Bioscience Evidence 2024, Vol.14, No.2, 39-43 http://bioscipublisher.com/index.php/be 41 by extracellular PtCAL-1a/PtCAL-2a compared to the GFP extracellular control over time. Each condition was tested in three replicates. Figure 3e assesses which enantiomer of 3 is used as a substrate in the formation of 9. Since the enantiomers of 3 cannot be directly observed, the chirality is inferred by measuring the enantiomer of 4 formed through the spontaneous decarboxylation of 3. The enantiomers were analyzed as N-acetylated derivatives, with the average ratio shown above each bar graph. Each condition was tested in three replicates. Figure 3f proposes the condensation reaction of 1 and 2 catalyzed by PtCAL-3, producing (S)-3. Figure 3g shows the co-expression of PtCAL-3 with the rest of the pathway required for the production of 9. Three leaves were infiltrated under each condition. Figure 3h displays the EICs for 3 (m/z 186.1125) produced by extracellular PtCAL-3 with different substrates. Figure 3i compares the formation of 3 by extracellular PtCAL-3 with 1 and 2 as substrates over time against the GFP extracellular control. Each condition was tested in three replicates. Figure 3j evaluates the enantiospecific formation of PtCAL-3 products by analyzing the enantiomers of 4 (after N-acetylation) through chiral LC-MS. Figure 3k presents the ratio of (S)-4 to (R)-4 over time in the extracellular PtCAL-3 reaction. Figure 3 Neofunctionalized CAL enzymes in Lycopodium alkaloid biosynthesis Figure 4 highlights the crucial role of CAL enzymes in the early biosynthesis of lycopodium alkaloids. Figure 4a presents the biosynthetic proposal for the early chemical transformations of lycopodium alkaloids, noting that the transmembrane transport of intermediates is speculative. Figure 4b is based on an evolutionary tree of CAH family proteins across multiple biological kingdoms (aligned using MUSCLE, with tree construction via the neighbor-joining method), where the bootstrap values (from 100 replicates) indicate support. The key active site residues of each aligned protein are also displayed, with numbering corresponding to human carbonic anhydrase 2 (HsCA2, UniProt ID: P00918). Changes in canonical/conserved sequences are highlighted with colored boxes. Stars mark proteins that have been validated to possess typical CAH activity. A more extensive alignment/evolutionary tree can be found in Supplementary Figure 2. This reflects the evolutionary and functional diversification of CAL enzymes in the biosynthesis of lycopodium alkaloids and their evolutionary journey across different forms of life.
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