Structure of Aspergillus fumigatus Cytosolic Thiolase: Trapped Tetrahedral Reaction Intermediates and Activation by Monovalent Cations

Andrew C. Marshall, Charles S. Bond, John B. Bruning

Research output: Contribution to journalArticle

Abstract

Cytosolic thiolase (CT) catalyzes the reversible Claisen condensation of two molecules of acetyl-CoA to produce acetoacetyl-CoA. The reaction cycle proceeds via a ping-pong mechanism involving an acetylated enzyme intermediate and two separate oxyanion holes which stabilize negatively charged reaction intermediates. This is the initial step in the synthesis of ergosterol in the prominent fungal pathogen Aspergillus fumigatus and is essential for the growth and survival of the organism. Here, we present crystal structures of A. fumigatus CT in liganded and apo forms and in a complex with different monovalent cations. Careful observation of the electron density at the active sites of two different afCT structures crystallized in the presence of acetyl-CoA shows that our crystals have trapped various stages of the thiolase catalytic cycle, including two tetrahedral reaction intermediates that have previously eluded structural characterization. Unexpectedly, we have also shown that afCT is activated by monovalent cations, a biochemical property previously thought to apply only to the mitochondrial biosynthetic thiolase, with a preference for potassium ions. Structures of fungal CT provide valuable insight into the thiolase reaction cycle and allosteric activation of members of this class of enzymes by monovalent cations.

LanguageEnglish
Pages1973-1989
Number of pages17
JournalACS Catalysis
Volume8
Issue number3
DOIs
StatePublished - 2 Mar 2018

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Monovalent Cations
Reaction intermediates
Aspergillus
Acetyl Coenzyme A
Positive ions
Chemical activation
Enzymes
Ergosterol
Pathogens
Carrier concentration
Potassium
Condensation
Crystal structure
Ions
Crystals
Molecules

Cite this

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title = "Structure of Aspergillus fumigatus Cytosolic Thiolase: Trapped Tetrahedral Reaction Intermediates and Activation by Monovalent Cations",
abstract = "Cytosolic thiolase (CT) catalyzes the reversible Claisen condensation of two molecules of acetyl-CoA to produce acetoacetyl-CoA. The reaction cycle proceeds via a ping-pong mechanism involving an acetylated enzyme intermediate and two separate oxyanion holes which stabilize negatively charged reaction intermediates. This is the initial step in the synthesis of ergosterol in the prominent fungal pathogen Aspergillus fumigatus and is essential for the growth and survival of the organism. Here, we present crystal structures of A. fumigatus CT in liganded and apo forms and in a complex with different monovalent cations. Careful observation of the electron density at the active sites of two different afCT structures crystallized in the presence of acetyl-CoA shows that our crystals have trapped various stages of the thiolase catalytic cycle, including two tetrahedral reaction intermediates that have previously eluded structural characterization. Unexpectedly, we have also shown that afCT is activated by monovalent cations, a biochemical property previously thought to apply only to the mitochondrial biosynthetic thiolase, with a preference for potassium ions. Structures of fungal CT provide valuable insight into the thiolase reaction cycle and allosteric activation of members of this class of enzymes by monovalent cations.",
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Structure of Aspergillus fumigatus Cytosolic Thiolase : Trapped Tetrahedral Reaction Intermediates and Activation by Monovalent Cations. / Marshall, Andrew C.; Bond, Charles S.; Bruning, John B.

In: ACS Catalysis, Vol. 8, No. 3, 02.03.2018, p. 1973-1989.

Research output: Contribution to journalArticle

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