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Catalytic Promiscuity Potentiated the Divergence of New Cytochrome bioRxiv preprint doi: https://doi.org/10.1101/398503; this version posted August 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Catalytic promiscuity potentiated the divergence of new cytochrome P450 2 enzyme functions in cyanogenic defense metabolism 3 4 Brenden Barco*(1), Lara Zipperer (2), and Nicole K. Clay (1) 5 6 *Correspondence should be addressed to B. Barco ([email protected]) 7 8 Current addresses: 9 (1) Department of Molecular, Cellular & Developmental Biology, Yale University, Kline 10 Biology Tower 734, 219 Prospect St., New Haven, CT 06511 11 (2) Department of Ecology & Evolutionary Biology, Yale University, New Haven, CT 12 06511 13 14 1 bioRxiv preprint doi: https://doi.org/10.1101/398503; this version posted August 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 15 Key words: enzyme promiscuity, regioselectivity, indole-3-cyanohydrin, camalexin, 16 cytochrome P450 monooxygenase, (regulatory) neofunctionalization, indole- 17 carbonylnitrile (ICN), substrate recognition site (SRS) 18 19 Abstract 20 Cytochrome P450 monooxygenases (P450s) constitute the largest metabolic enzyme 21 family in plants, responsible for synthesizing hundreds of thousands of specialized 22 metabolites with essential roles in chemical defenses against herbivores and 23 pathogens (Banks et al., 2011; Nelson and Werck-Reichhart, 2011; Wurtzel and 24 Kutchan, 2016). Substrate promiscuity has been documented to play a central role in 25 the evolution of plant specialized metabolic enzymes (Weng et al., 2012; Leong and 26 Last, 2017), however most plant P450s are highly substrate-specific (Verpoorte, 2013). 27 Here, we show the rapid inversion of primary and weak secondary (promiscuous) 28 catalytic activities between two distinct yet evolutionarily linked multifunctional P450s, 29 CYP71A12 and CYP71A13, based on intramolecular epistasis of two amino acid 30 residues under positive selection in CYP71A12. Furthermore, we uncover previously 31 undocumented catalytic activity during the inversion as well as naturally occurring 32 amino acid substitution patterns that could have been present in evolutionary 33 intermediates between the two enzymes. Comparative expression profiling and 34 homology modeling reveal that natural selection acted on the promoter of CYP71A13 35 and the substrate-recognition elements of CYP71A12 to improve the efficiencies of 36 their promiscuous reactions. The rise in catalytic promiscuity potentiated the 37 divergence of new P450 enzyme functions in cyanogenic defense metabolism. 38 Directed evolution of promiscuous reactions is one of the core technologies 39 underpinning the field of synthetic biology. Our results provide a more complete 40 understanding of how natural selection uses promiscuous reactions to generate new 41 enzymes in nature and chemical diversity in pathogen defense, as well as demonstrate 42 a novel strategy for identifying their molecular origins in highly divergent, related 43 enzymes. 44 2 bioRxiv preprint doi: https://doi.org/10.1101/398503; this version posted August 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 45 Main 46 Class II P450s from a monophyletic group of CYP71, CYP736 and CYP83 families have 47 been repeatedly recruited for the biosynthesis of cyanohydrins (α-hydroxynitriles) and 48 related structures from amino acid-derived oximes (Fig. 1a) (Bak et al., 1998; 49 Jørgensen et al., 2011; Nafisi et al., 2007; Nelson and Werck-Reichhart, 2011; Takos et 50 al., 2011; Irmisch et al., 2014; Rajniak et al., 2015; Yamaguchi et al., 2014, 2016). They 51 catalyze three consecutive reactions: an E-to-Z isomerization of the oxime substrate, 52 oxime dehydration, and hydroxylation at either the α-, β-, γ- or benzylic carbon (Bak et 53 al., 1998; Nafisi et al., 2007; Klein et al., 2013; Clausen et al., 2015; Knoch et al., 2016). 54 In A. thaliana, CYP83B1 lost the ability to isomerize tryptophan (Trp)-derived indole-3- 55 acetaldoxime (IAOx), using instead the E-oxime as its substrate in a pathway that 56 produces the defense metabolite 4-methoxy-indol-3yl-methylglucosinolate (4M-I3M) 57 (Fig. 1a) (Bak et al., 2001; Bednarek et al., 2009; Clay et al., 2009; Bednarek et al., 58 2011; Clausen et al., 2015). By contrast, CYP71A12 and CYP71A13 - which share 59 89.3% sequence identity - catalyze the conversion of IAOx to form α-hydroxy-indole-3- 60 acetonitrile (α-hydroxy-IAN), a novel cyanohydrin that is the precursor to the 61 cyanogenic defense metabolite 4-hydroxy-indole-3-carbonylnitrile (4OH-ICN) in 62 Arabidopsis (Fig. 1a) (Nafisi et al., 2007; Klein et al., 2013; Rajniak et al., 2015). 63 CYP71A13 alone additionally dehydrates α-hydroxy-IAN to form dehydro-IAN, 64 precursor to the non-cyanogenic defense metabolite camalexin in Arabidopsis (Klein et 65 al., 2013). 3 bioRxiv preprint doi: https://doi.org/10.1101/398503; this version posted August 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. (E/Z)-oxime acetonitrile cyanohydrin A barley CYP71C113 CYP79A8 CYP71L1 CYP71C113 Glc CYP79A12 HO O NH2 NOH CYP71U5 CYP71L1 N N N CO2H epiheterodendrin L-Leu CYP71C113 CYP71L1 β/γ-hydroxynitrile glycosides sorghum CYP71E1 Glc HO O NH2 CYP79A1 NOH N N N CO2H HO HO HO HO HO L-Tyr dhurrin Arabidopsis CYP71A12 CYP79B2 CYP71A13 CYP71A13 CYP79B3 HO NOH N N N N N N N H H H H L-Trp IAOx IAN α-OH-IAN dehydro-IAN camalexin CYP83B1 indole glucosinolates ICN 4OH-ICN B Capsella CYP71A28CYP71A13CYP71A12 Arabidopsis thaliana 8 months old Arabidopsis lyrata Boechera Olimarabidopsis cabulica Capsella rubella Super-tribe Crucihimalaya lasiocarpa n.d. Erysimum Boechera holboelii Polyctenium fremontii tr. Lineage I Erysimum chieri n.d. n.d. Cardamine hirsuta n.d. n.d. Brassica Lineage II Brassica rapa n.d. n.d. 0 50 100 150 0 5 15 25 35 nmol / mg DW nmol / mg DW camalexin (4OH-)ICN + products 66 Fig. 1. (a) (left) Images courtesy of https://plants.usda.gov. (right) Schematics of the three-branched pathway for cyanohydrin metabolism in H. vulgare, S. bicolor, and A. thaliana. Amino acids are N,N-hydroxylated and decarboxylated by CYP79 enzymes to produce aldoximes. CYP71 enzymes then perform a variety of subsequent reactions, including aldoxime isomerization, aldoxime dehydration, acetonitrile oxygenation, and cyanohydrin dehydration. Diverse enzyme families then tailor these compounds into cyanogenic glycosides (e.g. epiheterodendrin and dhurrin), cyanohydrin- derived antimicrobials (e.g. camalexin and 4OH-ICN), or side products not derived from a cyanohydrin (e.g. β/γ hydroxynitrile glycosides, indole glucosinolates). (b) (far left, top to bottom) Images of Capsella rubella, Boechera holboelii, Erysimum chieri, and Brassica rapa. (middle) Occurence of CYP71As denoted by yellow (CYP71A28), red (CYP71A13), and blue (CYP71A12) circles. Dashed boxes indicate hypothesized presence due to the absence of genomic sequence. (right) Camalexin and ICN metabolite levels in 10-day-old seedlings inoculated with Pto avrRpm1 for 30 h. Data represent the mean ± SE of four replicates of 13 to 17 seedlings each from a compilation of three independent experiments, each containing A. thaliana as a positive control. Experiments were repeated twice, producing similar results. Thick branches represent the Super-tribe. ICA-ME, breakdown product of ICN. Abbreviations: DW, dry weight; tr. trace; n.d., not detected, tr., trace. 4 bioRxiv preprint doi: https://doi.org/10.1101/398503; this version posted August 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 67 Fig. 1. Phylogenetic distribution of CYP71A12 and CYP71A13 orthologs and their 68 physiological functions. (a) Biosynthetic pathways of cyanohydrin-derived plant 69 defense metabolites. Images courtesy of https://plants.usda.gov. (b) Presence of 70 CYP71A12, CYP71A13, and CYP71A28 orthologs in the Brassicaceae, inferred from 71 shared synteny (solid circles) or relatedness to plants with sequenced orthologs 72 (dashed circles) See Methods for lists of contigs and chromosomes used. Phylogenetic 73 species tree (left). Levels of camalexin (middle) and (4OH-)ICN metabolites (right) in 10- 74 day-old seedlings inoculated with Psta for 30 h. Data represent mean ± SE of four 75 replicates of 13 to 17 seedlings each. Thick branches represent the Super-tribe. To 76 ensure robust sampling of certain lineages, unsequenced plant species were profiled, 77 and presence of orthologs in these species were predicted based on the available 78 sequences of close relatives. To ensure robust activation of defense metabolism in 79 sampled species, 4M-I3M and related metabolites were profiled, and 4M-I3M’s 80 signaling function was assayed (Extended Data Fig. 1a–b; Supplementary Note 1). 5 bioRxiv preprint doi: https://doi.org/10.1101/398503; this version posted August 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 81 To determine whether the CYP71A13 dehydration reaction is indicative of a functional 82 gain or loss, we investigated whether physiological functions of CYP71A12/A13 are 83 conserved outside of Arabidopsis.
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