Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs

Abstract

Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates the metabolism-regulating peptide hormone ghrelin at Ser3 and may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Protein octanoylation is unique to ghrelin in humans, and little is known about the mechanism of GOAT or of related protein-O-acyltransferases HHAT or PORC. In this study, we explored an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features. Measurement of Km for 10-mer, 27-mer, and synthetic Tat-peptide-containing ghrelin substrates provided evidence for a role of charge interactions in substrate binding. Ghrelin substrates with amino-alanine in place of Ser3 demonstrated that GOAT can catalyze the formation of an octanoyl-amide bond at a similar rate compared with the natural reaction. A pH-rate comparison of these substrates revealed minimal differences in acyltransferase activity across pH 6.0–9.0, providing evidence that these reactions may be relatively insensitive to the basicity of the substrate nucleophile. The conserved His338 residue was required both for Ser3 and amino-Ala3 ghrelin substrates, suggesting that His338 may have a key catalytic role beyond that of a general base.

Synthetic Triterpenoid Inhibition of Human Ghrelin O-Acyltransferase: The Involvement of a Functionally Required Cysteine Provides Mechanistic Insight into Ghrelin Acylation

“…62, 72, 74 Mutational analyses of the three protein-modifying members of the MBOAT family (Hhat, PORCN, and GOAT) have revealed functionally required residues but none have implicated cysteine residues as functionally essential. 30, 62, 65, 73, 75–77 While Hhat and PORCN contain palmitoylated cysteine residues, 73, 77 our findings provide the first evidence supporting an enzymatic cysteine residue involved in MBOAT-catalyzed protein acylation. One intriguing possibility involves formation of an octanoyl acyl-enzyme intermediate involving a cysteine residue within GOAT in the course of transferring the octanoyl group to ghrelin (Figure 7), similar to the ping-pong mechanism proposed for protein palmitoylation by DHHC-family palmitoyltransfersases.…”

“…32, 64 The majority of in vitro assays for GOAT activity utilize the mouse isoform of GOAT (mGOAT) in either an enzyme- and cell-based format, 15, 30, 32, 33, 62, 65–69 with mGOAT and hGOAT exhibiting a high level of amino acid sequence homology (79% identity, 92% similarity; Figure 6). Only one example of a direct comparison of inhibitor potency between these closely related enzyme isoforms has been reported, with the [Dap 3 ]octanoyl-ghrelin (1–5)-NH 2 inhibitor exhibiting similar activity against hGOAT and mGOAT.…”

“…Compounds containing the pharmacophores identified in this study can also be designed to combine aspects of these CDDO derivatives with elements involved in ghrelin recognition by GOAT to maximize both potency and specificity. 31, 52, 65 …”

The octanoylated energy regulating hormone ghrelin: An expanded view of ghrelin’s biological interactions and avenues for controlling ghrelin signaling

“…Several early investigations established the ability of ghrelin mimetic short peptides to serve as GOAT substrates and determined that the N-terminal sequence of ghrelin/proghrelin is essential for recognition by GOAT (Ohgusu et al, 2009;Yang et al, 2008b). Studies by our group and others have used these synthetic peptide substrates to characterize GOAT substrate selectivity, confirming the importance of the N-terminal sequence of ghrelin and establishing the contribution of each side chain in the first four amino acids of ghrelin for recognition by GOAT (Barnett et al, 2010;Darling et al, 2013Darling et al, , 2015Taylor et al, 2015). There is some tolerance for modification at the site of acylation as the mouse and human isoforms of GOAT accept substrates containing a threonine at the third residue, and bullfrog ghrelin has been confirmed to be octanoylated at a threonine (Darling et al, 2015;Kaiya et al, 2001Kaiya et al, , 2006Kaiya et al, , 2011Yang et al, 2008b).…”

Molecular architecture of a membrane-spanning hormone acyltransferase required for metabolic regulation

“…(10) While the unique chemistry and biology of ghrelin and GOAT has inspired continued interest in targeting this system for therapeutic benefit, the inability to purify active GOAT and determine its structure has hampered progress towards this goal. (11)(12)(13) In this work, we report the first structural model for a eukaryotic MBOAT family member. Our human GOAT (hGOAT) structure is highly consistent with a recently reported crystal structure for the D-alanyl transferase DltB, a bacterial MBOAT homolog.…”