Crystal Structure of Vinorine Synthase, the First Representative of the BAHD Superfamily

Abstract

Vinorine synthase is an acetyltransferase that occupies a central role in the biosynthesis of the antiarrhythmic monoterpenoid indole alkaloid ajmaline in the plant Rauvolfia. Vinorine synthase belongs to the benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl-, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase (BAHD) enzyme superfamily, members of which are involved in the biosynthesis of several important drugs, such as morphine, Taxol, or vindoline, a precursor of the anti-cancer drugs vincaleucoblastine and vincristine. The x-ray structure of vinorine synthase is described at 2.6-Å resolution. Despite low sequence identity, the two-domain structure of vinorine synthase shows surprising similarity with structures of several CoA-dependent acyltransferases such as dihydrolipoyl transacetylase, polyketide-associated protein A5, and carnitine acetyltransferase. All conserved residues typical for the BAHD family are found in domain 1. His 160 of the HXXXD motif functions as a general base during catalysis. It is located in the center of the reaction channel at the interface of both domains and is accessible from both sides. The channel runs through the entire molecule, allowing the substrate and co-substrate to bind independently. Asp 164 points away from the catalytic site and seems to be of structural rather than catalytic importance. Surprisingly, the DFGWG motif, which is indispensable for the catalyzed reaction and unique to the BAHD family, is located far away from the active site and seems to play only a structural role. Vinorine synthase represents the first solved protein structure of the BAHD superfamily.The acyl-CoA-dependent BAHD 1 superfamily is a fast growing enzyme family that has only recently been defined (1). The name BAHD is coined from the first four enzymes of the family isolated from plant species. The members of this family play an important role in the biosynthesis of a variety of secondary metabolites. The family might become significantly larger in the near future because ϳ70 BAHD-related genes have been identified recently in the Arabidopsis genome (2), and in most cases, their biochemical function still needs to be explored. Several BAHD members occurring in medicinal plants and fungi play very specific metabolic roles in biosynthetic pathways. The most prominent members are, for instance, those participating in the biosynthesis of the Catharanthus alkaloid vindoline (3), a precursor of the anti-cancer drugs vincaleucoblastine and vincristine, the Papaver alkaloid morphine (4), the diterpenoid alkaloid Taxol (5-7), anthocyanins (8 -10) as well as some phytoalexins (11), and enzymes involved in floral scent (12).A well-characterized enzyme of this family is vinorine synthase (VS; EC 2.3.1.160), which is of central importance in the endogenous formation of monoterpenoid indole alkaloids of the ajmalan type in the plant genus Rauvolfia. The synthase is located in the middle of the complex biosynthetic pathway that starts with tryptamine and the monoterpene s...

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.…”

“…When protein sequences of this clade were subjected to motif analysis using MEME (Bailey et al, 2015), we found the two BAHD family-conserved functional domains, HXXXD and DFGWG (Ma et al, 2005;D'Auria, 2006), and two clade IV-specific motifs (Supplemental Figures 7A and 7B). Considering the distinct specificity between clade IVa and IVb (exclusively using aliphatic or aromatic amines as acceptors, respectively; Tables 2 and 3), further motif searching coupled with visual inspection revealed a number of subclade-specific motifs.…”

“…Considering the distinct specificity between clade IVa and IVb (exclusively using aliphatic or aromatic amines as acceptors, respectively; Tables 2 and 3), further motif searching coupled with visual inspection revealed a number of subclade-specific motifs. Three motifs were observed in clade IVa, and two of them (VDGRAR and FMPSYLP) were located near the GN and DFGWG domain that compose a solvent channel for substrate binding (Ma et al, 2005; Supplemental Figures 7C and 7D). Similarly, two conserved motifs were highlighted in clade IVb (Supplemental Figures 7E and 7F).…”

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

“…HCT belongs to the BAHD family of plant acyl-CoAdependent acyltransferases, which possess a conserved HxxxD motif (Burhenne et al, 2003;Ma et al, 2005;D'Auria, 2006). 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).…”