Hedgehog acyltransferase catalyzes a random sequential reaction and utilizes multiple fatty acyl-CoA substrates Journal Article
| Authors: | Schonbrun, A. R.; Resh, M. D. |
| Article Title: | Hedgehog acyltransferase catalyzes a random sequential reaction and utilizes multiple fatty acyl-CoA substrates |
| Abstract: | Sonic hedgehog (Shh) signaling is a key component of embryonic development and is a driving force in several cancers. Hedgehog acyltransferase (Hhat), a member of the membrane-bound O-acyltransferase family of enzymes, catalyzes the attachment of palmitate to the N-terminal cysteine of Shh, a posttranslation modification critical for Shh signaling. The activity of Hhat has been assayed in cells and in vitro, and cryo-EM structures of Hhat have been reported, yet several unanswered questions remain regarding the enzyme's reaction mechanism, substrate specificity, and the impact of the latter on Shh signaling. Here, we present an in vitro acylation assay with purified Hhat that directly monitors attachment of a fluorescently tagged fatty acyl chain to Shh. Our kinetic analyses revealed that the reaction catalyzed by Hhat proceeds through a random sequential mechanism. We also determined that Hhat can utilize multiple fatty acyl-CoA substrates for fatty acid transfer to Shh, with comparable affinities and turnover rates for myristoyl-CoA, palmitoyl-CoA, palmitoleoyl-CoA, and oleoyl-CoA. Furthermore, we investigated the functional consequence of differential fatty acylation of Shh in a luciferase-based Shh reporter system. We found that the potency of the signaling response in cells was higher for Shh acylated with saturated fatty acids compared to monounsaturated fatty acids. These findings demonstrate that Hhat can attach fatty acids other than palmitate to Shh and suggest that heterogeneous fatty acylation has the potential to impact Shh signaling in the developing embryo and/or cancer cells. © 2022 The Authors |
| Keywords: | signal transduction; controlled study; unclassified drug; human cell; binding affinity; protein function; protein analysis; protein binding; in vitro study; endoplasmic reticulum; medical imaging; catalysis; amino acids; enzyme kinetics; protein structure; enzyme specificity; sonic hedgehog; substrates; acyltransferase; cancer cells; acylation; cell signaling; palmitoylation; hedgehog acyltransferase; hydrophobicity; acyltransferases; acyl coenzyme a; palmitic acid; ultracentrifugation; fatty acyl-coa; human; article; hedgehog signaling; acyl-coa; sonic hedgehogs; mboat; saturated fatty acids; acylations; enzymes kinetics; hedgehog signalling |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 298 |
| Issue: | 10 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2022-10-01 |
| Start Page: | 102422 |
| Language: | English |
| DOI: | 10.1016/j.jbc.2022.102422 |
| PUBMED: | 36030053 |
| PROVIDER: | scopus |
| PMCID: | PMC9513256 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 1 November 2022 -- Source: Scopus |
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