Structure and dynamics of human and bacterial acyl carrier proteins and their interactions with fatty acid synthesis proteins.

Acyl carrier protein (ACP) is highly conserved across taxa and plays key roles in the fatty acid synthesis system by mediating acyl group delivery and shuttling. Here, we compared the structural and dynamic features of human type Ι ACP (hACP) and Escherichia coli type II ACP (EcACP). Analysis of chemical shift perturbations upon octanoyl group attachment showed perturbations in hACP only near acyl-group attachment sites, whereas EcACP showed the perturbation at residues in the hydrophobic cavity. This difference confirmed that hACP does not sequester the acyl chain in the hydrophobic cavity, which is blocked by hydrophobic triad residues (L34, L39, and V64). Moreover, hACP showed more flexible backbone dynamics than EcACP, especially in the front of α1α2 loop. We further investigated the interactions of hACP with Streptomyces coelicolor ACP synthase (ScAcpS), which is used to convert apo mammalian ACP to the holo form. Similar to protein-protein interface (PPI) found in hACP-hAcpS crystal structure, docking simulation and binding affinity measurements showed that the hydrophobic residues in universal recognition helix II of hACP contribute mainly to ScAcpS binding with binding affinity of 9.2 ±â€¯9.1 × 104 M. In contrast, interaction found in EcACP-EcAcpS crystal structure is dominated by electrostatic interactions. These results suggest that ScAcpS has relatively relaxed substrate specificity and a similar charge distribution to hAcpS. These fundamental differences of the charge distribution in hAcpS, ScAcpS and EcAcpS largely affect the interaction with hACP. These findings can provide a useful resource for development of novel antibiotics inhibiting PPI in bacterial FAS proteins with specificity.

Target Proteins of Phloretin for Its Anti-Inflammatory and Antibacterial Activities Against Propionibacterium acnes-Induced Skin Infection.

Phloretin is a natural chalcone with antibacterial and anti-inflammatory effects. This study investigated the anti-acne activity of phloretin against Propionibacterium acnes-induced skin infection and the potential target proteins of its anti-inflammatory and antibacterial effects. Phloretin potently inhibited the growth of P. acnes and P. acnes-induced Toll-like receptor (TLR) 2-mediated inflammatory signaling in human keratinocytes. Secreted embryonic alkaline phosphatase assay confirmed that the anti-inflammatory activity of phloretin is associated with the P. acnes-stimulated TLR2-mediated NF-κB signaling pathway. Phloretin significantly decreased the level of phosphorylated c-Jun N-terminal kinase (JNK), showing a binding affinity of 1.184 × 10-5 M-1. We also found that phloretin binds with micromolar affinity to P. acnes ß-ketoacyl acyl carrier protein (ACP) synthase III (KAS III), an enzyme involved in fatty acid synthesis. Conformation-sensitive native polyacrylamide gel electrophoresis showed that phloretin reduced KAS III-mediated 3-ketoacyl ACP production by over 66%. A docking study revealed that phloretin interacts with the active sites of JNK1 and KAS III, suggesting their involvement in P. acnes-induced inflammation and their potential as targets for the antibacterial activity of phloretin. These results demonstrate that phloretin may be useful in the prevention or treatment of P. acnes infection.

Structural basis of branched-chain fatty acid synthesis by Propionibacterium acnes ß-ketoacyl acyl Carrier protein synthase.

Propionibacterium acnes is an anaerobic gram-positive bacterium found in the niche of the sebaceous glands in the human skin, and is a causal pathogen of inflammatory skin diseases as well as periprosthetic joint infection. To gain effective control of P. acnes, a deeper understanding of the cellular metabolism mechanism involved in its ability to reside in this unique environment is needed. P. acnes exhibits typical cell membrane features of gram-positive bacteria, such as control of membrane fluidity by branched-chain fatty acids (BCFAs). Branching at the iso- or anteiso-position is achieved by incorporation of isobutyryl- or 2-methyl-butyryl-CoA via ß-ketoacyl acyl carrier protein synthase (KAS III) from fatty acid synthesis. Here, we determined the crystal structure of P. acnes KAS III (PaKAS III) at the resolution of 1.9 Šfor the first time. Conformation-sensitive urea polyacrylamide gel electrophoresis and tryptophan fluorescence quenching experiments confirmed that PaKAS III prefers isobutyryl-CoA as the acetyl-CoA, and the unique shape of the active site cavity complies with incorporation of branched-short chain CoAs. The determined structure clearly illustrates how BCFA synthesis is achieved in P. acnes. Moreover, the unique shape of the cavity required for the branched-chain primer can be invaluable in designing novel inhibitors of PaKAS III and developing new specifically targeted antibiotics.