Probing the Phosphopantetheine Arm Conformations of Acyl Carrier Proteins Using Vibrational Spectroscopy

Abstract

Acyl carrier proteins (ACPs) are universal and highly conserved domains central to both fatty acid and polyketide biosynthesis. These proteins tether reactive acyl intermediates with a swinging 4′-phosphopantetheine (Ppant) arm and interact with a suite of catalytic partners during chain transport and elongation while stabilizing the growing chain throughout the biosynthetic pathway. The flexible nature of the Ppant arm and the transient nature of ACP–enzyme interactions impose a major obstacle to obtaining structural information relevant to understanding polyketide and fatty acid biosynthesis. To overcome this challenge, we installed a thiocyanate vibrational spectroscopic probe on the terminal thiol of the ACP Ppant arm. This site-specific probe successfully reported on the local environment of the Ppant arm of two ACPs previously characterized by solution NMR, and was used to determine the solution exposure of the Ppant arm of an ACP from 6-deoxyerythronolide B synthase (DEBS). Given the sensitivity of the probe’s CN stretching band to conformational distributions resolved on the picosecond time scale, this work lays a foundation for observing the dynamic action-related structural changes of ACPs using vibrational spectroscopy.

Acyl Carrier Protein Cyanylation Delivers a Ketoacyl Synthase–Carrier Protein Cross-Link

“…AcpP-SCN exhibited a mode frequency of 2161 cm −1 , which based on prior results from model molecules 6 and proteins 2 suggests that the Ppant arm is mainly solvent exposed in the absence of a catalytic partner (Figure 1D). …”

“…The infrared CN stretching band of AcpP-SCN (black) absorbs at 2161 cm −1 , a frequency consistent with the label being water exposed. 2 When AcpP SCN was titrated with FabF a new peak at 2120 cm −1 (arrow) was observed.…”

Reinvigorating natural product combinatorial biosynthesis with synthetic biology

“…Recent structural studies of NRPS 31 and PKS assembly lines 32,33 have provided insight into some of the intramolecular contacts and motions of these proteins (see page xxx). A recently developed thiocyanate vibrational spectroscopic probe installed on the terminal thiol of the ACP’s 4′-phosphopantetheine arm 34 , which enables the observation of ACP conformational changes during biosynthesis, will also be a useful tool to comprehend PKS operating principles and to design productive hybrid PKSs. Although developing universal rules to bring together disparate enzymatic parts and design new products is still a major challenge, this provides the opportunity to utilize new basic knowledge to develop engineering strategies.…”

Using Modern Tools To Probe the Structure–Function Relationship of Fatty Acid Synthases

“…[19] Sequestration has been proposed to shape ACP for subsequent protein-protein interactions, define substrate length, and/or protect the labile thioester bond from hydrolytic cleavage. [20,21] Recent work exploiting solvatochromic probes [22] and vibrational probes [23] appended to the PPant arm of ACPs have allowed direct observation of both sequestered and non-sequestered microstates. Type I ACPs are not thought to sequester, as observed by a lack of structural perturbations by NMR of the excised ACP [24] and solvatochromic probes.…”