Dynamic Conformational States Dictate Selectivity toward the Native Substrate in a Substrate-Permissive Acyltransferase

Abstract

Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) is an essential acyltransferase that mediates flux through plant phenylpropanoid metabolism by catalyzing a reaction between p-coumaroyl CoA and shikimate, yet it also exhibits broad substrate permissiveness in vitro. How do enzymes like HCT avoid functional derailment by cellular metabolites that qualify as non-native substrates? Here, we combine X-ray crystallography and molecular dynamics to reveal distinct dynamic modes of HCT under native versus non-native catalysis. We find that essential electrostatic and hydrogen-bonding interactions between the ligand and active site residues, enabled by active site plasticity, are elicited more effectively by shikimate than by other non-native substrates. This work provides a structural basis for how dynamic conformational states of HCT favor native over non-native catalysis by reducing the number of futile encounters between the enzyme and shikimate.

Promiscuity, impersonation and accommodation: evolution of plant specialized metabolism

“…This theme is exemplified by the hydroxycinnamoyltransferases (HCTs), which play a role in phenylpropanoid biosynthesis [23–26]. HCTs are involved in the conjugation of p -coumaroyl CoA to shikimate to form p -coumaroyl shikimate, and can be highly promiscuous [23–26]. For example, the A. thaliana HCT (AtHCT) uses nine substrates besides the native substrate shikimate – some better than shikimate (Figure 2C) – to produce a diversity of products in vitro .…”

“…Crystallography and molecular dynamics analysis of AtHCT highlighted two mechanisms by which HCT maintains its in vivo function while being such a promiscuous enzyme [26]. First, comparison of AtHCT apoenzyme:substrate crystal structure and molecular dynamics revealed that this enzyme undergoes a conformational change upon p -coumaroyl-CoA or p -coumaroylshikimate binding: this reduces the volume of the active site and induces changes in the position of residues involved in substrate binding and catalysis [26].…”

“…First, comparison of AtHCT apoenzyme:substrate crystal structure and molecular dynamics revealed that this enzyme undergoes a conformational change upon p -coumaroyl-CoA or p -coumaroylshikimate binding: this reduces the volume of the active site and induces changes in the position of residues involved in substrate binding and catalysis [26]. Second, a conserved Arg ‘handle’ in the active site forms electrostatic interactions with the shikimate carboxyl group, orienting the 5-hydroxyl towards the catalytic center (Figure 2D) [26]. An HCT structure from Coleus blumei (CbHCT) was used to corroborate both features [26].…”

“…Second, a conserved Arg ‘handle’ in the active site forms electrostatic interactions with the shikimate carboxyl group, orienting the 5-hydroxyl towards the catalytic center (Figure 2D) [26]. An HCT structure from Coleus blumei (CbHCT) was used to corroborate both features [26]. CbHCT was crystallized as a ternary complex with p -coumaroyl-CoA and 3-hydroxyacetophenone (Figure 2C), an uncharged non-native substrate [26].…”

“…An HCT structure from Coleus blumei (CbHCT) was used to corroborate both features [26]. CbHCT was crystallized as a ternary complex with p -coumaroyl-CoA and 3-hydroxyacetophenone (Figure 2C), an uncharged non-native substrate [26]. The CbHCT structure revealed that the active site does not shrink when bound to 3-hydroxyacetophenone [26].…”