title: The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase.
abstract: Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a Vmax of 12 µmol/min/mg protein and a Km of 45 μM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5-7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.
authors.affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA. Electronic address: jurkowitz.1@osu.edu.
authors.forename: Marianne S
authors.initials: MS
authors.lastname: Jurkowitz
authors.affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Aalapi
authors.initials: A
authors.lastname: Patel
authors.affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Lai-Chu
authors.initials: LC
authors.lastname: Wu
authors.affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Annalise
authors.initials: A
authors.lastname: Krautwater
authors.affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Pfeiffer
authors.affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA; Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Bell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017124:Conserved Sequence; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D016952:Legionella pneumophila / Q000737:chemistry* / Q000201:enzymology; D008246:Lysophospholipids / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D008246:Lysophospholipids; D011993:Recombinant Fusion Proteins; C021215:lysoplasmalogens; D049452:Green Fluorescent Proteins; D006867:Hydrolases; C118173:lysoplasmalogenase; D010450:Endopeptidases; C099488:TEV protease
keywords: Legionella pneumophila; Lysophospholipase; Lysoplasmalogenase; Plasmalogen/lysoplasmalogen; Vinyl ether bond; YhhN gene Lpg1991