Activity of Fatty Acid Biosynthesis Initiating Ketosynthase FabH with Acetyl/Malonyl-oxa/aza(dethia)CoAs.

Autor: Boram TJ; Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, Indiana 47907, United States., Benjamin AB; Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, Indiana 47907, United States., Silva de Sousa A; Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, Indiana 47907, United States., Stunkard LM; Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, Indiana 47907, United States., Stewart TA; Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, Indiana 47907, United States., Adams TJ; Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, Indiana 47907, United States., Craft NA; Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, Indiana 47907, United States., Velázquez-Marrero KG; Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, Indiana 47907, United States., Ling J; Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, Indiana 47907, United States., Nice JN; Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, Indiana 47907, United States., Lohman JR; Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, Indiana 47907, United States.
Jazyk: angličtina
Zdroj: ACS chemical biology [ACS Chem Biol] 2023 Jan 20; Vol. 18 (1), pp. 49-58. Date of Electronic Publication: 2023 Jan 10.
DOI: 10.1021/acschembio.2c00667
Abstrakt: Fatty acid and polyketide biosynthetic enzymes exploit the reactivity of acyl- and malonyl-thioesters for catalysis. A prime example is FabH, which initiates fatty acid biosynthesis in many bacteria and plants. FabH performs an acyltransferase reaction with acetyl-CoA to generate an acetyl- S -FabH acyl-enzyme intermediate and subsequent decarboxylative Claisen-condensation with a malonyl-thioester carried by an acyl carrier protein (ACP). We envision that crystal structures of FabH with substrate analogues can provide insight into the conformational changes and enzyme/substrate interactions underpinning the distinct reactions. Here, we synthesize acetyl/malonyl-CoA analogues with esters or amides in place of the thioester and characterize their stability and behavior as Escherichia coli FabH substrates or inhibitors to inform structural studies. We also characterize the analogues with mutant FabH C112Q that mimics the acyl-enzyme intermediate allowing dissection of the decarboxylation reaction. The acetyl- and malonyl-oxa(dethia)CoA analogues undergo extremely slow hydrolysis in the presence of FabH or the C112Q mutant. Decarboxylation of malonyl-oxa(dethia)CoA by FabH or C112Q mutant was not detected. The amide analogues were completely stable to enzyme activity. In enzyme assays with acetyl-CoA and malonyl-CoA (rather than malonyl-ACP) as substrates, acetyl-oxa(dethia)CoA is surprisingly slightly activating, while acetyl-aza(dethia)CoA is a moderate inhibitor. The malonyl-oxa/aza(dethia)CoAs are inhibitors with K i 's near the K m of malonyl-CoA. For comparison, we determine the FabH catalyzed decomposition rates for acetyl/malonyl-CoA, revealing some fundamental catalytic traits of FabH, including hysteresis for malonyl-CoA decarboxylation. The stability and inhibitory properties of the substrate analogues make them promising for structure-function studies to reveal fatty acid and polyketide enzyme/substrate interactions.
Databáze: MEDLINE
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