Ghrelin is a 28-amino acid peptide hormone that has been shown to have several physiological impacts such as stimulating the secretion of growth hormone from the pituitary, energy regulation, control of glucose metabolism, along with modulating other neurological functions. Upon secretion from X/A endocrine cells in the stomach, ghrelin undergoes a unique posttranslational modification in which a N-terminal serine, Ser-3, residue is acylated by an octanoate lipid moiety for biological functioning. The enzyme responsible for this modification is ghrelin <i>O</i>-acyltransferase (GOAT), a member of the membrane-bound O-acyltransferase (MBOAT) enzyme family. Ghrelin is believed to be the only substrate for GOAT in the human proteome, making it a potential therapeutic target for disorders such as diabetes and Prader-Willi syndrome. Investigations into the nature and location of the active site of GOAT using peptide mimetics of the N-terminal sequence of ghrelin uncovered principal interactions in GOAT for ghrelin recognition and octanoylation. However, the identity of functionally essential residues within the enzyme were not well established. To investigate this, we developed a cross-species database of ghrelin and GOAT protein sequences across five classes of vertebrates for residue conservation analysis. The knowledge gained from this database provided novel sites for mutagenesis that altered GOAT substrate catalysis and selectivity with small ghrelin peptide mimics. Particularly of interest were single site cysteine mutations to serine which largely reduced enzyme activity. We further probed the enzyme in the presence of cysteine alkylating agents to determine the necessity of cysteine(s) on enzyme function. Using both bioinformatics and biochemical analyses, this study has provided a general chemical map of the enzyme by identifying important regions within GOAT for ghrelin modification and areas that can be further perturbed using small molecules and/or fluorescent probes.

Investigation of the catalytic mechanism for ghrelin acylation within ghrelin <i>O</i>-acyltransferase (GOAT)

256th National Meeting (2018)

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Investigation of the catalytic mechanism for ghrelin acylation within ghrelin O-acyltransferase (GOAT)