Research Article: Expression of a recombinant, 4′-Phosphopantetheinylated, active M. tuberculosis fatty acid synthase I in E. coli

Date Published: September 24, 2018

Publisher: Public Library of Science

Author(s): Szilvia Baron, Yoav Peleg, Jacob Grunwald, David Morgenstern, Nadav Elad, Moshe Peretz, Shira Albeck, Yishai Levin, John T. Welch, Kim A. DeWeerd, Alon Schwarz, Yigal Burstein, Ron Diskin, Zippora Shakked, Oren Zimhony, Laurent Kremer.

http://doi.org/10.1371/journal.pone.0204457

Abstract

Fatty acid synthase 1 (FAS I) from Mycobacterium tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4′-Phosphopantetheine (4′-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4′-PP transferase, termed acyl carrier protein synthase (AcpS).

In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid. We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography.

Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM.

Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structure-function analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

Partial Text

M. tuberculosis (Mtb) possesses a unique lipid-rich cell wall that confers virulence, persistence and drug resistance. The important components of Mtb cell wall are α-alkyl-β-hydroxyl C80-90 fatty acids termed mycolic acids, and several polyketides (Pk) [1–4]. Several steps in mycolic acid biosynthesis were identified as drug targets for first-line anti-tuberculosis agents [5]. Mycobacteria unlike other prokaryotes or eukaryotes possess two types of fatty acid synthase systems, FAS I and FAS II. FAS I elongates acetyl- CoA to C16:0–26:0 fatty acids, the substrate for all subsequent steps of elongation and modifications.

This study describes the expression of a 4′-phosphopantetheinylated active M. tuberculosis fatty acid synthase I in E. coli following sequential expression of Mtb FAS I after its Mtb PPTase enzyme, AcpS.

 

Source:

http://doi.org/10.1371/journal.pone.0204457

 

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