Fig. 5: GhDP1 and GhAKR13D2 collaboratively facilitate extracellular hydroxylation under oxidative condition. | Nature Communications

Fig. 5: GhDP1 and GhAKR13D2 collaboratively facilitate extracellular hydroxylation under oxidative condition.

From: A dirigent protein redirects extracellular terpenoid metabolism for defense against biotic challenges

Fig. 5

a Structural model of the GhDP1–GhAKR13D2 heterodimeric complex associated with hemigossypol (1). The structure was predicted by AlphaFold3, with GhDP1_A1 shown in green and GhAKR13D2_A3 in purple. 1 (yellow) is modeled in the active site of GhDP1_A1, positioned in close proximity to two key residues, R145 and Y102. The lower panel highlights the predicted hydrogen bonding interactions between 1 and the catalytic residues, with distances (in Å) indicated in red. Gray numbers indicate the positions of carbon atoms in 1. b Characterization of dirigent activities in vitro using apoplastic fluids from N. benthamiana leaves transiently expressing GhDP1_A1, GhDP1_A1 mutants, and control (empty vector), with 1 as the substrate. Extracted ion chromatograms (EICs) at m/z 277.1067 and m/z 519.2013 indicate 3 and 4, respectively. c, d Functional characterization of GhAKR13D2_A3 and GhDP1_A1 in the presence of Cu2+ by in vitro assays. Chromatographic peaks corresponding to 2 (c) and 3 (d) were detected at EICs m/z 277.1067 and m/z 275.0914, respectively, in positive mode. e LC-MS detection of hydroxylation products of 1 by GhDP1_A1, GhAKR13D2_A3 and homologous proteins of GhAKR13D2_A3 in vitro, in the presence of Cu²⁺. The hydroxylation product 2 was detected by LC-MS with an EIC at m/z 277.1067. The homologous proteins of GhAKR13D2_A3 were derived from Escherichia coli (NP_414953.2), Saccharomyces cerevisiae (NP_014068.1), Chlamydomonas reinhardtii (Cre11.g467622.t1.1), Physcomitrella patens (Pp3c6_770V3.1), Selaginella moellendorffii (270369), A. thaliana (AT1G60690.1), Solanum lycopersicum (Solyc01g097390.2.1), Drosophila melanogaster (NP_996068.1), Danio rerio (XP_021329604.1), Mus musculus (XP_006501119.1), and Homo sapiens (NP_751892.1). The right panel shows quantitative comparisons of hydroxylation product (2) levels, with and without AKRs. Data are presented as mean ± s.d. (n = 3 independent experiments); significance was determined by unpaired two-tailed t-tests. The product level in the group without AKR protein was set to 1. f Phylogenetic analysis and amino acid sequence alignment of GhAKR13D2 proteins across different kingdoms including microorganisms, animals and plants. Conserved catalytic sites are marked by red asterisks, while black asterisks denote NADP(H) binding sites. Source data are provided as a Source Data file.

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