Abstract
The crystal structure of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) from Pseudomonas fluorescens AN103 has been solved to 1.8 A resolution. HCHL is a member of the crotonase superfamily and catalyses the hydration of the acyl-CoA thioester of ferulic acid [3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid] and the subsequent retro-aldol cleavage of the hydrated intermediate to yield vanillin (4-hydroxy-3-methoxy-benzaldehyde). The structure contains 12 molecules in the asymmetric unit, in which HCHL assumes a hexameric structure of two stacked trimers. The substrate, feruloyl-CoA, was modelled into the active site based on the structure of enoyl-CoA hydratase bound to the feruloyl-CoA-like substrate 4-(N,N-dimethylamino)-cinnamoyl-CoA (PDB code 1ey3). Feruloyl-CoA was bound in this model between helix 3 of the A subunit and helix 9 of the B subunit. A highly ordered structural water in the HCHL structure coincided with the thioester carbonyl of feruloyl-CoA in the model, suggesting that the oxyanion hole for stabilization of a thioester-derived enolate, characteristic of coenzyme-A dependent members of the crotonase superfamily, is conserved. The model also suggested that a strong hydrogen bond between the phenolic hydroxyl groups of feruloyl-CoA and BTyr239 may be an important determinant of the enzyme's ability to discriminate between the natural substrate and cinnamoyl-CoA, which is not a substrate.
Highlights
The microbial degradation of phenolic compounds from plant residues is of major biological and economic importance since it is an essential process in the decay of wood, in the disposal and recycling of plant wastes and in the industrial production of several commercially significant substances, such as the flavour and aroma compound vanillin (4-hydroxy-3-methoxybenzaldehyde), by biotransformation
The acyl-CoA thioester was transformed to vanillin (4) by the action of a single enzyme, hydroxycinnamoyl-CoA hydratase-lyase (HCHL), which first catalyses the hydration of the double bond to yield 4-hydroxy-3-methoxy-phenyl- -hydroxy-propionyl-CoA (3); the retro-aldol cleavage of (3) gives (4) and acetyl-CoA
We present a solution of the structure of HCHL, solved by molecular-replacement techniques using the enoyl-CoA hydratase (ECH) from Thermus thermophilus (PDB code 1uiy) as a model
Summary
The microbial degradation of phenolic compounds from plant residues is of major biological and economic importance since it is an essential process in the decay of wood, in the disposal and recycling of plant wastes and in the industrial production of several commercially significant substances, such as the flavour and aroma compound vanillin (4-hydroxy-3-methoxybenzaldehyde), by biotransformation. The acyl-CoA thioester was transformed to vanillin (4) by the action of a single enzyme, hydroxycinnamoyl-CoA hydratase-lyase (HCHL), which first catalyses the hydration of the double bond to yield 4-hydroxy-3-methoxy-phenyl- -hydroxy-propionyl-CoA (3); the retro-aldol cleavage of (3) gives (4) and acetyl-CoA. The enzymatic transformation of (2) into (4) represents an interesting mode of enzymatic activity that is reminiscent of the hydration of double bonds in enoyl-CoA and related substrates in fatty-acid oxidation pathways by the enzyme enoyl-CoA hydratase (ECH), sometimes known as crotonase (Engel et al, 1996). Crotonase superfamily members have been observed to catalyse a number of interesting chemical reactions, including the stereospecific hydration of double bonds performed by ECH and dehalogenation (Benning et al, 1996), double-bond isomerization in fatty acids (Modis et al, 1998; Mursula et al, 2001) and cyclization/aromatization reactions in the synthesis of vitamin K intermediates (Truglio et al, 2003). A model of the natural substrate, feruloyl-CoA, in the active site, is strongly indicative of a catalytic role for Glu143 and a substrate-specificity determining role for Tyr239 from the neighbouring monomer
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