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Structural Insights on PHA Binding Protein PhaP from Aeromonas hydrophila.

ABSTRACT: Phasins or PhaPs are a group of amphiphilic proteins that are found attached to the surface of microbial polyhydroxyalkanoate (PHA) granules. They have both structural and regulatory functions and can affect intracellular PHA accumulation and mediate protein folding. The molecular basis for the diverse functions of the PhaPs has not been fully understood due to the lack of the structural knowledge. Here we report the structural and biochemical studies of the PhaP cloned from Aeromonas hydrophila (PhaPAh), which is utilized in protein and tissue engineering. The crystal structure of PhaPAh was revealed to be a tetramer with 8 ?-helices adopting a coiled-coil structure. Each monomer has a hydrophobic and a hydrophilic surface, rendering the surfactant properties of the PhaPAh monomer. Based on the crystal structure, we predicted three key amino acid residues and obtained mutants with enhanced stability and improved emulsification properties. The first PhaP crystal structure, as reported in this study, is an important step towards a mechanistic understanding of how PHA is formed in vivo and why PhaP has such unique surfactant properties. At the same time, it will facilitate the study of other PhaP members that may have significant biotechnological potential as bio-surfactants and amphipathic coatings.

SUBMITTER: Zhao H

PROVIDER: S-EPMC5180188 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Structural Insights on PHA Binding Protein PhaP from Aeromonas hydrophila.

Zhao Hongyu H Wei Hui H Liu Xi X Yao Zhenyu Z Xu Manyu M Wei Daixu D Wang Jiawei J Wang Xinquan X Chen Guo-Qiang GQ

Scientific reports 20161223

Phasins or PhaPs are a group of amphiphilic proteins that are found attached to the surface of microbial polyhydroxyalkanoate (PHA) granules. They have both structural and regulatory functions and can affect intracellular PHA accumulation and mediate protein folding. The molecular basis for the diverse functions of the PhaPs has not been fully understood due to the lack of the structural knowledge. Here we report the structural and biochemical studies of the PhaP cloned from Aeromonas hydrophila ...[more]

PMID: 28009010

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Project description:BackgroundAeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS). It uses N-acyl-homoserine lactone (AHL) quorum sensing signals to coordinate biofilm formation, motility, and virulence gene expression. The AHL signaling pathway is therefore considered to be a therapeutic target against pathogenic A. hydrophila infection. In A. hydrophila, AHL autoinducers biosynthesis are specifically catalyzed by an ACP-dependent AHL synthase AhyI using the precursors SAM and acyl-ACP. Our previously reported AhyI was heterologously expressed in E. coli, which showed the production characteristics of medium-long chain AHLs. This contradicted the prevailing understanding that AhyI was only a short-chain C4/C6-HSL synthase.ResultsIn this study, six linear acyl-ACP proteins with C-terminal his-tags were synthesized in Vibrio harveyi AasS using fatty acids and E. coli produced active holo-ACP proteins, and in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs were performed. UPLC-MS/MS analyses indicated that AhyI can synthesize short-, medium- and long-chain AHLs from SAM and corresponding linear acyl-ACP substrates. Kinetic parameters measured using a DCPIP colorimetric assay, showed that there was a notable decrease in catalytic efficiency with acyl-chain lengths above C6, and hyperbolic or sigmoidal responses in rate curves were observed for varying acyl-donor substrates. Primary sequence alignment of the six representative AHL synthases offers insights into the structural basis for their specific acyl substrate preference. To further understand the acyl chain length preference of AhyI for linear acyl-ACP, we performed a structural comparison of three ACP-dependent LuxI homologs (TofI, BmaI1 and AhyI) and identified three key hydrophobic residues (I67, F125 and L157) which confer AhyI to selectively recognize native C4/C6-ACP substrates. These predictions were further supported by a computational Ala mutation assay.ConclusionsIn this study, we have redefined AhyI as a multiple short- to long-chain AHL synthase which uses C4/C6-ACP as native acyl substrates and longer acyl-ACPs (C8 ~ C14) as non-native ones. We also theorized that the key residues in AhyI would likely drive acyl-ACP selective recognition.

Dataset Information

Structural Insights on PHA Binding Protein PhaP from Aeromonas hydrophila.

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Structural Insights on PHA Binding Protein PhaP from Aeromonas hydrophila.

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