O‐Methyltransferases involved in biphenyl and dibenzofuran biosynthesis

Abstract

SUMMARYBiphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O-methylation reactions. cDNAs encoding the O-methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab-causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5-dihydroxybiphenyl, supplied by the first pathway-specific enzyme, biphenyl synthase (BIS). 3,5-Dihydroxybiphenyl underwent a single methylation reaction in the presence of S-adenosyl-L-methionine (SAM). The second enzyme, SaOMT2, exhibited its highest affinity for noraucuparin, however the turnover rate was greater with 5-hydroxyferulic acid. Both substrates were only methylated at the meta-positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of the substrates with SAM also took place at a position other than the sulfur group. Expression of SaOMT1, SaOMT2 and SaBIS3 was transiently induced in rowan cell cultures by the addition of the fungal elicitor. While the immediate SaOMT1 products were not detectable in elicitor-treated cell cultures, noraucuparin and noreriobofuran accumulated transiently, followed by increasing levels of the SaOMT2 products aucuparin and eriobofuran. SaOMT1, SaOMT2 and SaBIS3 were N-and C-terminally fused with the super cyan fluorescent protein and a modified yellow fluorescent protein, respectively. All the fluorescent reporter fusions were localized to the cytoplasm of Nicotiana benthamiana leaf epidermis cells. A revised biosynthetic pathway of biphenyls and dibenzofurans in the Malinae is presented.

Genes Involved in Stress Response and Especially in Phytoalexin Biosynthesis Are Upregulated in Four Malus Genotypes in Response to Apple Replant Disease

“…So far, the ratelimiting steps of biphenyl and dibenzofuran biosynthesis remain unknown. Compared to the BIS genes, the fold changes in the expression of B4Ha and B4Hb were markedly lower, and genes for O-methyltransferases (Khalil et al, 2015) were not among the upregulated genes. Due to the incomplete examination of the phytoalexin biosynthetic pathway, some genes remain to be identified, including the gene coding for the enzyme that converts aucuparin to 2'-hydroxyaucuparin ( Figure S2).…”

Molecular architectures of benzoic acid-specific type III polyketide synthases

“…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.…”

Cinnamate‐CoA ligase is involved in biosynthesis of benzoate‐derived biphenyl phytoalexin in Malus × domestica ‘Golden Delicious’ cell cultures

“…In planta , BZL activity has been biochemically identified in two plants other than Malus , namely Clarkia breweri and Hypericum androsaemum (Abd El‐Mawla and Beerhues, ; Beuerle and Pichersky, 2002a). Although the core biphenyl biosynthetic pathway is well studied in the Malinae (Chizzali et al ., 2012a; Khalil et al ., ; Sircar et al ., ; Sarkate et al ., ) , the formation of the precursor, benzoyl‐CoA, is not well understood.…”