Image Credit: Woo Cheol Lee, Sungjae Choi, Ahjin Jang, Kkabi Son & Yangmee Kim. Structural comparison of Acinetobacter baumannii β-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis. Scientific Reports 11, Article number: 7945 (2021). DOI: 10.1038/s41598-021-86997-3. Figure 2. a.

Abstract

Some Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the β-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the β-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure–function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host–pathogen interaction mechanisms and novel antibiotics discovery.

Crystallization and structure determination

AbApeQ was crystallized using the PEG Ion screen kit (Hampton Research, USA). Crystals appeared several days after crystallization with the crystallization buffer containing 50 mM HEPES pH 7, 12% (w/v) PEG 3350, and 1% (w/v) tryptone. For crystallization of the complex of the protein with NADPH, a 1:3 molar ratio of NADPH sodium salt (Sigma Aldrich, St. Louis, MO, USA) was added to the protein solution, followed by incubation on ice for 1 h before crystallization. Crystals were observed following addition of the crystallization buffer, which was composed of 0.2 M potassium sodium tartrate and 20% (w/v) PEG 3350. Apo AbFabG crystals were obtained using crystallization condition of 0.1 M sodium formate pH 7.0 and 12% (w/v) PEG 3350.

Prior to data collection, the crystals were immersed in the crystallization buffer supplemented with 25% (v/v) ethylene glycol to prevent ice formation and then flash-cooled with liquid nitrogen. X-ray diffraction data sets were collected using the beamline 7A system at the Pohang Accelerator Laboratory at a wavelength of 0.975 Å. Diffraction images were integrated and scaled using the XDS program44. The structure of AbApeQ was determined by molecular replacement using the coordinates of a putative FabG structure from E. coli CFT073 (PDB ID: 4IIU) with the program Phaser45. Modeling and refinement of the AbApeQ structures were performed using the programs Coot and Phenix, respectively46,47. The data collection and refinement statistics are listed in Supplementary Table S2. The three-dimensional structural coordinates of the structures have been deposited in the Protein Data Bank under accession numbers: 7CAW (apo AbApeQ), 7CAX (AbApeQ-NADPH complex) and 7CAZ (apo AbFabG).