Characterization and heterologous expression of plasmalogen synthase MeHAD from Megasphaera elsdenii |
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| Affiliation: | 1. State Key Laboratory Incubation Base for Green Processing of Chemical Engineering, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832003, China;2. College of Life Sciences, Zhejiang University, Hangzhou 310058, China;3. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;1. Nanoscopy, Istituto Italiano Tecnologia, Via Enrico Melen 83, 16152, Genova, Italy;2. Department of Earth, Environment and Life Sciences (DISTAV), University of Genova, Corso Europa 26, 16132, Genova, Italy;3. Department of Physics (DIFILAB), University of Genoa, Via Dodecaneso 33, 16146, Genoa, Italy;4. DIMES, Department of Experimental Medicine, University of Genoa, Genoa, Italy;5. IRCCS Ospedale Policlinico San Martino, Genoa, Italy;6. Clinica Medica “A. Murri”, Department of Biomedical Sciences and Human Oncology, University of Bari, Medical School, Piazza Giulio Cesare 11, 70124, Bari, Italy;1. Department of Physiology, School of Basic Medical Sciences, Shenzhen University Medical School, Shenzhen University, Shenzhen 518055, Guangdong, China;2. GuangZhou Laboratory, No.9 XingDaoHuanBei Road, Guangzhou International Bio Island, Guangzhou 510005, Guangdong, China;1. Department of Chemistry and Pharmacy, University of Sassari, Italy;2. STS on contract to Department of Aeromedical Research, United States Air Force School of Aerospace Medicine, USA;3. Air Force Research Laboratory, 711th HPW/RHXBC, Wright Patterson Air Force Base, OH 45433, USA;4. Faculty of Chemistry, University of Wroclaw, Poland;1. Department of Medical Biotechnology and Translational Medicine, University of Milano, 20054 Segrate, Milano, Italy;2. UK Dementia Research Institute at UCL, London, UK;3. Neuro-Sys, 410 Chemin Départemental 60, 13120 Gardanne, France;4. Department of Immunology, St. Jude Children''s Research Hospital, Memphis, TN 38105, USA;5. Department of Chemistry, University of Milano, Milan, Italy;1. Group on Molecular and Cell Biology of Lipids, University of Alberta, Alberta, Canada;2. Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Alberta, Canada;3. Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Alberta, Canada |
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| Abstract: | Plasmalogens (Pls) are vinyl-ether bond-containing glycerophospholipids or glycosyl diradyl glycerols, and are of great importance in the physiological functions and stability of cell membrane. Here, we identified and characterized that the plasmalogen synthase MeHAD from anaerobic Megasphaera elsdenii was responsible for vinyl-ether bond formation. Different from the 2-hydroxyacyl-CoA dehydratase (HAD) family plasmalogen synthase PlsA-PlsR which are encoded by two genes in Clostridium perfringens, the HAD homolog (MeHAD) encoded by a single gene MELS_0169 was found in M. elsdenii. By heterologous expression of the MeHAD gene into a nonplasmalogen-producing Escherichia coli strain, the expressed MeHAD was found to be located in the cell membrane region. Plasmalogens were detected in the recombinant strain using GC–MS and LC-MS, demonstrating that MeHAD was the key enzyme for plasmalogen synthesis. Moreover, the synthesized plasmalogens could enhance the oxidative stress-resistance and osmotic pressure-resistance of the recombinant strain, probably due to the ROS scavenging and decreased membrane permeability by the plasmalogens, respectively. The four-cysteine (Cys125, Cys164, Cys445 and Cys484) site-mutant of MeHAD, which were predicted binding to the [4Fe-4S] cluster, was unable to synthesize plasmalogens, indicating that the cysteines are important for the catalytic activity of MeHAD. Our results revealed the single gene encoded plasmalogen synthase in M. elsdenii and established a recombinant E. coli strain with plasmalogen production potential. |
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