Visualizing the Chain‐Flipping Mechanism in Fatty‐Acid Biosynthesis

Abstract

In the fatty acid biosynthesis of plants and bacteria, the acyl carrier protein (ACP) is known to sequester elongating products within its hydrophobic core, but this dynamic mechanism remains poorly understood. In this paper we exploit solvatochromic pantetheine probes attached to ACP that fluoresce when sequestered. Addition of a catalytic partner lures the cargo out of the ACP and into the active site of the enzyme, enhancing fluorescence to reveal the elusive chain-flipping mechanism. This activity is confirmed by demonstration of a dual solvatochromic-crosslinking probe and solution-phase NMR. The chain-flipping mechanism can be visualized by single molecule fluorescent techniques, demonstrating specificity between the Escherichia coli ACP and its ketoacyl synthase catalytic partner KASII. Keywordssolvatochromism; acyl carrier protein; chain-flipping mechanism; fatty acid synthase; fatty acids Primary and secondary acetate metabolites are synthesized by dedicated fatty acid and polyketide synthases. Both families are subdivided in type I and type II architectures, based on whether they are encoded on one multidomain polypeptide chain or as separate proteins, respectively. These are often represented as molecular assembly lines with domains that perform chemistry on the growing metabolite while tethered to an acyl carrier protein (ACP), [1] which shuttles its cargo from one enzyme to the next. Although synthetic biologists have ambition to swap domains and modules between these synthases to create new molecular architectures, these efforts have been tempered by the discovery that both protein-substrate and protein-protein interactions play an important role in both iterative and

A Turnstile Mechanism for the Controlled Growth of Biosynthetic Intermediates on Assembly Line Polyketide Synthases

“…These modified proteins, designated crypto -ACPs, are excellent probes of protein–protein interactions between ACP domains and their partner enzymes in fatty acid and polyketide synthases. 17 , 18 Synthesis of crypto -ACP2 and crypto -ACP3 is described in the Supporting Information . (A) crypto -ACP2 (250 μM; with its C-terminal flanking peptide, as explained in Figure S1 ) was incubated with KS3AT3 (50 μM) in the absence (top) and presence (bottom) of 5 mM diketide 1 , which competitively acylates the KS active site.…”

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

“…Analogous carrier proteins from polyketide synthetases can sequester their molecular cargo,although the biochemical relevance of this mechanism is not known. [31][32][33] In the case of PCPs,the Ppant arm does not appear to interact appreciably with the protein core nor to alter the tertiary structure of the PCP in asignificant way. [21,34] This observation supports the "swinging arm hypothesis", in which the flexible Ppant arm delivers substrates to adjacent domains and the PCP domain serves as al argely rigid and chemically inert platform.…”

The Conserved Modular Elements of the Acyl Carrier Proteins of Lipid Synthesis Are Only Partially Interchangeable

“…3 for review). This process is called chain flipping, the dynamics of which have been recently explored by indirect means (23). Following enzyme catalysis, the chain must be "reverse flipped" back into the ACP fourhelix sheath for presentation to the next enzyme in the pathway.…”