Structural stability of Cutibacterium acnes acyl carrier protein studied using CD and NMR spectroscopy

Ahjin Jang,Eunha Hwang,Dasom Cheon,Yangmee Kim

doi:10.1186/s40543-021-00310-5

Abstract

To survive in diverse environments, bacteria adapt by changing the composition of their cell membrane fatty acids. Compared with aerobic bacteria, Cutibacterium acnes has much greater contents of branched-chain fatty acids (BCFAs) in the cell membrane, which helps it survive in anaerobic environments. To synthesize BCFAs, C. acnes acyl carrier protein (CaACP) has to transfer growing branched acyl intermediates from its hydrophobic cavity to fatty acid synthases. CaACP contains an unconserved, distinctive Cys50 in its hydrophobic pocket, which corresponds to Leu in other bacterial acyl carrier proteins (ACPs). Herein, we investigated the substrate specificity of CaACP and the importance of Cys50 in its structural stability. We mutated Cys50 to Leu (C50L mutant) and measured the melting temperatures (Tms) of both CaACP and the C50L mutant by performing circular dichroism experiments. The Tm of CaACP was very low (49.6 °C), whereas that of C50L mutant was 55.5 °C. Hydrogen/deuterium exchange experiments revealed that wild-type CaACP showed extremely fast exchange rates within 50 min, whereas amide peaks of the C50L mutant in the heteronuclear single quantum coherence spectrum remained up to 200 min, thereby implying that Cys50 is the key residue contributing to the structural stability of CaACP. We also monitored chemical shift perturbations upon apo to holo, apo to butyryl, and apo to isobutyryl conversion, confirming that CaACP can accommodate isobutyryl BCFAs. These results provide a preliminary understanding into the substrate specificity of CaACPs for the production of BCFAs necessary to maintain cell membrane fluidity under anaerobic environments.

Highlights

Cutibacterium acnes, previously known as Propionibacterium acnes, is an important member of the human skin microbiota
Straight chain saturated fatty acids (SFAs) are stacked tightly, making the bilayer more stable and rigid; when cis-unsaturated fatty acids (UFAs) or Branched-chain fatty acids (BCFA) add to the chain, the order of the bilayer is interrupted, which increases the permeability of membrane and lowers the transition temperature
We investigated the importance of Cys50 in the structural stability of C. acnes acyl carrier protein (CaACP) using circular dichroism (CD) and hydrogen/deuterium (H/D) exchange experiments

Summary

Introduction

Cutibacterium acnes, previously known as Propionibacterium acnes, is an important member of the human skin microbiota. C. acnes thrives in anaerobic conditions and produces various metabolites (Aubin et al 2017). It plays important roles in the production of vitamin B12 as well as probiotics used commonly in the food and cosmetic industries. Membrane lipid homeostasis is crucial for the survival of bacteria. By adjusting their membrane composition, bacteria can adapt to environmental changes (Zhang and Rock 2008). Bacteria are exposed to toxic organic compounds under anaerobic environments. The membrane fluidity of C. acnes supports cell viability and the degradation of toxic compounds (Duldhardt et al 2010; Kaspar 1982). C. acnes has a high content of branched-chain fatty acids (BCFAs) in its membrane, which provides fluidity for adaptation to anaerobic growth environments. In whole cells of various Cutibacteria strains, C15 BCFA is the most abundant fatty acid (~ 50%); 12-methyltetradecanoic acid (a-C15) and 13-methyltetradecanoic acid (i-C15) are present in different proportions (Moss et al 1969)

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Conclusion