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Structure and reactivity of Bacillus subtilis MenD catalyzing the first committed step in menaquinone biosynthesis.

Dawson A, Chen M, Fyfe PK, Guo Z, Hunter WN - J. Mol. Biol. (2010)

Bottom Line: Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of alpha-ketoglutarate.Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position.These data allow for a detailed description of the structure-reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

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A model for the post-decarboxylation covalent adduct formed after ThDP C2 has reacted with α-ketoglutarate and then isochorismate. A stereoview into the active site with atoms colored as follows: C of ThDP, black; C of protein, gray; O, red; N, blue; P, orange; Mn2+, purple. The eight key residues discussed in the text are labeled. Two water molecules are depicted as gray spheres and ethane-1,2-diol is shown as gray sticks, labeled EDO.
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fig6: A model for the post-decarboxylation covalent adduct formed after ThDP C2 has reacted with α-ketoglutarate and then isochorismate. A stereoview into the active site with atoms colored as follows: C of ThDP, black; C of protein, gray; O, red; N, blue; P, orange; Mn2+, purple. The eight key residues discussed in the text are labeled. Two water molecules are depicted as gray spheres and ethane-1,2-diol is shown as gray sticks, labeled EDO.

Mentions: The narrow crevice where substrates bind is likewise formed by a dimer. One subunit contributes three α-helical segments (α10, α14, and α17), which line one side of the cavity. Three non-helical segments (the β1–α2 turn, the loop between β6 and β7, and the loop following β4) are provided by the partner subunit. The active site is polar, primarily basic due to the presence of four arginine residues (32A, 106A, 409B, and 428B) and a lysine (299B). A hydrophobic patch is formed by Ile489B, Phe490B, and Leu493B (Fig. 6). Seven of these eight amino acids are strictly conserved, and one, Lys299, is conserved or replaced by arginine in more than 90% of MenD sequences.


Structure and reactivity of Bacillus subtilis MenD catalyzing the first committed step in menaquinone biosynthesis.

Dawson A, Chen M, Fyfe PK, Guo Z, Hunter WN - J. Mol. Biol. (2010)

A model for the post-decarboxylation covalent adduct formed after ThDP C2 has reacted with α-ketoglutarate and then isochorismate. A stereoview into the active site with atoms colored as follows: C of ThDP, black; C of protein, gray; O, red; N, blue; P, orange; Mn2+, purple. The eight key residues discussed in the text are labeled. Two water molecules are depicted as gray spheres and ethane-1,2-diol is shown as gray sticks, labeled EDO.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2914249&req=5

fig6: A model for the post-decarboxylation covalent adduct formed after ThDP C2 has reacted with α-ketoglutarate and then isochorismate. A stereoview into the active site with atoms colored as follows: C of ThDP, black; C of protein, gray; O, red; N, blue; P, orange; Mn2+, purple. The eight key residues discussed in the text are labeled. Two water molecules are depicted as gray spheres and ethane-1,2-diol is shown as gray sticks, labeled EDO.
Mentions: The narrow crevice where substrates bind is likewise formed by a dimer. One subunit contributes three α-helical segments (α10, α14, and α17), which line one side of the cavity. Three non-helical segments (the β1–α2 turn, the loop between β6 and β7, and the loop following β4) are provided by the partner subunit. The active site is polar, primarily basic due to the presence of four arginine residues (32A, 106A, 409B, and 428B) and a lysine (299B). A hydrophobic patch is formed by Ile489B, Phe490B, and Leu493B (Fig. 6). Seven of these eight amino acids are strictly conserved, and one, Lys299, is conserved or replaced by arginine in more than 90% of MenD sequences.

Bottom Line: Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of alpha-ketoglutarate.Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position.These data allow for a detailed description of the structure-reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

Show MeSH