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Characterization of bacterial homologs of prostaglandin H 2 synthase using GC/MS
Author(s) -
Nichols Caytlin,
Butchy Margaret,
Selinsky Barry
Publication year - 2016
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.30.1_supplement.1075.4
Subject(s) - biochemistry , orfs , mutagenesis , enzyme , biology , mutant , arachidonic acid , atp synthase , site directed mutagenesis , microbiology and biotechnology , open reading frame , peptide sequence , gene
Prostaglandins are formed by the enzyme prostaglandin H 2 synthase (PGHS), which has been found in all vertebrates whose genomic sequence is known. In the peroxidase‐cyclooxygenase superfamily, several archaeal, bacterial, fungal, and plant open reading frames (ORFs) were predicted to have structures similar to mammalian PGHS. Our laboratory has subcloned and expressed several bacterial ORFs encoding for proteins resembling mammalian PGHS. This report describes the development of a GC/MS assay to follow substrate utilization in these bacterial enzymes. The method was tested using ovine prostaglandin H 2 synthase (which uses arachidonic acid), and the linoleate 10S dioxygenase, expressed from an ORF copied from Nostoc punctiforme . The lipid preference of both enzymes was confirmed by our GC/MS results. Using site directed mutagenesis, active site variants will be made in attempt to convert the Nostoc 10S‐dioxygenase into an enzyme that will use arachidonic acid as a substrate, as seen in mammalian PGHS. The success of our mutagenesis and enzymatic analysis of the mutants will be discussed.