Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes

Abstract

Convergent evolution is a process that has occurred throughout the tree of life, but the historical genetic and biochemical context promoting the repeated independent origins of a trait is rarely understood. The well-known stimulant caffeine, and its xanthine alkaloid precursors, has evolved multiple times in flowering plant history for various roles in plant defense and pollination. We have shown that convergent caffeine production, surprisingly, has evolved by two previously unknown biochemical pathways in chocolate, citrus, and guaraná plants using either caffeine synthase- or xanthine methyltransferase-like enzymes. However, the pathway and enzyme lineage used by any given plant species is not predictable from phylogenetic relatedness alone. Ancestral sequence resurrection reveals that this convergence was facilitated by co-option of genes maintained over 100 million y for alternative biochemical roles. The ancient enzymes of the Citrus lineage were exapted for reactions currently used for various steps of caffeine biosynthesis and required very few mutations to acquire modern-day enzymatic characteristics, allowing for the evolution of a complete pathway. Future studies aimed at manipulating caffeine content of plants will require the use of different approaches given the metabolic and genetic diversity revealed by this study.

Keywords: caffeine biosynthesis; convergent evolution; enzyme evolution; paleomolecular biology.

Fig. 1.

Caffeine biosynthetic network has 12 potential paths. The only path characterized from plants is shown by solid black arrows and involves sequential methylation of xanthosine at N-7, 7-methylxanthine at N-3, and theobromine at N-1 of the heterocyclic ring. Each methylation step is performed by a separate xanthine alkaloid methyltransferase in Coffea. In contrast, Camellia employs the distantly related caffeine synthase enzyme, TCS1, for both the second and third methylation steps, whereas the enzyme that catalyzes the first reaction remains uncharacterized. Other potential biochemical pathways to caffeine are shown by dashed arrows, but enzymes specialized for those conversions are unknown. Cleavage of ribose from 7-methylxanthosine is not shown, but may occur concomitantly with N-7 methylation of xanthosine. CF, caffeine; PX, paraxanthine; TB, theobromine; TP, theophylline; X, xanthine; 1X, 1-methylxanthine; 3X, 3-methylxanthine; 7X, 7-methylxanthine; XR, xanthosine.

Fig. 2.

Caffeine has convergently evolved in five flowering plant species using different combinations of genes and pathways. (A) Phylogenetic relationships among orders of Rosids and Asterids show multiple origins of caffeine biosynthesis. Lime-green lineages trace the ancient CS lineage of enzymes that has been independently recruited for use in caffeine-accumulating tissues in Theobroma, Paullinia, and Camellia. Turquoise lineages trace the ancient XMT lineage that was independently recruited in Citrus and Coffea. (B and C) Theobroma and Paullinia have converged upon similar biosynthetic pathways catalyzed by CS-type enzymes. (D) Citrus has evolved a different pathway catalyzed by XMT-type enzymes, despite its close relationship to Paullinia. (E and F) Camellia and Coffea catalyze the same pathway using different enzymes. Proposed biochemical pathways are based on relative enzyme activities shown by corresponding bar charts that indicate mean relative activities (from 0 to 1) with eight xanthine alkaloid substrates. CisXMT1 and TCS1 catalyze more than one reaction in the proposed pathways. XMT and CS have recently and independently duplicated in each of the five lineages (see Fig. S1 for a detailed gene tree). ^#Data taken from the literature; *substrate not assayed.

Fig. S1.

Phylogenetic relationships among 356 SABATH protein sequences. Sequences were extracted from 11 complete genomes of land plants in addition to selected CS and XMT transcriptome sequences from the oneKP database. Lineages with functionally characterized sequences are labeled by enzyme name, whereas those without known functions are arbitrarily numbered from MT1 to MT6. Bootstrap support values are shown for selected nodes that define major enzyme lineages. Enzymes from Camellia (CS) or Coffea (XMT) known to be involved in caffeine biosynthesis are shown in lime-green and turquoise, respectively. Sequences expressed in Theobroma and Paullinia fruits are clearly orthologous to CS sequences from Camellia. Sequences expressed in Citrus flowers are clearly orthologous to XMT sequences of Coffea. Arrows point to CS and XMT lineages to show recent duplication events within Theobroma, Paullinia, Camellia, Citrus, and Coffea. Nodes for which ancestral resurrected proteins were studied are labeled A–C. Accession numbers for the oneKP and GenBank databases are shown before and after relevant sequences, respectively.

Fig. S2.

(A) The closely related TcCS1 and TcCS2 of Theobroma cacao are highly represented in fruits where theobromine and caffeine accumulate. EST counts in various tissues are shown for all full-length SABATH genes from the genome of T. cacao (Matina). Relationships among the sequences are shown below the chart. GenBank accession numbers are as follows: IAMT (Thecc1EG030787), FAMTa (Thecc1EG019318), FAMTb (Thecc1EG019315), FAMTc (Thecc1EG019314), MT4a (Thecc1EG011287), MT4b (Thecc1EG011286), MT4c (Thecc1EG011290), MT4d (Thecc1EG011291), MT1a (Thecc1EG045368), MT1b (Thecc1EG045372), MT1c (Thecc1EG045370), MT5a (Thecc1EG012604), MT5b (Thecc1EG031006), BAMTa (Thecc1EG000331), BAMTb (Thecc1EG000328), BAMTc (Thecc1EG040854), XMT (Thecc1EG006850), MT3 (Thecc1EG000336), JMTa (Thecc1EG034091), JMTb (Thecc1EG034089), SAMTa (Thecc1EG000326), SAMTb (Thecc1EG000324), BTS (Thecc1EG042576), TcCS1 (Thecc1EG042578), and TcCS2 (Thecc1EG042587, Thecc1EG042590). The two accession numbers for TcCS2 are indistinguishable in the ORFs and are therefore classified together in this chart. (B) The closely related PcCS1 to PcCS5 are highly represented in fruits of Paullinia cupana var. sorbilis, where caffeine accumulates. EST counts in fruit tissues are shown for all full-length SABATH genes. SABATH sequences from the Citrus genome were used to BLAST for ESTs from Paullinia, because a genome is not yet characterized for it. Relationships among the sequences are shown below the chart. Only CS-type sequences were found from Paullinia ESTs. GenBank accession numbers are as follows: GAMT (Citrus: 1g014333m), IAMT (Citrus: 1g016644m), FAMT (Citrus: 1g017702m), MT4 (Citrus: 1g040129m), MT2 (Citrus: 1g018119m), XMT (Citrus: 1g044727m), SAMT (Citrus: 1g017514m), JMT (Citrus: XM_006478399), MT3 (Citrus: 1g017747m), PcCS3 (EC763988, EC777706, EC777629, EC777248, EC774687, EC768101, EC769415, EC769184, EC769690, EC773071, EC764367), PcCS1 (59), PcCS4 (EC765512, EC766603, EC766748, EC777652, EC769966, EC775308, EC766876), PcCS5 (EC765614, EC764433, EC776114, EC764220, EC778019, EC768317, EC764348, EC774623, EC765624, EC772805), and PcCS2 (EC774880, EC768886, EC775438, EC772015, EC764433, EC771794, EC774462, EC772993, EC770731, EC773205, EC765633, EC764023, 776855, EC767182, EC770596). Enzyme activities of PcCS 1 and 4 were highly comparable, as were PcCS2 and 5, so we only present data for one of each in the manuscript. PcCS3 had no detectable activity with any xanthine alkaloid substrate tested. (C) The closely related TCS1 and TCS2 of Camellia sinensis are highly represented in leaves and buds where caffeine accumulates. EST counts in various tissues are shown for representatives of each SABATH gene lineage. Full-length genes from each major lineage of the SABATH family in the Mimulus genome were used to BLAST for ESTs from Camellia, because its genome is not yet characterized. Relationships among the sequences are shown below the chart. Sequences used for BLAST/phylogenetic analysis are as follows: Mimulus IAMT (Phytozome accession no. M01704), Camellia sinensis MT4 (GenBank accession no. GB-GBBZ01008401), Mimulus MT1 (Phytozome accession no. H02148), Mimulus FAMT (Phytozome accession no. H00254), Camellia sinensis SAMT (GenBank accession no. KA284044), Camellia sinensis JMT (GenBank accession no. KA286401), Camellia sinensis MT3 (GenBank accession no. FS950428), Mimulus BAMT (Phytozome accession no. N01694), Camellia sinensis TCS2 (GenBank accession no. AB031281), and Camellia sinensis TCS1 (GenBank accession no. AB031280). No XMT ortholog is known from Mimulus or Ericales.

Fig. S2.

(A) The closely related TcCS1 and TcCS2 of Theobroma cacao are highly represented in fruits where theobromine and caffeine accumulate. EST counts in various tissues are shown for all full-length SABATH genes from the genome of T. cacao (Matina). Relationships among the sequences are shown below the chart. GenBank accession numbers are as follows: IAMT (Thecc1EG030787), FAMTa (Thecc1EG019318), FAMTb (Thecc1EG019315), FAMTc (Thecc1EG019314), MT4a (Thecc1EG011287), MT4b (Thecc1EG011286), MT4c (Thecc1EG011290), MT4d (Thecc1EG011291), MT1a (Thecc1EG045368), MT1b (Thecc1EG045372), MT1c (Thecc1EG045370), MT5a (Thecc1EG012604), MT5b (Thecc1EG031006), BAMTa (Thecc1EG000331), BAMTb (Thecc1EG000328), BAMTc (Thecc1EG040854), XMT (Thecc1EG006850), MT3 (Thecc1EG000336), JMTa (Thecc1EG034091), JMTb (Thecc1EG034089), SAMTa (Thecc1EG000326), SAMTb (Thecc1EG000324), BTS (Thecc1EG042576), TcCS1 (Thecc1EG042578), and TcCS2 (Thecc1EG042587, Thecc1EG042590). The two accession numbers for TcCS2 are indistinguishable in the ORFs and are therefore classified together in this chart. (B) The closely related PcCS1 to PcCS5 are highly represented in fruits of Paullinia cupana var. sorbilis, where caffeine accumulates. EST counts in fruit tissues are shown for all full-length SABATH genes. SABATH sequences from the Citrus genome were used to BLAST for ESTs from Paullinia, because a genome is not yet characterized for it. Relationships among the sequences are shown below the chart. Only CS-type sequences were found from Paullinia ESTs. GenBank accession numbers are as follows: GAMT (Citrus: 1g014333m), IAMT (Citrus: 1g016644m), FAMT (Citrus: 1g017702m), MT4 (Citrus: 1g040129m), MT2 (Citrus: 1g018119m), XMT (Citrus: 1g044727m), SAMT (Citrus: 1g017514m), JMT (Citrus: XM_006478399), MT3 (Citrus: 1g017747m), PcCS3 (EC763988, EC777706, EC777629, EC777248, EC774687, EC768101, EC769415, EC769184, EC769690, EC773071, EC764367), PcCS1 (59), PcCS4 (EC765512, EC766603, EC766748, EC777652, EC769966, EC775308, EC766876), PcCS5 (EC765614, EC764433, EC776114, EC764220, EC778019, EC768317, EC764348, EC774623, EC765624, EC772805), and PcCS2 (EC774880, EC768886, EC775438, EC772015, EC764433, EC771794, EC774462, EC772993, EC770731, EC773205, EC765633, EC764023, 776855, EC767182, EC770596). Enzyme activities of PcCS 1 and 4 were highly comparable, as were PcCS2 and 5, so we only present data for one of each in the manuscript. PcCS3 had no detectable activity with any xanthine alkaloid substrate tested. (C) The closely related TCS1 and TCS2 of Camellia sinensis are highly represented in leaves and buds where caffeine accumulates. EST counts in various tissues are shown for representatives of each SABATH gene lineage. Full-length genes from each major lineage of the SABATH family in the Mimulus genome were used to BLAST for ESTs from Camellia, because its genome is not yet characterized. Relationships among the sequences are shown below the chart. Sequences used for BLAST/phylogenetic analysis are as follows: Mimulus IAMT (Phytozome accession no. M01704), Camellia sinensis MT4 (GenBank accession no. GB-GBBZ01008401), Mimulus MT1 (Phytozome accession no. H02148), Mimulus FAMT (Phytozome accession no. H00254), Camellia sinensis SAMT (GenBank accession no. KA284044), Camellia sinensis JMT (GenBank accession no. KA286401), Camellia sinensis MT3 (GenBank accession no. FS950428), Mimulus BAMT (Phytozome accession no. N01694), Camellia sinensis TCS2 (GenBank accession no. AB031281), and Camellia sinensis TCS1 (GenBank accession no. AB031280). No XMT ortholog is known from Mimulus or Ericales.

Fig. S2.

(A) The closely related TcCS1 and TcCS2 of Theobroma cacao are highly represented in fruits where theobromine and caffeine accumulate. EST counts in various tissues are shown for all full-length SABATH genes from the genome of T. cacao (Matina). Relationships among the sequences are shown below the chart. GenBank accession numbers are as follows: IAMT (Thecc1EG030787), FAMTa (Thecc1EG019318), FAMTb (Thecc1EG019315), FAMTc (Thecc1EG019314), MT4a (Thecc1EG011287), MT4b (Thecc1EG011286), MT4c (Thecc1EG011290), MT4d (Thecc1EG011291), MT1a (Thecc1EG045368), MT1b (Thecc1EG045372), MT1c (Thecc1EG045370), MT5a (Thecc1EG012604), MT5b (Thecc1EG031006), BAMTa (Thecc1EG000331), BAMTb (Thecc1EG000328), BAMTc (Thecc1EG040854), XMT (Thecc1EG006850), MT3 (Thecc1EG000336), JMTa (Thecc1EG034091), JMTb (Thecc1EG034089), SAMTa (Thecc1EG000326), SAMTb (Thecc1EG000324), BTS (Thecc1EG042576), TcCS1 (Thecc1EG042578), and TcCS2 (Thecc1EG042587, Thecc1EG042590). The two accession numbers for TcCS2 are indistinguishable in the ORFs and are therefore classified together in this chart. (B) The closely related PcCS1 to PcCS5 are highly represented in fruits of Paullinia cupana var. sorbilis, where caffeine accumulates. EST counts in fruit tissues are shown for all full-length SABATH genes. SABATH sequences from the Citrus genome were used to BLAST for ESTs from Paullinia, because a genome is not yet characterized for it. Relationships among the sequences are shown below the chart. Only CS-type sequences were found from Paullinia ESTs. GenBank accession numbers are as follows: GAMT (Citrus: 1g014333m), IAMT (Citrus: 1g016644m), FAMT (Citrus: 1g017702m), MT4 (Citrus: 1g040129m), MT2 (Citrus: 1g018119m), XMT (Citrus: 1g044727m), SAMT (Citrus: 1g017514m), JMT (Citrus: XM_006478399), MT3 (Citrus: 1g017747m), PcCS3 (EC763988, EC777706, EC777629, EC777248, EC774687, EC768101, EC769415, EC769184, EC769690, EC773071, EC764367), PcCS1 (59), PcCS4 (EC765512, EC766603, EC766748, EC777652, EC769966, EC775308, EC766876), PcCS5 (EC765614, EC764433, EC776114, EC764220, EC778019, EC768317, EC764348, EC774623, EC765624, EC772805), and PcCS2 (EC774880, EC768886, EC775438, EC772015, EC764433, EC771794, EC774462, EC772993, EC770731, EC773205, EC765633, EC764023, 776855, EC767182, EC770596). Enzyme activities of PcCS 1 and 4 were highly comparable, as were PcCS2 and 5, so we only present data for one of each in the manuscript. PcCS3 had no detectable activity with any xanthine alkaloid substrate tested. (C) The closely related TCS1 and TCS2 of Camellia sinensis are highly represented in leaves and buds where caffeine accumulates. EST counts in various tissues are shown for representatives of each SABATH gene lineage. Full-length genes from each major lineage of the SABATH family in the Mimulus genome were used to BLAST for ESTs from Camellia, because its genome is not yet characterized. Relationships among the sequences are shown below the chart. Sequences used for BLAST/phylogenetic analysis are as follows: Mimulus IAMT (Phytozome accession no. M01704), Camellia sinensis MT4 (GenBank accession no. GB-GBBZ01008401), Mimulus MT1 (Phytozome accession no. H02148), Mimulus FAMT (Phytozome accession no. H00254), Camellia sinensis SAMT (GenBank accession no. KA284044), Camellia sinensis JMT (GenBank accession no. KA286401), Camellia sinensis MT3 (GenBank accession no. FS950428), Mimulus BAMT (Phytozome accession no. N01694), Camellia sinensis TCS2 (GenBank accession no. AB031281), and Camellia sinensis TCS1 (GenBank accession no. AB031280). No XMT ortholog is known from Mimulus or Ericales.

Fig. S3.

TcCS2 converts xanthine to 7-methylxanthine. Eight mass spectrometry scans show parent ion–fragment ion pairs that are largely unique for each xanthine alkaloid product detected in enzyme assays supplied with 100 µM xanthine. Scans for authentic standards are shown below to verify product identity.

Fig. S4.

(A) The closely related CisXMT1 and CisXMT2 of Citrus sinensis are highly represented in flowers where caffeine accumulates. EST counts in various tissues are shown for all full-length SABATH genes from the genome of Citrus sinensis. Relationships among the sequences are shown below the chart. GenBank accession numbers are as follows: GAMT (1g014333m), IAMT (1g016644m), MT4a (1g040129m), MT4b (1g044174m), MT2 (1g018119m), FAMTa (1g017702m), FAMTb (1g037735m), FAMTc (1g018250m), FAMTd (1g017363m), FAMTe (017439m), CS (1g18139m), MT3 (1g017747m), CisXMT5 (1g036911m), CisXMT4 (XM_006469416), CisXMT3 (1g045960m), CisXMT2 (1g047625m), CisXMT1 (1g044727m), JMT (XM_006478399), and SAMT (1g017514m). (B) Relative enzyme activity profile for SAMT from Citrus sinensis. Bar charts show relative enzyme activities (from 0 to 1) for three substrates. AA, anthranilic acid; BA, benzoic acid; SA, salicylic acid.

Fig. S5.

Mass spectrometry scans show that Citrus CisXMT1 can form 1-methylxanthine, 3-methylxanthine, and theophylline from xanthine alone. Citrus CisXMT1 converts xanthine to both 1-methylxanthine and 3-methylxanthine, as indicated by the presence of parent ion–fragment ion peaks unique for both compounds. The presence of the theophylline peak likely is a result of methylation of both 1-methylxanthine and 3-methylxanthine, although which may be preferred cannot be discerned from these traces alone. However, it should be noted that the catalytic efficiency by which 3-methylxanthine is converted to theophylline is ca. four times lower than that of 1-methylxanthine (Table S1). Scans for authentic standards are shown below to confirm product identities.

Fig. S6.

Citrus flower buds appear to accumulate xanthine, 1-methylxanthine, theophylline, and caffeine as well as minor amounts of 3-methylxanthine. Mass spectrometry scans show parent ion–fragment ion pairs unique for each xanthine alkaloid product detected in Citrus flower buds. The presence of these metabolites is consistent with the enzyme assays (Fig. 2D and Fig. S5) and suggests that the primary pathways by which caffeine is produced in Citrus flowers are those shown in Fig. 2D. Table S2 shows parent ion–fragment ion masses for xanthine alkaloid standards.

Fig. 3.

Resurrected ancestral XMT proteins reveal the historical context for convergent evolution of caffeine biosynthesis. Bar charts show mean relative enzyme activities (from 0 to 1) for 10 substrates. BA, benzoic acid; SA, salicylic acid; all others are as in Fig. 1. Node A shows the resurrected enzyme of the >100-My-old ancestor of Rosids and Asterids that exhibits high relative activity with benzoic and salicylic acid. Although those ancestral activities were maintained in CisAncXMT1 at node B and modern-day Mangifera, they were eventually replaced by increased relative preference for xanthine alkaloid methylation as seen at node C and its descendants. CisAncXMT2 mutants (P25S and H150N indicated on lineages C′ and C′′, respectively) show that very few amino acid replacements are necessary to re-evolve modern-day enzyme activity patterns and form a complete caffeine biosynthetic pathway. Product formation from assays and implied pathway connections are shown by color-coded dots (Insets). For example, a connection between TP (green) and CF (black) implies that the enzyme in question converts theophylline to caffeine. Unshaded rectangles exhibit complete metabolic connections to caffeine, whereas shaded rectangles do not. Average site-specific posterior probabilities are shown for each resurrected ancestral enzyme. Select substrate structures are shown to specify the atom to which a methyl group is transferred.

Fig. S7.

(A) CisAncXMT2 converts 1-methylxanthine to paraxanthine. Mass spectrometry scans show parent ion–fragment ion pairs largely unique for each xanthine alkaloid product detected in enzyme assays supplied with 100 µM 1-methylxanthine. Scans for authentic standards are shown below to confirm product identity. (B) CisAncXMT2 converts 3-methylxanthine to theophylline. Mass spectrometry scans show parent ion–fragment ion pairs largely unique for each xanthine alkaloid product detected in enzyme assays supplied with 100 µM 3-methylxanthine. Scans for authentic standards are shown below to confirm product identity. (C) CisAncXMT2 converts theophylline to caffeine. Mass spectrometry scans show parent ion–fragment ion pairs largely unique for each xanthine alkaloid product detected in enzyme assays supplied with 100 µM theophylline. Scans for authentic standards are shown below to confirm product identity.

Fig. S7.

(A) CisAncXMT2 converts 1-methylxanthine to paraxanthine. Mass spectrometry scans show parent ion–fragment ion pairs largely unique for each xanthine alkaloid product detected in enzyme assays supplied with 100 µM 1-methylxanthine. Scans for authentic standards are shown below to confirm product identity. (B) CisAncXMT2 converts 3-methylxanthine to theophylline. Mass spectrometry scans show parent ion–fragment ion pairs largely unique for each xanthine alkaloid product detected in enzyme assays supplied with 100 µM 3-methylxanthine. Scans for authentic standards are shown below to confirm product identity. (C) CisAncXMT2 converts theophylline to caffeine. Mass spectrometry scans show parent ion–fragment ion pairs largely unique for each xanthine alkaloid product detected in enzyme assays supplied with 100 µM theophylline. Scans for authentic standards are shown below to confirm product identity.

Fig. S7.

(A) CisAncXMT2 converts 1-methylxanthine to paraxanthine. Mass spectrometry scans show parent ion–fragment ion pairs largely unique for each xanthine alkaloid product detected in enzyme assays supplied with 100 µM 1-methylxanthine. Scans for authentic standards are shown below to confirm product identity. (B) CisAncXMT2 converts 3-methylxanthine to theophylline. Mass spectrometry scans show parent ion–fragment ion pairs largely unique for each xanthine alkaloid product detected in enzyme assays supplied with 100 µM 3-methylxanthine. Scans for authentic standards are shown below to confirm product identity. (C) CisAncXMT2 converts theophylline to caffeine. Mass spectrometry scans show parent ion–fragment ion pairs largely unique for each xanthine alkaloid product detected in enzyme assays supplied with 100 µM theophylline. Scans for authentic standards are shown below to confirm product identity.

Fig. S8.

HPLC analyses show the evolution of product formation in ancestral and modern-day Citrus XMT enzymes. CisAncXMT2 converts xanthine to 1-methylxanthine but not 3-methylxanthine (Insets). In contrast, CisAncXMT2 P25S and CisXMT1 methylate xanthine to both 1-methylxanthine and 3-methylxanthine, which are further converted into theophylline. Enzyme assays were conducted using 2 mM xanthine, and products were measured by absorbance at 272 nm. mAU, milliabsorption units.

Fig. S9.

Aligned protein sequences show that very few sites differ between ancestral and modern-day enzymes and that mutations at very few sites account for functional change. (A) Amino acid alignment of functionally characterized modern-day XMT and CS sequences. Also shown are resurrected ancestral XMT protein sequences. Amino acids that were experimentally replaced to recapitulate evolutionary changes in the Citrus lineage are highlighted in turquoise. Alternative ancestral alleles were generated by mutations shown in green. (B) Posterior probabilities of original and mutated sites are shown for the four alternative ancestral alleles generated and assayed. (C) Bar charts show relative enzyme activities of additional site-directed mutants made to recapitulate evolutionary changes from CisAncXMT2 to either CisXMT1 or CisXMT2. Mutated amino acid positions are highlighted in magenta.

Fig. S9.

Aligned protein sequences show that very few sites differ between ancestral and modern-day enzymes and that mutations at very few sites account for functional change. (A) Amino acid alignment of functionally characterized modern-day XMT and CS sequences. Also shown are resurrected ancestral XMT protein sequences. Amino acids that were experimentally replaced to recapitulate evolutionary changes in the Citrus lineage are highlighted in turquoise. Alternative ancestral alleles were generated by mutations shown in green. (B) Posterior probabilities of original and mutated sites are shown for the four alternative ancestral alleles generated and assayed. (C) Bar charts show relative enzyme activities of additional site-directed mutants made to recapitulate evolutionary changes from CisAncXMT2 to either CisXMT1 or CisXMT2. Mutated amino acid positions are highlighted in magenta.

Fig. S9.

Aligned protein sequences show that very few sites differ between ancestral and modern-day enzymes and that mutations at very few sites account for functional change. (A) Amino acid alignment of functionally characterized modern-day XMT and CS sequences. Also shown are resurrected ancestral XMT protein sequences. Amino acids that were experimentally replaced to recapitulate evolutionary changes in the Citrus lineage are highlighted in turquoise. Alternative ancestral alleles were generated by mutations shown in green. (B) Posterior probabilities of original and mutated sites are shown for the four alternative ancestral alleles generated and assayed. (C) Bar charts show relative enzyme activities of additional site-directed mutants made to recapitulate evolutionary changes from CisAncXMT2 to either CisXMT1 or CisXMT2. Mutated amino acid positions are highlighted in magenta.

Fig. S10.

HPLC analyses show that CisAncXMT2 P25S converts 1-methylxanthine to theophylline instead of paraxanthine, like its ancestor, CisAncXMT2. Enzyme assays were conducted using 200 µM 1-methylxanthine, and products were measured by absorbance at 272 nm. Unlabeled peaks in Middle and Lower are unidentified molecules found in both the enzyme assay and negative control to which no xanthine alkaloids were added.