Structural basis for inverting the enantioselectivity of arylmalonate decarboxylase revealed by the structural analysis of the Gly74Cys/Cysl88Ser mutant in the liganded form

Rika Obata, Masayoshi Nakasako

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Arylmalonate decarboxylase catalyzes the enantioselective decarboxylation of α-aryl-α-methylmalonate to produce optically pure α-arylpropionate. The enzyme is comprised of two α/β domains and contains an active site situated between the two domains. The site is formed by Tyr48, Gly74-Thr75-Ser76, Tyr126, and Cys188-Gly189-Gly190 residues. Since it has been observed that the Gly74Cys/Cys188Ser mutation inverts the enantioselectivity of the enzyme, we determined the crystal structure of the Gly74Cys/ Cys188Ser mutant in the liganded form at a resolution of 1.45 Å to understand the structural basis for this inversion. The overall structure of the enzyme overlapped well with that of the benzylphosphonate-associated wild-type enzyme, and the mutations had little effect on the structure of the active site. A ligand molecule bound to the active site in an unusual semiplanar conformation resembling the planar enediolate reaction intermediate could be assigned as phenyl acetate. The inversion in enantioselectivity by the paired mutation is explained by the mirror symmetry between Cys74 in the mutant and Cys188 of the wild type with respect to the carbon atom in the ligand to be protonated. Comparison of the wild-type and Gly74Cys mutant crystal structures suggested that ligand binding induces a positional shift of the Cys188-Gly189-Gly190 region toward the Gly74-Thr75 pair which provides two oxyanion holes necessary to stabilize the negatively charged enediolate reaction intermediate. The ligand binding also simultaneously induces the formation of a hydrophobic cluster over the active site cleft. Thus, AMDase is proposed to have "open" and "closed" conformations of the active site that are regulated by ligand binding. These results may provide an effective strategy for the rational design to invert the enantioselectivity of enzymes.

Original languageEnglish
Pages (from-to)1963-1969
Number of pages7
JournalBiochemistry
Volume49
Issue number9
DOIs
Publication statusPublished - 2010 Mar 9

Fingerprint

Enantioselectivity
Structural analysis
Catalytic Domain
Ligands
Enzymes
Reaction intermediates
Mutation
Conformations
Crystal structure
Decarboxylation
Mirrors
Carbon
malonate decarboxylase
Atoms
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{b81b5bcd49b048fc881c5dd0a7493ba5,
title = "Structural basis for inverting the enantioselectivity of arylmalonate decarboxylase revealed by the structural analysis of the Gly74Cys/Cysl88Ser mutant in the liganded form",
abstract = "Arylmalonate decarboxylase catalyzes the enantioselective decarboxylation of α-aryl-α-methylmalonate to produce optically pure α-arylpropionate. The enzyme is comprised of two α/β domains and contains an active site situated between the two domains. The site is formed by Tyr48, Gly74-Thr75-Ser76, Tyr126, and Cys188-Gly189-Gly190 residues. Since it has been observed that the Gly74Cys/Cys188Ser mutation inverts the enantioselectivity of the enzyme, we determined the crystal structure of the Gly74Cys/ Cys188Ser mutant in the liganded form at a resolution of 1.45 {\AA} to understand the structural basis for this inversion. The overall structure of the enzyme overlapped well with that of the benzylphosphonate-associated wild-type enzyme, and the mutations had little effect on the structure of the active site. A ligand molecule bound to the active site in an unusual semiplanar conformation resembling the planar enediolate reaction intermediate could be assigned as phenyl acetate. The inversion in enantioselectivity by the paired mutation is explained by the mirror symmetry between Cys74 in the mutant and Cys188 of the wild type with respect to the carbon atom in the ligand to be protonated. Comparison of the wild-type and Gly74Cys mutant crystal structures suggested that ligand binding induces a positional shift of the Cys188-Gly189-Gly190 region toward the Gly74-Thr75 pair which provides two oxyanion holes necessary to stabilize the negatively charged enediolate reaction intermediate. The ligand binding also simultaneously induces the formation of a hydrophobic cluster over the active site cleft. Thus, AMDase is proposed to have {"}open{"} and {"}closed{"} conformations of the active site that are regulated by ligand binding. These results may provide an effective strategy for the rational design to invert the enantioselectivity of enzymes.",
author = "Rika Obata and Masayoshi Nakasako",
year = "2010",
month = "3",
day = "9",
doi = "10.1021/bi9015605",
language = "English",
volume = "49",
pages = "1963--1969",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "9",

}

TY - JOUR

T1 - Structural basis for inverting the enantioselectivity of arylmalonate decarboxylase revealed by the structural analysis of the Gly74Cys/Cysl88Ser mutant in the liganded form

AU - Obata, Rika

AU - Nakasako, Masayoshi

PY - 2010/3/9

Y1 - 2010/3/9

N2 - Arylmalonate decarboxylase catalyzes the enantioselective decarboxylation of α-aryl-α-methylmalonate to produce optically pure α-arylpropionate. The enzyme is comprised of two α/β domains and contains an active site situated between the two domains. The site is formed by Tyr48, Gly74-Thr75-Ser76, Tyr126, and Cys188-Gly189-Gly190 residues. Since it has been observed that the Gly74Cys/Cys188Ser mutation inverts the enantioselectivity of the enzyme, we determined the crystal structure of the Gly74Cys/ Cys188Ser mutant in the liganded form at a resolution of 1.45 Å to understand the structural basis for this inversion. The overall structure of the enzyme overlapped well with that of the benzylphosphonate-associated wild-type enzyme, and the mutations had little effect on the structure of the active site. A ligand molecule bound to the active site in an unusual semiplanar conformation resembling the planar enediolate reaction intermediate could be assigned as phenyl acetate. The inversion in enantioselectivity by the paired mutation is explained by the mirror symmetry between Cys74 in the mutant and Cys188 of the wild type with respect to the carbon atom in the ligand to be protonated. Comparison of the wild-type and Gly74Cys mutant crystal structures suggested that ligand binding induces a positional shift of the Cys188-Gly189-Gly190 region toward the Gly74-Thr75 pair which provides two oxyanion holes necessary to stabilize the negatively charged enediolate reaction intermediate. The ligand binding also simultaneously induces the formation of a hydrophobic cluster over the active site cleft. Thus, AMDase is proposed to have "open" and "closed" conformations of the active site that are regulated by ligand binding. These results may provide an effective strategy for the rational design to invert the enantioselectivity of enzymes.

AB - Arylmalonate decarboxylase catalyzes the enantioselective decarboxylation of α-aryl-α-methylmalonate to produce optically pure α-arylpropionate. The enzyme is comprised of two α/β domains and contains an active site situated between the two domains. The site is formed by Tyr48, Gly74-Thr75-Ser76, Tyr126, and Cys188-Gly189-Gly190 residues. Since it has been observed that the Gly74Cys/Cys188Ser mutation inverts the enantioselectivity of the enzyme, we determined the crystal structure of the Gly74Cys/ Cys188Ser mutant in the liganded form at a resolution of 1.45 Å to understand the structural basis for this inversion. The overall structure of the enzyme overlapped well with that of the benzylphosphonate-associated wild-type enzyme, and the mutations had little effect on the structure of the active site. A ligand molecule bound to the active site in an unusual semiplanar conformation resembling the planar enediolate reaction intermediate could be assigned as phenyl acetate. The inversion in enantioselectivity by the paired mutation is explained by the mirror symmetry between Cys74 in the mutant and Cys188 of the wild type with respect to the carbon atom in the ligand to be protonated. Comparison of the wild-type and Gly74Cys mutant crystal structures suggested that ligand binding induces a positional shift of the Cys188-Gly189-Gly190 region toward the Gly74-Thr75 pair which provides two oxyanion holes necessary to stabilize the negatively charged enediolate reaction intermediate. The ligand binding also simultaneously induces the formation of a hydrophobic cluster over the active site cleft. Thus, AMDase is proposed to have "open" and "closed" conformations of the active site that are regulated by ligand binding. These results may provide an effective strategy for the rational design to invert the enantioselectivity of enzymes.

UR - http://www.scopus.com/inward/record.url?scp=77749259029&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77749259029&partnerID=8YFLogxK

U2 - 10.1021/bi9015605

DO - 10.1021/bi9015605

M3 - Article

C2 - 20136121

AN - SCOPUS:77749259029

VL - 49

SP - 1963

EP - 1969

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 9

ER -