A ternary complex of hydroxycinnamoyl-CoA hydratase–lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism

Abstract

HCHL (hydroxycinnamoyl-CoA hydratase-lyase) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and is exploited in whole-cell systems for the bioconversion of ferulic acid into natural equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active-site residues identified using the crystal structure of HCHL revealed that while Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the K(M) for the substrate ferulic acid, fulfilling its anticipated role as a factor in substrate binding. Structures of WT (wild-type) HCHL and of the S123A mutant, each of which had been co-crystallized with feruloyl-CoA, reveal a subtle helix movement upon ligand binding, the consequence of which is to bring the phenolic hydroxyl of Tyr-239 into close proximity to Tyr-75 from a neighbouring subunit in order to bind the phenolic hydroxyl of the product vanillin, for which electron density was observed. The active-site residues of ligand-bound HCHL display a remarkable three-dimensional overlap with those of a structurally unrelated enzyme, vanillyl alcohol oxidase, that also recognizes p-hydroxylated aromatic substrates related to vanillin. The data both explain the observed substrate specificity of HCHL for p-hydroxylated cinnamate derivatives and illustrate a remarkable convergence of the molecular determinants of ligand recognition between the two otherwise unrelated enzymes.

Mechanism of the Intramolecular Claisen Condensation Reaction Catalyzed by MenB, a Crotonase Superfamily Member

“…A similar strategy is employed by hydroxycinnamoyl-CoA hydratase–lyase (HCHL), another crotonase superfamily member that uses two Tyr residues as a molecular “pincer” to recognize the substrate, initiate deprotonation and exert strain on the substrate. The two Tyr residues in HCHL are located on analogous loops to those found in MenB (Figure 4), one of which is also only observed when the analogous A-loop in HCHL becomes ordered (48). …”

“…Throughout the MenB family, this residue is either an Asp or a Gly (Figure 4). Although G156 or the water molecule does not appear to play a major role during α-deprotonation or stabilization of the two oxyanion intermediates based on our ecMenB structure, a glutamate or water in a similar position has been proposed to perform acid/base catalysis in a number of crotonase superfamily members including ECH (62), dienoyl-CoA isomerase (63), ECI, (64), HCHL, (48) and DpgC (39). The bicarbonate dependence in the MenB activities of E. coli , S. aureus and B. subtilis and the presence of bicarbonate in the crystal structure of stMenB also has led to a proposal that D185 is used for α-deprotonation in mtMenB and that a bicarbonate cofactor performs the same function when the Asp is replaced by a Gly as in ecMenB, saMenB and bsMenB (21).…”

“…The CoA portion of the ligand is bound in the U-shaped conformation that is seen in the structures of other crotonase family members (8, 38, 42–48). In addition, the OSB portion is bound in such a way that the C7 carboxylate and C4 carbonyl are clearly out of the plane of the benzene ring, which is also the case when OSB is bound in OSB synthase from Amycolatopsis sp.…”

“…The disorder of the A-loop is a common observation among crotonase superfamily members (enoyl-CoA hydratase (ECH) (46, 57–58), methylmalonyl CoA decarboxylase (MMCD) (45), Δ 3 - Δ 2 -enoyl-CoA isomerase (ECI) (59), and hydroxycinnamoyl-CoA hydratase-lyase (HCHL) (48)), while in MenB the flexibility of the B-loop and C-loop has also been observed (8, 22). These loops together form the interface between the two trimers in the hexamer that buries more than 3450 Å, or 24% of the total surface area, and which is likely the reason for the stability of the mtMenB structure (22).…”

Biotechnology of flavours—the next generation

“…A labelling study using deuterated ferulic acid and Pycnoporus cinnabarinus showed that, by analogy to fatty acid degradation, the mechanism comprised the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant b-hydroxy thioester by a retro-aldol reaction (Krings et al 2001). Recently, the crystal structure of a substrate bound hydroxycinnamoyl-CoA hydrataselyase was reported (Bennett et al 2008). Only a few flavour formation pathways have received this high degree of attention.…”