate cycloisomerases are due to a number of features that can be changed independently of each other.
John W Kozarich - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of 3 carboxy cis cis Muconate lactonizing enzyme from pseudomonas putida a fumarase class ii type cycloisomerase enzyme evolution in parallel pathways
Biochemistry, 2004Co-Authors: Jian Yang, Y Wang, Elisa M Woolridge, Vandana Arora, Gregory A Petsko, John W Kozarich, Dagmar RingeAbstract:3-Carboxy-cis,cis-Muconate lactonizing enzymes (CMLEs), the key enzymes in the protocatechuate branch of the β-ketoadipate pathway in microorganisms, catalyze the conversion of 3-carboxy-cis,cis-Muconate to muconolactones. We have determined the crystal structure of the prokaryotic Pseudomonas putida CMLE (PpCMLE) at 2.6 A resolution. PpCMLE is a homotetramer and belongs to the fumarase class II superfamily. The active site of PpCMLE is formed largely by three regions, which are moderately conserved in the fumarase class II superfamily, from three respective monomers. It has been proposed that residue His141, which is highly conserved in all fumarase class II enzymes and forms a charge relay with residue Glu275 (both His141 and Glu275 are in adenylosuccinate lyase numbering), acts as the general base in most fumarase class II superfamily members. However, this charge relay pair is broken in PpCMLE. The residues corresponding to His141 and Glu275 are Trp153 and Ala289, respectively, in PpCMLE. The structur...
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the structure of neurospora crassa 3 carboxy cis cis Muconate lactonizing enzyme a β propeller cycloisomerase
Structure, 2002Co-Authors: Tommi Kajander, John W Kozarich, Michael C Merckel, A Thompson, Ashley M Deacon, Paul Mazur, Adrian GoldmanAbstract:Abstract Muconate lactonizing enzymes (MLEs) convert cis,cis -Muconates to muconolactones in microbes as part of the β-ketoadipate pathway; some also dehalogenate Muconate derivatives of xenobiotic haloaromatics. There are three different MLE classes unrelated by evolution. We present the X-ray structure of a eukaryotic MLE, Neurospora crassa 3-carboxy- cis,cis -Muconate lactonizing enzyme ( Nc CMLE) at 2.5 A resolution, with a seven-bladed β propeller fold. It is related neither to bacterial MLEs nor to other β propeller enzymes, but is structurally similar to the G protein β subunit. It reveals a novel metal-independent cycloisomerase motif unlike the bacterial metal cofactor MLEs. Together, the bacterial MLEs and Nc CMLE structures comprise a striking structural example of functional convergence in enzymes for 1,2-addition-elimination of carboxylic acids. Nc CMLE and bacterial MLEs may enhance the reaction rate differently: the former by electrophilic catalysis and the latter by electrostatic stabilization of the enolate.
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characterization of two crystal forms of 3 carboxy cis cis Muconate lactonizing enzyme from pseudomonas putida
Acta Crystallographica Section D-biological Crystallography, 1997Co-Authors: Miriam S Hasson, Elisa M Woolridge, Gregory A Petsko, John W Kozarich, Ilme Schlichting, Margaret M Mcgowen, Dagmar RingeAbstract:Two crystal forms of 3-carboxy-cis,cis-Muconate lactonizing enzyme from Pseudomonas putida have been characterized. Form A is in space group P6, with unit-cell dimensions a = b = 232, c = 79 A, alpha = beta = 90, gamma = 120 degrees. Form B is orthorhombic, with cell dimensions a = 163, b = 139, c = 90 A alpha = beta = gamma = 90 degrees.
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crystallization and preliminary crystallographic analysis of 3 carboxy cis cis Muconate lactonizing enzyme from neurospora crassa
Acta Crystallographica Section D-biological Crystallography, 1996Co-Authors: T Glumoff, John W Kozarich, Paul Mazur, S Helin, Adrian GoldmanAbstract:Crystals of 3-carboxy-cis,cis-Muconate lactonizing enzyme (CMLE; E.C. 5.5.1.5) from Neurospora crassa that diffract to high resolution have been obtained. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit-cell dimensions a = 92.1, b = 159.7, c = 236.6 A (at 103 K) and diffract at most to 2 A resolution. The asymmetric unit of the crystals appears to contain two tetrameric CMLE molecules making up a total of 328 kDa per asymmetric unit. Both cross-linking with glutaraldehyde and cryo-cooling to 103 K have been used to facilitate data collection because the crystals are unstable in the X-ray beam; both techniques extend the crystal lifetime but cryo-cooling, unlike glutaraldehyde cross-linking, does not lower the quality of the diffraction pattern.
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3 carboxy cis cis Muconate lactonizing enzyme from neurospora crassa an alternate cycloisomerase motif
Journal of Bacteriology, 1994Co-Authors: Paul Mazur, W J Henzel, Seema Mattoo, John W KozarichAbstract:3-Carboxy-cis,cis-Muconate lactonizing enzyme (CMLE; EC 5.5.1.5) from Neurospora crassa catalyzes the reversible gamma-lactonization of 3-carboxy-cis,cis-Muconate by a syn-1,2 addition-elimination reaction. The stereochemical and regiochemical course of the reaction is (i) opposite that of CMLE from Pseudomonas putida (EC 5.5.1.2) and (ii) identical to that of cis,cis-Muconate lactonizing enzyme (MLE; EC 5.5.1.1) from P. putida. In order to determine the mechanistic and evolutionary relationships between N. crassa CMLE and the procaryotic cycloisomerases, we have purified CMLE from N. crassa to homogeneity and determined its nucleotide sequence from a cDNA clone isolated from a p-hydroxybenzoate-induced N. crassa cDNA library. The deduced amino acid sequence predicts a protein of 41.2 kDa (365 residues) which does not exhibit sequence similarity with any of the bacterial cycloisomerases. The cDNA encoding N. crassa CMLE was expressed in Escherichia coli, and the purified recombinant protein exhibits physical and kinetic properties equivalent to those found for the isolated N. crassa enzyme. We also report that N. crassa CMLE possesses substantially reduced yet significant levels of MLE activity with cis,cis-Muconate and, furthermore, does not appear to be dependent on divalent metals for activity. These data suggest that the N. crassa CMLE may represent a novel eucaryotic motif in the cycloisomerase enzyme family.
Akikazu Matsumoto - One of the best experts on this subject based on the ideXlab platform.
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epitaxial crystal growth and solid state polymerization of piperonyl Muconate on the 001 surface of kcl crystal for controlling polymer chain alignment
ACS Applied Materials & Interfaces, 2012Co-Authors: Katsuya Onodera, Chiaki Tanioku, Akikazu MatsumotoAbstract:We investigated the crystal growth of piperonyl (E,E)-Muconate [bis(3,4-methylenedioxybenzyl) (E,E)-Muconate, MDO] on inorganic crystalline substrates during vapor deposition for the control of polymer chain alignment by the subsequent solid-state photopolymerization of the MDO monomer thin films deposited on the substrate. We controlled the arrangement of the MDO molecules and the polymer chains produced on the substrate, depending on the lattice parameters of the substrate surfaces used. The epitaxial crystal growth of MDO on the {001} plane of a KCl single crystal was observed under the condition that the crystal lattice lengths of MDO agreed well with the specific space distance of the substrate; i.e., the KCl cubic crystal resulted in a d110 value of 4.45 A, which was very close to the value of the monomer stacking distance in the MDO crystal (ds = 4.43 A). On the other hand, slightly large and too small d110 values for KBr and NaCl, respectively, resulted in the less controlled and no epitaxial crys...
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single crystal to single crystal transformation of di isopropylammonium z z Muconate into the e e Muconate during one way photoisomerization in the solid state
Crystal Growth & Design, 2011Co-Authors: Natsuko Nishizawa, Junya Nakamura, Akikazu MatsumotoAbstract:The solid-state EZ photoisomerization of di(isopropylammonium) (Z,Z)-Muconate (ZZ-1a) into the corresponding EE isomer (EE-1a) via a single-crystal-to-single-crystal reaction process was investigated. The molecular motion based on a bicycle-pedal model was directly observed during the photoisomerization in the crystals by an X-ray single crystal structure analysis. The photoirradiation of ZZ-1a provided EE-1a in a quantitative yield via a one-way isomerization mechanism, being different from the solution products in the photostationary state. The isomerization reactivity was discussed for the ammonium and ester derivatives of muconic acid.
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direct observation of change in the molecular structure of benzyl z z Muconate during photoisomerization in the solid state
Chemical Communications, 2008Co-Authors: Daisuke Furukawa, Seiya Kobatake, Akikazu MatsumotoAbstract:For the solid-state photoisomerization of benzyl (Z,Z)-Muconate to the corresponding (E,E)-Muconate, the direct observation of a change in the crystal structure has revealed that the isomerization occurs by a topochemical reaction process according to a bicycle-pedal model and is finally accompanied by a phase transition to a stable crystal structure.
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photodimerization mechanism of bis 3 4 5 trifluorobenzyl e e Muconate in a columnar assembly in the crystalline state
Chemistry Letters, 2007Co-Authors: Yutaka Mori, Akikazu MatsumotoAbstract:We have revealed the photoreaction mechanism for the single-crystal-to-single-crystal transformation of bis(3,4,5-trifluorobenzyl) (E,E)-Muconate (1) to the corresponding dimer. The [2 + 2] photodi...
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synthesis of new stereoregular host polymers for organic intercalation by solid state hydrolysis using layered syndiotactic polymer crystals
Chemistry Letters, 2005Co-Authors: Shinya Oshita, Toshihiro Tanaka, Akikazu MatsumotoAbstract:meso- and racemo-disyndiotactic Muconate polymers, which were prepared by the solid-state polymerization of the substituted benzyl esters of muconic acid, were converted to the corresponding stereo...
Gregg T Beckham - One of the best experts on this subject based on the ideXlab platform.
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Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase
2019Co-Authors: Ramesh K. Jha, Christopher W Johnson, Gregg T Beckham, Niju Narayanan, Naresh Pandey, Jeremy M. Bingen, Theresa L. Kern, Charlie E. M. Strauss, Scott P. Hennelly, Taraka DaleAbstract:Product inhibition is a frequent bottleneck in industrial enzymes, and testing mutations to alleviate product inhibition via traditional methods remains challenging as many variants need to be tested against multiple substrate and product concentrations. Further, traditional screening methods are conducted in vitro, and resulting enzyme variants may perform differently in vivo in the context of whole-cell metabolism and regulation. In this study, we address these two problems by establishing a high-throughput screening method to alleviate product inhibition in an industrially relevant enzyme, chorismate pyruvate-lyase (UbiC). First, we engineered a highly specific, genetically encoded biosensor for 4-hydroxybenzoate (4HB) in an industrially relevant host, Pseudomonas putida KT2440. We subsequently applied the biosensor to detect the activity of a heterologously expressed UbiC that converts chorismate into 4HB and pyruvate. By using benzoate as a product surrogate that inhibits UbiC without activating the biosensor, we were able to efficiently create and screen a diversified library for UbiC variants with reduced product inhibition. Introduction of the improved UbiC enzyme variant into an experimental production strain for the industrial precursor cis,cis-muconic acid (Muconate), enabled a >2-fold yield improvement for glucose to Muconate conversion when the new UbiC variant was expressed from a plasmid and a 60% yield increase when the same UbiC variant was genomically integrated into the strain. Overall, this work demonstrates that by coupling a library of enzyme variants to whole-cell catalysis and biosensing, variants with reduced product inhibition can be identified, and that this improved enzyme can result in increased titers of a downstream molecule of interest
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iodine catalyzed isomerization of dimethyl Muconate
Chemsuschem, 2018Co-Authors: Gregg T Beckham, Amy E Settle, Laura Berstis, Shuting Zhang, Nicholas A Rorrer, Ryan M Richards, Michael F Crowley, Derek R. VardonAbstract:cis,cis-Muconic acid is a platform bio-based chemical that can be upgraded to drop-in commodity and novel monomers. Among the possible drop-in products, dimethyl terephthalate can be synthesized via esterification, isomerization, Diels-Alder cycloaddition, and dehydrogenation. The isomerization of cis,cis-dimethyl Muconate (ccDMM) to the trans,trans-form (ttDMM) can be catalyzed by iodine; however, studies have yet to address (i) the mechanism and reaction barriers unique to DMM, and (ii) the influence of solvent, potential for catalyst recycle, and recovery of high-purity ttDMM. To address this gap, we apply a joint computational and experimental approach to investigate iodine-catalyzed isomerization of DMM. Density functional theory calculations identified unique regiochemical considerations owing to the large number of halogen-diene coordination schemes. Both transition state theory and experiments estimate significant barrier reductions with photodissociated iodine. Solvent selection was critical for rapid kinetics, likely because of solvent complexation with iodine. Under select conditions, ttDMM yields of 95 % were achieved in 98 %) with crystallization. Lastly, post-reaction iodine can be recovered and recycled with minimal loss of activity. Overall, these findings provide new insight into the mechanism and conditions necessary for DMM isomerization with iodine to advance the state-of-the-art for bio-based chemicals.
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eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to Muconate in pseudomonas putida kt2440
Metabolic Engineering Communications, 2017Co-Authors: Christopher W Johnson, Paul E Abraham, Jeffrey G Linger, Payal Khanna, Robert L Hettich, Gregg T BeckhamAbstract:Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural environments, but may hinder their performance as domesticated microbial cell factories. In a Pseudomonas putida KT2440 strain engineered to convert lignin-derived aromatic monomers such as p-coumarate and ferulate to Muconate, a precursor to bio-based nylon and other chemicals, metabolic intermediates including 4-hydroxybenzoate and vanillate accumulate and subsequently reduce productivity. We hypothesized that these metabolic bottlenecks may be, at least in part, the effect of carbon catabolite repression caused by glucose or acetate, more preferred substrates that must be provided to the strain for supplementary energy and cell growth. Using mass spectrometry-based proteomics, we have identified the 4-hydroxybenzoate hydroxylase, PobA, and the vanillate demethylase, VanAB, as targets of the Catabolite Repression Control (Crc) protein, a global regulator of carbon catabolite repression. By deleting the gene encoding Crc from this strain, the accumulation of 4-hydroxybenzoate and vanillate are reduced and, as a result, Muconate production is enhanced. In cultures grown on glucose, the yield of Muconate produced from p-coumarate after 36 h was increased nearly 70% with deletion of the gene encoding Crc (94.6 ± 0.6% vs. 56.0 ± 3.0% (mol/mol)) while the yield from ferulate after 72 h was more than doubled (28.3 ± 3.3% vs. 12.0 ± 2.3% (mol/mol)). The effect of eliminating Crc was similar in cultures grown on acetate, with the yield from p-coumarate just slightly higher in the Crc deletion strain after 24 h (47.7 ± 0.6% vs. 40.7 ± 3.6% (mol/mol)) and the yield from ferulate increased more than 60% after 72 h (16.9 ± 1.4% vs. 10.3 ± 0.1% (mol/mol)). These results are an example of the benefit that reducing carbon catabolite repression can have on conversion of complex feedstocks by microbial cell factories, a concept we posit could be broadly considered as a strategy in metabolic engineering for conversion of renewable feedstocks to value-added chemicals.
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enhancing muconic acid production from glucose and lignin derived aromatic compounds via increased protocatechuate decarboxylase activity
Metabolic Engineering Communications, 2016Co-Authors: Christopher W Johnson, Payal Khanna, Davinia Salvachúa, Holly Smith, Darren J. Peterson, Gregg T BeckhamAbstract:The conversion of biomass-derived sugars and aromatic molecules to cis,cis-muconic acid (referred to hereafter as muconic acid or Muconate) has been of recent interest owing to its facile conversion to adipic acid, an important commodity chemical. Metabolic routes to produce Muconate from both sugars and many lignin-derived aromatic compounds require the use of a decarboxylase to convert protocatechuate (PCA, 3,4-dihydroxybenzoate) to catechol (1,2-dihydroxybenzene), two central aromatic intermediates in this pathway. Several studies have identified the PCA decarboxylase as a metabolic bottleneck, causing an accumulation of PCA that subsequently reduces Muconate production. A recent study showed that activity of the PCA decarboxylase is enhanced by co-expression of two genetically associated proteins, one of which likely produces a flavin-derived cofactor utilized by the decarboxylase. Using entirely genome-integrated gene expression, we have engineered Pseudomonas putida KT2440-derived strains to produce Muconate from either aromatic molecules or sugars and demonstrate in both cases that co-expression of these decarboxylase associated proteins reduces PCA accumulation and enhances Muconate production relative to strains expressing the PCA decarboxylase alone. In bioreactor experiments, co-expression increased the specific productivity (mg/g cells/h) of Muconate from the aromatic lignin monomer p-coumarate by 50% and resulted in a titer of >15 g/L. In strainsmore » engineered to produce Muconate from glucose, co-expression more than tripled the titer, yield, productivity, and specific productivity, with the best strain producing 4.92+/-0.48 g/L Muconate. Furthermore, this study demonstrates that overcoming the PCA decarboxylase bottleneck can increase Muconate yields from biomass-derived sugars and aromatic molecules in industrially relevant strains and cultivation conditions.« less
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Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity
Elsevier, 2016Co-Authors: Christopher W Johnson, Payal Khanna, Davinia Salvachúa, Holly Smith, Darren J. Peterson, Gregg T BeckhamAbstract:The conversion of biomass-derived sugars and aromatic molecules to cis,cis-muconic acid (referred to hereafter as muconic acid or Muconate) has been of recent interest owing to its facile conversion to adipic acid, an important commodity chemical. Metabolic routes to produce Muconate from both sugars and many lignin-derived aromatic compounds require the use of a decarboxylase to convert protocatechuate (PCA, 3,4-dihydroxybenzoate) to catechol (1,2-dihydroxybenzene), two central aromatic intermediates in this pathway. Several studies have identified the PCA decarboxylase as a metabolic bottleneck, causing an accumulation of PCA that subsequently reduces Muconate production. A recent study showed that activity of the PCA decarboxylase is enhanced by co-expression of two genetically associated proteins, one of which likely produces a flavin-derived cofactor utilized by the decarboxylase. Using entirely genome-integrated gene expression, we have engineered Pseudomonas putida KT2440-derived strains to produce Muconate from either aromatic molecules or sugars and demonstrate in both cases that co-expression of these decarboxylase associated proteins reduces PCA accumulation and enhances Muconate production relative to strains expressing the PCA decarboxylase alone. In bioreactor experiments, co-expression increased the specific productivity (mg/g cells/h) of Muconate from the aromatic lignin monomer p-coumarate by 50% and resulted in a titer of >15 g/L. In strains engineered to produce Muconate from glucose, co-expression more than tripled the titer, yield, productivity, and specific productivity, with the best strain producing 4.92±0.48 g/L Muconate. This study demonstrates that overcoming the PCA decarboxylase bottleneck can increase Muconate yields from biomass-derived sugars and aromatic molecules in industrially relevant strains and cultivation conditions. Keywords: Protocatechuate decarboxylase, Pseudomonas Putida KT2440, cis,cis-Muconate, Muconic acid, Lignin valorizatio