Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins Michael P. Torrens-Spencea, Ying-Chih Chiangb,1, Tyler Smitha,c, Maria A. Vicenta,d, Yi Wangb, and Jing-Ke Wenga,c,2 aWhitehead Institute for Biomedical Research, Cambridge, MA 02142; bDepartment of Physics, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong; cDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and dDepartment of Biology, Williams College, Williamstown, MA 01267 Edited by Richard A. Dixon, University of North Texas, Denton, TX, and approved March 28, 2020 (received for review November 14, 2019) Radiation of the plant pyridoxal 5′-phosphate (PLP)-dependent aro- such as dopamine and serotonin, from their respective aromatic matic L-amino acid decarboxylase (AAAD) family has yielded an ar- L-amino acid precursors (5). AAAD family enzymes have radi- ray of paralogous enzymes exhibiting divergent substrate preferences ated in plants and insects to yield a number of paralogous en- and catalytic mechanisms. Plant AAADs catalyze either the decar- zymes with variation in both substrate preference and catalytic boxylation or decarboxylation-dependent oxidative deamination mechanism (Fig. 1 A–C and SI Appendix, Fig. S1) (6). In plants, of aromatic L-amino acids to produce aromatic monoamines or L-tryptophan decarboxylases (TDCs) and L-tyrosine decarbox- aromatic acetaldehydes, respectively. These compounds serve as ylases (TyDCs) are canonical AAADs that supply the aromatic key precursors for the biosynthesis of several important classes of monoamine precursors tryptamine and tyramine for the bio- plant natural products, including indole alkaloids, benzylisoquino- line alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde- synthesis of monoterpene indole alkaloids (MIAs) and benzyli- derived floral volatiles, and tyrosol derivatives. Here, we present soquinoline alkaloids (BIAs), respectively (SI Appendix, Fig. S2) the crystal structures of four functionally distinct plant AAAD (7, 8). In contrast, phenylacetaldehyde synthases (PAASs) and paralogs. Through structural and functional analyses, we identify 4-hydroxyphenylacetaldehyde synthases (4HPAASs) are mechanistically BIOCHEMISTRY variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward Significance indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently Plants biosynthesize their own proteinogenic aromatic L-amino arising mutations in AAAD paralogs leading to the convergent acids, namely L-phenylalanine, L-tyrosine, and L-tryptophan, evolution of the derived aldehyde synthase activity. Applying not only for building proteins but also for the production of a knowledge learned from this study, we successfully engineered plethora of aromatic amino acid-derived natural products. a shortened benzylisoquinoline alkaloid pathway to produce (S)- Pyridoxal 5′-phosphate (PLP)-dependent aromatic L-amino acid norcoclaurine in yeast. This work highlights the pliability of the decarboxylase (AAAD) family enzymes play important roles in AAAD fold that allows change of substrate selectivity and access channeling various aromatic L-amino acids into diverse down- to alternative catalytic mechanisms with only a few mutations. stream specialized metabolic pathways. Through comparative structural analysis of four functionally divergent plant AAAD enzyme evolution | AAAD | aromatic amino acid metabolism | specialized proteins together with biochemical characterization and mo- metabolism lecular dynamics simulations, we reveal the structural and mechanistic basis for the rich divergent and convergent evo- lants are sessile organisms that produce a dazzling array of lutionary development within the plant AAAD family. Knowl- Pspecialized metabolites as adaptive strategies to mitigate edge learned from this study aids our ability to engineer high- multitudes of abiotic and biotic stressors. Underlying plants’ value aromatic L-amino acid-derived natural product biosynthesis in remarkable chemodiversity is the rapid evolution of the requisite heterologous chassis organisms. specialized metabolic enzymes and pathways (1). Genome-wide comparative analysis across major green plant lineages has Author contributions: M.P.T.-S. and J.-K.W. designed research; M.P.T.-S., Y.-C.C., T.S., M.A.V., Y.W., and J.-K.W. performed research; M.P.T.-S., Y.-C.C., T.S., M.A.V., Y.W., and revealed pervasive and progressive expansions of discrete en- J.-K.W. analyzed data; and M.P.T.-S., Y.-C.C., Y.W., and J.-K.W. wrote the paper. zyme families mostly involved in specialized metabolism, Competing interest statement: J.-K.W. is a cofounder, a member of the Scientific Advisory wherein new enzymes emerge predominantly through gene du- Board, and a shareholder of DoubleRainbow Biosciences, which develops biotechnologies plication followed by subfunctionalization or neofunctionalization related to natural products. (2). Newly evolved enzyme functions usually entail altered sub- This article is a PNAS Direct Submission. strate specificity and/or product diversity without changes in the Published under the PNAS license. ancestral catalytic mechanism. In rare cases, adaptive mutations Data deposition: The sequences of P. somniferum, C. roseus, and E. grandis genes re- occur in a progenitor protein fold—either catalytic or non- ported in this article are deposited into National Center for Biotechnology Information catalytic—that give rise to new enzymatic mechanisms and novel GenBank under the following accession numbers: PsTyDC (MG748690), CrTDC biochemistry (3, 4). Resolving the structural and mechanistic (MG748691), and EgPAAS (MG786260). The crystal structures of CrTDC in complex with ʟ-tryptophan, PsTyDC in complex with ʟ-tyrosine, AtPAAS in complex with ʟ-phenylala- bases for these cases is an important step toward understand- nine, and unbound Rr4HPAAS have been deposited in the Protein Data Bank with the ID ing the origin and evolution of functionally disparate enzyme codes 6EEW, 6EEM, 6EEI, and 6EEQ, respectively. families. 1Present address: School of Chemistry, University of Southampton, SO17 1BJ Southamp- Aromatic amino acid decarboxylases (AAADs) are an ancient ton, United Kingdom. group of pyridoxal 5′-phosphate (PLP)-dependent enzymes with 2To whom correspondence may be addressed. Email: [email protected]. primary functions associated with amino acid metabolism. This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Mammals possess a single AAAD, dopa decarboxylase (DDC), doi:10.1073/pnas.1920097117/-/DCSupplemental. responsible for the synthesis of monoamine neurotransmitters, www.pnas.org/cgi/doi/10.1073/pnas.1920097117 PNAS Latest Articles | 1of12 Downloaded by guest on September 27, 2021 ACO O O O Bacterial and chlorophyte A. halleri A. lyrata OH Basal OH OH OH A. thaliana NH TyDC N 2 NH2 NH2 NH2 H HO HO TDC C. grandiflora L-tryptophan L-tyrosine L-phenylalanine L-tyrosine C. rubella Brassicacaeae B. stricta H2O + O2 H2O + O2 CrTDC PsTyDC AtPAAS Rr4HPAAS B. rapa FPsc B. oleracea CO CO CO +NH CO +NH 2 2 2 3 2 3 E. salsugineum +H2O2 +H2O2 C. papaya NH NH2 2 O O G. raimondii T. cacao N H H H HO HO C. clementina tryptamine tyramine phenylacetaldehyde 4-hydroxy- Malvidae C. sinensis phenylacetaldehyde P. trichocarpa S. purpurea L. usitatissimum HO M. esculenta O O N OH O O HO O R. communis O OH M. domestica OH OH OH P. persica N phenylethyl O OH F. vesca N alcohol OH C. sativus O G. max O morphine H O P. vulgaris N salidroside Rosid M. truncatula OH T. pratense N E. grandis vinblastine V. vinifera K. laxiflora Pentapetalae K. fedtschenkoi Bacterial and S. lycopersicum B chlorophyte clade S. tuberosum OsTDC *AtPAAS Eudicot M. guttatus CaTDC D. carota AtTyDC A. hypochondriacus A. coerulea S. italica Basal Clade S. viridis TDC Os P. hallii P. virgatum Z. mays Z. mays PH207 S. bicolor O thomaeum B. distachyon TDC Clade Angiosperm B. stacei TyDC Clade O. sativa A. comosus CrTDC Tracheophyte M. acuminata PsTyDC S. polyrhiza CaTDC Z. marina A. trichopoda Embryophyte S. moellendorfii P. patens *PhPAAS S. fallax *Pc4HPAAS PAAS M. polymorpha **Eg C. reinhardtii V. carteri D. salina Chlorophyte C. subellipsoidea C-169 R r 4 H P A A S M. pusilla CCMP1545 BdTyDC *Rr4HPAAS Micromonas sp RCC299 O. lucimarinus Fig. 1. Function, phylogeny, and taxonomic distribution of plant AAADs. (A) Biochemical functions of four representative plant AAADs in the context of their native specialized metabolic pathways. The dashed arrows indicate multiple catalytic steps. (B) A simplified maximum likelihood phylogenetic tree of bacteria, chlorophyte, and plant AAADs. A fully annotated tree is shown in SI Appendix, Fig. S3. The bacterial/chlorophyte, basal, TyDC, and TDC clades are colored in yellow, green, blue, and pink, respectively. Functionally characterized enzymes are labeled at the tree branches. The four AAADs for which their crystal structures were resolved in this study are denoted in bold. The EgPAAS identified and characterized in this study is underlined; * and ** denote two mechanistic classes of AASs that harbor naturally occurring substitutions at the large-loop catalytic tyrosine or the small-loop catalytic histidine, respectively. (C) Taxonomic distribution of plant AAADs across major lineages of green plants. The tree illustrates the phylogenetic relationship among