Date Published: December 01, 2017
Publisher: International Union of Crystallography
Author(s): Charles Stewart, Kate Woods, Greg Macias, Andrew C. Allan, Roger P. Hellens, Joseph P. Noel.
The structures of biphenyl synthase, of biphenyl synthase complexed with benzoyl-CoA and of benzophenone synthase are compared with that of a chalcone synthase homolog. These structures reveal that benzoic acid-specific type III polyketide synthases contain distinct structural elements, including a novel pocket, which underlie their evolutionary emergence.
Benzoic acid-specific type III polyketide synthases (PKSs) constitute a distinct clade within the type III PKS family that use benzoic acid-derived substrates (for example benzoyl-CoA, 3-hydroxybenzoyl-CoA and salicoyl-CoA) to produce phytoalexins and pharmacologically active compounds (Beerhues & Liu, 2009 ▸; Fig. 1 ▸). Biphenyl synthase (BIS) generates the core chemical scaffolds of biphenyl and dibenzofuran phytoalexins commonly found in the Pyrinae subtribe (Rosaceae; Liu et al., 2007 ▸; Khalil et al., 2013 ▸). The Pyrinae contain several economically important species, including apple (Malus × domestica), pear (Pyrus communis) and mountain ash (Sorbus aucuparia). Apples increased their expression of BIS after inoculation with the fireblight bacterium Erwinia amylovora (Chizzali et al., 2011 ▸). Furthermore, BIS transcripts as well as biphenyl and dibenzofuran compounds have been isolated from the transition zone between necrotic and healthy tissues in both apples and pears after inoculation with the fireblight bacterium (Chizzali et al., 2012 ▸, 2016 ▸). Additionally, when challenged with Venturia inaequalis, the causative fungus of apple scab, cell cultures of S. aucuparis and a scab-resistant M. domestica cultivar produced biphenyl and dibenzofuran metabolites (Hüttner et al., 2010 ▸; Khalil et al., 2013 ▸; Hrazdina & Borejsza-Wysocki, 2003 ▸). Additionally, the promiscuous in vitro activity of BIS with salicoyl-CoA was exploited to develop an artificial metabolic system in Escherichia coli that is capable of producing 4-hydroxycoumarin, an immediate precursor to synthetic anticoagulants (for example warfarin; Liu et al., 2010 ▸; Lin et al., 2013 ▸). In contrast to BIS, benzophenone synthase (BPS) generates the core chemical scaffolds of xanthones, guttiferones and sampsoniones that are prominently found in the closely related Hypericaceae and Clusiaceae families (Liu et al., 2003 ▸; Nualkaew et al., 2012 ▸). Xanthones are associated with diverse biological functions in Hypericum spp., including antimicrobials, UV pigments and antioxidants (Gronquist et al., 2001 ▸). Cell cultures of H. calycinum produced xanthones in response to yeast elicitation (Gaid et al., 2012 ▸). Similarly, elicitation of H. perforatum cell cultures with Agrobacterium tumefaciens led to an increase in BPS transcripts and xanthone accumulation (Franklin et al., 2009 ▸). Lastly, polyisoprenylated benzophenone derivatives are pharmacologically active and have served as lead compounds for drug development (Acuña et al., 2009 ▸; Wang et al., 2016 ▸).
The displaced solvent-exposed loop of MdBIS3 and HaBPS has also been noted in the structures of 2-pyrone synthase and stilbene synthase (Jez, Austin et al., 2000 ▸; Austin, Bowman et al., 2004 ▸). In the CoA complexes of MdBIS3 and 2-pyrone synthase, their displaced loops provide an additional hydrogen bond and van der Waals contacts to CoA ligands. The displaced loops of MdBIS3 and HaBPS correspond to the region B loop of pine STS described by Austin et al. (2004 ▸). Mutagenesis of residues in the pine STS region B loop by Austin et al. (2004 ▸) did not alter the cyclization specificity of pine STS. Interestingly, the region B loop in grapevine STS contains a Pro residue (Pro269) that exhibits a strong positive-selection pattern in a dN/dS analysis; an indicator that this residue and loop played a significant role in the emergence of the large stilbene synthase gene family in grapevine (Parage et al., 2012 ▸). Pro269 of grapevine STS is not conserved in MdBIS3 or HaBPS and there does not appear to be a consensus sequence for the displaced loop. Nonetheless, displacement of the region B loop is not observed in any available CHS structures. Furthermore, structural comparisons indicate that the rotameric state of the gatekeeper residue Phe265 (CHS numbering) consistently differs between CHS and functionally divergent type III PKSs (non-CHS). Thus, it appears that rotameric differences of Phe265 and the associated displacement of the three-residue solvent-exposed loop are likely to affect the kinetics and/or thermodynamics of acyl-CoA binding during type III PKS catalysis.
In conclusion, we report here the first structural analyses of benzoic acid-specific type III PKSs. Benzoic acid-specific type III PKSs catalyse the committed steps in the biosynthesis of benzophenone and biphenyl metabolites in plants. The products of BIS and BPS, 3,5-dihydroxybiphenyl and phlorobenzophenone, respectively, are subsequently modified via an assortment of tailoring reactions (for example, hydroxylation, O-methylation and prenylation) to yield the final repertoire of biphenyl, dibenzofuran and phlorobenzophenone metabolites found in plants (Khalil et al., 2015 ▸; El-Awaad et al., 2016 ▸; Sircar et al., 2015 ▸; Fiesel et al., 2015 ▸). BIS and BPS contain several small-to-large mutations which block the binding of 4-coumaroyl-CoA and provide hydrophobic surfaces for smaller hydrophobic starter CoAs and intermediates. Significantly, BIS and BPS contain a novel pocket in their active-site cavities associated with their chain-elongation and cyclization reactions. These structural idiosyncrasies underlie the preference of BIS and BPS for benzoic acid-derived substrates. Furthermore, the promiscuous nature of BIS and BPS may have contributed to the emergence of a type III PKS involved in alkaloid biosynthesis. The renowned promiscuity of type III PKSs is likely to underpin their ability to evolve as their host organisms interacted with constantly changing environments (Weng & Noel, 2012 ▸). The structural snapshots presented in this paper deepen our understanding of structure–function relationships in the type III PKS family and lay a foundation for ongoing efforts to exploit the biosynthetic potential of plant metabolic enzymes.