Hedgehog acyltransferase catalyzes a random sequential reaction and utilizes multiple fatty acyl-CoA substrates

Sonic hedgehog (Shh) signaling plays a central role in embryonic development and is implicated in various cancers. A key modification in this pathway involves the attachment of palmitate to Shh, mediated by Hedgehog acyltransferase (Hhat). While prior research has established the importance of this modification, the exact mechanism of Hhat's activity remains unclear. Studies have explored Hhat in both cellular and in vitro settings, and cryo-EM structures have been reported. However, gaps persist in understanding the enzyme's kinetic behavior and substrate preferences. Researchers have yet to determine whether Hhat can use alternative fatty acyl-CoA substrates and how these might influence signaling outcomes. Additionally, the functional consequences of different fatty acyl modifications on Shh activity remain unresolved. This uncertainty has driven recent investigations into the enzyme’s mechanism and substrate flexibility. Understanding these aspects could provide insights into how Shh signaling is modulated in development and disease.

This study aimed to clarify the reaction mechanism of Hhat and its substrate specificity. Researchers sought to determine whether Hhat can utilize fatty acyl-CoA substrates beyond palmitoyl-CoA and how this affects Shh signaling. A purified in vitro acylation assay was developed to directly monitor Hhat activity using fluorescently labeled fatty acyl chains. The goal was to assess kinetic parameters and identify the reaction mechanism. Additionally, the team investigated the functional impact of different fatty acyl modifications on Shh signaling. By comparing saturated and unsaturated fatty acids, they aimed to evaluate how acylation type influences signaling potency. The study also aimed to test whether Hhat’s substrate flexibility could lead to heterogeneous acylation of Shh. This approach allowed for a direct assessment of enzyme behavior and signaling outcomes in a controlled setting.

The researchers developed a fluorescently tagged in vitro acylation assay using purified Hhat and Shh. This setup enabled direct monitoring of fatty acyl chain transfer to Shh. They tested multiple fatty acyl-CoA substrates, including myristoyl-CoA, palmitoyl-CoA, palmitoleoyl-CoA, and oleoyl-CoA. Kinetic analyses were performed to determine reaction rates and affinities for each substrate. A luciferase-based reporter system was used to assess the functional consequences of different fatty acyl modifications on Shh signaling. The team compared signaling potency between saturated and unsaturated fatty acid modifications. Cryo-EM structures were referenced to contextualize the findings. The experimental design allowed for precise measurement of Hhat activity and signaling outcomes.

Kinetic analyses revealed that Hhat catalyzes a random sequential reaction mechanism. The enzyme exhibited comparable affinities and turnover rates for myristoyl-CoA, palmitoyl-CoA, palmitoleoyl-CoA, and oleoyl-CoA. This suggests that Hhat is not limited to palmitoyl-CoA as previously assumed. The fluorescent assay confirmed direct fatty acyl transfer to Shh in vitro. The luciferase-based reporter system showed that Shh modified with saturated fatty acids induced a stronger signaling response than those modified with monounsaturated fatty acids. These findings indicate that Hhat can utilize multiple fatty acyl-CoA substrates. The enzyme’s flexibility in substrate use may lead to heterogeneous acylation of Shh. This heterogeneity could influence signaling outcomes in both developmental and pathological contexts.

The study demonstrates that Hhat can catalyze fatty acid transfer using multiple acyl-CoA substrates. The enzyme follows a random sequential reaction mechanism, which was previously uncharacterized for this family of enzymes. The findings suggest that Hhat is not strictly specific for palmitoyl-CoA. Instead, it can utilize myristoyl-CoA, palmitoleoyl-CoA, and oleoyl-CoA with similar efficiency. The researchers observed that Shh modified with saturated fatty acids activates signaling more potently than monounsaturated modifications. This implies that the type of fatty acyl modification may influence signaling strength. The results support the idea that heterogeneous fatty acylation of Shh could modulate signaling in developmental and cancer contexts. These conclusions align with the authors’ hypothesis that Hhat’s substrate flexibility may have functional implications for Shh activity.