A Structural Basis for the Biosynthesis of the Major Chlorogenic Acids Found in Coffee    

Abstract

(A.A.M.) Chlorogenic acids (CGAs) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee (Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5-O-dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in finetuning the levels of shikimate and quinate esters (CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds.

Maize Homologs of CCoAOMT and HCT, Two Key Enzymes in Lignin Biosynthesis, Form Complexes with the NLR Rp1 Protein to Modulate the Defense Response

“…The residue His (H) in the HxxxD motif is important for the enzymatic activity of HCT, and mutation of this amino acid greatly reduces its catalytic activity (Lallemand et al, 2012;Walker et al, 2013). We have shown that mutations at this conserved H residue in HCT1806/4918 did not reduce their ability to suppress Rp1-D21-induced HR (Wang et al, 2015c).…”

Evolutionarily Distinct BAHD N-acyltransferases are Responsible for Natural Variation of Aromatic Amine Conjugates in Rice

“…responsible for the biosynthesis and natural variation of benzoylated tryptamine/serotonin. Previous crystal studies have identified several amino acid residues in the N-and C-terminal regions of BAHD acyltransferases important for differential acylacceptor specificity (Ma et al, 2005;Unno et al, 2007;Lallemand et al, 2012;Walker et al, 2013). Here, comparative sequence analysis with in vitro assays using mutant enzymes led to the identification of a RRRR motif that is sufficient to transform agmatine utilization ( Figures 9A and 9B), which is similar to the conserved arginine-rich motifs in RNA binding proteins (Lazinski et al, 1989;Zapp et al, 1991;Yuryev et al, 1996).…”

“…The biological significance underpinning the convergent evolution of aryldiamine acyltransferases from both BAHD and GNAT families of proteins, especially within Gramineae, requires further investigation. The 3D structures of some BAHD acyltransferases have been resolved (Ma et al, 2005;Unno et al, 2007;Garvey et al, 2008;Lallemand et al, 2012;Manjasetty et al, 2012;Walker et al, 2013), but no structures are yet available for N-acyltransferases. Elucidation the structure of BAHD N-acyltransferase will greatly facilitate understanding these divergent and convergent evolutionary paths.…”

Maize Homologs of HCT, a Key Enzyme in Lignin Biosynthesis, Bind the NLR Rp1 Proteins to Modulate the Defense Response

“…BAHD family members share relatively low sequence identity, but nearly all members possess a conserved HxxxD motif (Burhenne et al, 2003). The His in the HxxxD motif is important for the catalytic activity of HCT, and mutation of this residue greatly reduces its activity (Lallemand et al, 2012;Walker et al, 2013). Mutations at this conserved His motif in HCT1806 and HCT4918 did not reduce their ability to inhibit Rp1-D21-induced HR, suggesting that the catalytic activity of HCT1806 and HCT4918 may not be required for their function in regulating Rp1-D21.…”

“…4A). The mutation in the His residue of sorghum or coffee (Coffea arabica) HCTs greatly reduced their enzymatic activities (Lallemand et al, 2012;Walker et al, 2013). We thus generated the corresponding mutation in the maize HCT4918: HCT4918(H152A).…”