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Substrate recognition by β-ketoacyl-ACP synthases.

Janine G Borgaro1, Andrew Chang, Carl A Machutta

  • 1Institute for Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, United States.

Biochemistry
|October 25, 2011
PubMed
Summary
This summary is machine-generated.

This study investigates β-Ketoacyl-ACP synthase (KAS) enzyme substrate specificity in fatty acid biosynthesis. KASI enzymes require an ACP substrate for acyl donation, impacting drug targets in Mycobacterium tuberculosis.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Fatty acid biosynthesis relies on β-Ketoacyl-ACP synthase (KAS) enzymes, which catalyze essential Claisen condensation reactions.
  • KAS enzymes exhibit distinct substrate preferences, with KASIII using acyl-CoA and KASI/KASII utilizing acyl-ACP as donor substrates.
  • The essentiality of KasA in Mycobacterium tuberculosis, unlike its E. coli counterpart, suggests alternative substrate utilization, potentially involving CoA donors.

Purpose of the Study:

  • To elucidate the substrate specificity of KASI enzymes, particularly KasA from Mycobacterium tuberculosis, and ecFabB/ecFabF from E. coli.
  • To investigate the role of carrier molecules like phosphopantetheine, CoA, and ACP in KAS-catalyzed reactions.
  • To understand the implications of substrate specificity for the mycobacterial fatty acid synthesis pathway and potential drug development.

Main Methods:

  • Enzyme kinetics assays were performed using various acyl donor and acceptor substrates based on phosphopantetheine, CoA, and ACP mimics.
  • The catalytic efficiency (kcat/Km) was determined for different substrate combinations with KASI enzymes (ecFabB, KasA) and KASII (ecFabF).
  • Structural insights were proposed regarding enzyme conformational changes upon substrate binding.

Main Results:

  • KASI enzymes (ecFabB, KasA) demonstrated an absolute requirement for an acyl-ACP substrate as the acyl donor.
  • A 14-residue malonyl-phosphopantetheine peptide efficiently substituted malonyl-ACP as an acceptor substrate for ecFabF.
  • While ecFabF could utilize CoA for both substrates, KASI enzymes showed strict dependence on ACP for acyl donation, with acceptor carrier variation having minimal impact on catalytic efficiency.

Conclusions:

  • The study confirms the essential acyl donor role of ACP for KASI enzymes, irrespective of the acceptor carrier.
  • A conformational change model is proposed where ACP binding to KASI enzymes facilitates malonyl substrate binding.
  • The observed substrate inhibition with palmitoyl-CoA in KasA highlights the importance of understanding these enzymes in mycobacterial fatty acid biosynthesis.