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Mutational analysis of the Arabidopsis thaliana lysoglycerophospholipid acyltransferase AT1g78690
Author(s) -
Nag Saparja,
Garrett Teresa A.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.781.10
Subject(s) - biochemistry , acyltransferase , biology , arabidopsis thaliana , histidine , threonine , acyltransferases , enzyme , mutant , microbiology and biotechnology , chemistry , serine , biosynthesis , gene
Headgroup‐acylated glycerophospholipids (HAGPLs) are a class of poorly understood lipids responsible for supporting cell division and maintenance of the outer cell membrane in Escherichia coli . Overexpression of AT1g78690 in E. coli results in the accumulation of the HAGPL acylphosphatidylglycerol (acyl‐PG). However, this peripheral membrane protein, native to Arabidopsis thaliana , does not directly acylate PG but in vitro is a lysophospholipid acyltransferase that catalyzes the formation of di‐acylated glycerophospholipids, PE or PG, from mono‐acylated glycerophospholipid substrates, 1‐acyl lyso PE or 1‐acyl lyso PG respectively. AT1g78690 bears 39% and 41% sequence identity to human and Drosophila tafazzin, respectively, a monolysocardiolipin remodeling enzyme implicated in Barth Syndrome. Mutations to tafazzin are implicated in altered cardiolipin metabolism found in Barth Syndrome patients. To understand the catalytic and kinetic properties of AT1g78690, several amino acids that are 100% conserved among members of the lysophospholipid acyltransferase (LPLAT) superfamily, threonine‐63, asparagine‐66, histidine‐67, aspartate‐72, and arginine‐92 were targeted for mutation to alanine. Histidine‐67 and arginine‐92 are particularly interesting because mutation of analogous amino acids in tafazzin are associated with Barth Syndrome. Following cloning into pET28a for overexpression in E. coli , membranes will be isolated and used as the enzyme source for a thin layer chromatography based in vitro enzyme assay that follows the transfer of radiolabeled palmitate from palmitoyl Co‐A to lyso PE to form PE. Kinetic parameters, including the Michaelis‐Menten constant (K m ) and catalytic efficiency (k cat ) as well as substrate specificity will be examined to better understand the catalytic mechanism of AT1g78690 and its homologs. Support or Funding Information National Science Foundation Research at Undergraduate Institutions Grant #1516805 and #1152463