1d6f
From Proteopedia
(New page: 200px<br /><applet load="1d6f" size="450" color="white" frame="true" align="right" spinBox="true" caption="1d6f, resolution 1.69Å" /> '''CHALCONE SYNTHASE C1...) |
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| - | [[Image:1d6f.jpg|left|200px]]<br /><applet load="1d6f" size=" | + | [[Image:1d6f.jpg|left|200px]]<br /><applet load="1d6f" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1d6f, resolution 1.69Å" /> | caption="1d6f, resolution 1.69Å" /> | ||
'''CHALCONE SYNTHASE C164A MUTANT'''<br /> | '''CHALCONE SYNTHASE C164A MUTANT'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid | + | Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases. |
==About this Structure== | ==About this Structure== | ||
| - | 1D6F is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Medicago_sativa Medicago sativa] with SO4 and B3P as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Naringenin-chalcone_synthase Naringenin-chalcone synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.3.1.74 2.3.1.74] Full crystallographic information is available from [http:// | + | 1D6F is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Medicago_sativa Medicago sativa] with <scene name='pdbligand=SO4:'>SO4</scene> and <scene name='pdbligand=B3P:'>B3P</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Naringenin-chalcone_synthase Naringenin-chalcone synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.3.1.74 2.3.1.74] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1D6F OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Naringenin-chalcone synthase]] | [[Category: Naringenin-chalcone synthase]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Bowman, M | + | [[Category: Bowman, M E.]] |
| - | [[Category: Dixon, R | + | [[Category: Dixon, R A.]] |
| - | [[Category: Ferrer, J | + | [[Category: Ferrer, J L.]] |
| - | [[Category: Jez, J | + | [[Category: Jez, J M.]] |
| - | [[Category: Noel, J | + | [[Category: Noel, J P.]] |
[[Category: B3P]] | [[Category: B3P]] | ||
[[Category: SO4]] | [[Category: SO4]] | ||
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[[Category: site- directed mutant]] | [[Category: site- directed mutant]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:13:27 2008'' |
Revision as of 10:13, 21 February 2008
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CHALCONE SYNTHASE C164A MUTANT
Overview
Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.
About this Structure
1D6F is a Single protein structure of sequence from Medicago sativa with and as ligands. Active as Naringenin-chalcone synthase, with EC number 2.3.1.74 Full crystallographic information is available from OCA.
Reference
Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase., Jez JM, Ferrer JL, Bowman ME, Dixon RA, Noel JP, Biochemistry. 2000 Feb 8;39(5):890-902. PMID:10653632
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