#1: ジャーナル: ACS Chem Biol / 年: 2018 タイトル: Biosynthesis of t-Butyl in Apratoxin A: Functional Analysis and Architecture of a PKS Loading Module. 著者: Meredith A Skiba / Andrew P Sikkema / Nathan A Moss / Andrew N Lowell / Min Su / Rebecca M Sturgis / Lena Gerwick / William H Gerwick / David H Sherman / Janet L Smith / 要旨: The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, ...The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (ΨGNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the ΨGNAT and provides the initial acyl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module.
温度: 293 K / 手法: 蒸気拡散法, シッティングドロップ法 詳細: 11mg/mL protein in 50mM Tris pH7.4, 50mM NaCl, 10%(V/V) glycerol, 1mM S-adenosyl-L-homocysteine (SAH), and 5mM MnCl2. Well solution: 15% PEG 3350, 0.1M di-methyl-malonate Protein to well solution ratio is 2:2