Metabolic pathways and phylogeny analysis of SerDC and AlaDC in plants.

(A) The decarboxylation of serine and alanine in plants. SerDC, serine decarboxylase; AlaDC, alanine decarboxylase. (B) Phylogenetic analysis of the SerDC homologous proteins in Algal, Pteridohyta, Bryophyta, Dicots and Monocots. The phylogenetic tree was performed using the neighbor-joining method in MAGA 7.0 software and bootstrap values based on 1000 replicates were displayed at branch points. These proteins were represented by their GenBank accession number. (C) Multiple alignment of the amino acid sequences of AlaDC and SerDCs. Primary (100%), secondary (80%), and tertiary (60%) conserved percent of similar amino acid residues were shaded in deep blue, light blue and cheer red, respectively. The multiple alignment was constructed by MAGA 7.0 software by ClustalW method, and GeneDOC software was used to visualize multiple sequence alignments. “*” indicated amino acid residues mutated only in CsAlaDC.

Purification and characterization of CsAlaDC, AtSerDC, and CsSerDC.

(A) Identification of CsAlaDC, AtSerDC, and CsSerDC using SDS-PAGE. (B) Detection of enzyme activities of CsAlaDC, AtSerDC, and CsSerDC by UPLC. (C) Reaction rates of substrates with different concentrations catalyzed by CsAlaDC, AtSerDC, and CsSerDC.

Kinetic parameters of CsAlaDC, AtSerDC and CsSerDC

Crystal structures of CsAlaDC and AtSerDC.

(A) Dimer structure of CsAlaDC. The color display of the N-terminal domain, large domain, and C-terminal domains of chain A is shown in light pink, khaki and sky blue, respectively. Chain B is shown in spring green. The PLP molecule is shown as a sphere model. The zinc finger structure at the C-terminus of CsAlaDC is indicated by the red box. The gray spheres represent zinc ions, while the red dotted line depicts the coordination bonds formed by zinc ions with cysteine and histidine. (B) The 2Fo-Fc electron density maps of K309-PLP-EA (contoured at 1σ level). The PLP is shown in violet, the K309 is shown in spring green, and the EA is shown in lightblue. (C) Active center of the CsAlaDC-EA complex, with hydrogen bonds denoted by black dotted lines. “*” denotes the amino acids on adjacent subunits. (D) Dimer structure of AtSerDC. The color display of the N-terminal domain, large domain, and C-terminal domains of chain A is shown in light pink, khaki and sky blue, respectively. Chain B is shown in cyan. The PLP molecule is shown as a sphere model. The zinc finger structure at the C-terminus of AtSerDC is indicated by the red box. The gray spheres represent zinc ions, while the red dotted line depicts the coordination bonds formed by zinc ions with cysteine and histidine. (E) Active center of the AtSerDC, with hydrogen bonds denoted by black dotted lines. “*” denotes the amino acids on adjacent subunits. (F) The monomer of CsAlaDC and AtSerDC are superimposed. CsAlaDC is depicted in spring green, while AtSerDC is shown in cyan. The conserved amino acid catalytic ring is indicated by the red box (G) Amino acid residues of the active center in CsAlaDC apo and CsAlaDC-EA complex are superimposed. CsAlaDC apo is shown in floral white, while CsAlaDC-EA complex is shown in spring green. (H) The relative activity of wild-type CsAlaDC and its Y336F mutant (left), as well as wild-type AtSerDC and its Y341F mutant (right) is shown. (I) The effects of DTT on the activity of CsAlaDC and AtSerDC are depicted. Three independent experiments were conducted.

Key amino acid residues for substrate recognition.

(A) Superposition of substrate binding pocket amino acid residues in CsAlaDC and AtSerDC. The amino acid residues of CsAlaDC are shown in spring green, the amino acid residues of AtSerDC are shown in cyan, with the substrate specificity-related amino acid residue highlighted in a red ellipse. (B) Active-site-lining residues from Embryophyta AlaDC were identified. The height of the residue label displays the relative amino acid frequency. (C) Histogram showing the distribution of the number of key motifs. (D) Histogram showing the number of key motifs in different plant orders. (E) Relative enzyme activities of wild-type CsAlaDC and mutant protein CsAlaDCF106Y against Ala substrate (columns 1 and 2), and enzyme activities of wild-type AtSerDC and various AtSerDC mutant proteins against Ala substrate (columns 3-9) are presented. The percentage graph shows the relative activity of each protein compared to wild-type CsAlaDC activity (taken as a 100% benchmark). (F) Relative enzyme activities of wild-type AtSerDC and AtSerDC mutant proteins (columns 1-7) against Ser substrates, and enzyme activities of wild-type CsAlaDC and mutant protein CsAlaDCF106Y against Ser substrates(columns 8, 9) were measured. The percentage graph shows the relative activity of each protein compared to the wild-type AtSerDC (taken as a 100% benchmark). Three independent experiments were conducted. (G) The EA contents of AtSerDC and its mutant AtSerDCY111F in N. benthamiana. (H) The EA contents of CsAlaDC and its mutant CsAlaDCF106Y in N. benthamiana. The significance of the difference (P<0.05) was labeled with different letters according to Duncan’s multiple range test.

Mutations enhance CsAlaDC enzyme activity and theanine synthesis in vitro.

(A) Relative enzyme activities of CsAlaDC mutant proteins against Ala substrate. (B) Relative enzyme activities of CsAlaDCL110F, CsAlaDCP114A, and CsAlaDCL110F/P114A against Ala substrate. (C) Histogram showing the relative content of theanine resulting from different combinations of alanine decarboxylase and theanine synthetase. Three independent experiments were conducted.

Evolutionary analysis of CsAlaDC in Embryophyta.

(A) The presented diagram depicts an evolutionary tree of CsAlaDC, which is devoid of a root and solely portrays the topological structure of the tree without including distance information. The color of the inner ring corresponds to various orders, while the outer ring’s leaf nodes are colored based on the motif types that the sequence exhibits. (B) Diversity of serine decarboxylase-like proteins in Embryophyta (196 species). Colored scatter spots on the right side of leaf nodes correspond to the respective motifs shown in Figure A.

The relative mRNA levels of AtSerDC and its mutant Y111F (A), CsAlaDC and its mutant F106Y (B) in N. benthamiana leaves were measured by two primers. WT, wild type of N. benthamiana; EV, empty vector control; NbGAPDH was used as an internal control. Data represent mean ± SD (n=3). The significance of the difference (P<0.05) was labeled with different letters according to Duncan’s multiple range test.

Multiple sequence alignment of CsAlaDC, AtSerDC, MetDC, HisDC1, TrpDC, HisDC2, TyrDC, and GluDC were generated using MUSCLE and visualized with ESPript 3.x. Conserved amino acid residues in all eight proteins are highlighted with red backgrounds. The magenta box marks key amino acid residues involved in substrate recognition for CsAlaDC and AtSerDC. The Lys residue covalently bound to the PLP cofactor is denoted by a red star, while the green triangle indicates the Tyr residue associated with enzymatic activity. Amino acid residues involved in the CsAlaDC substrate binding pocket are marked with blue circles.

Structures of HisDC2, MetDC, TrpDC, AspDC, HisDC1 and GluDC.

(A) The Overall Structures of HisDC2, MetDC, TryDC, AspDC, HisDC1 and GluDC. Chain A is shown in khaki, chain B is shown in cyan. (B) Amino acid residues in the substrate binding pocket of HisDC2, MetDC, TryDC, AspDC, HisDC1 and GluDC. The amino acid residues in chain A are shown in khaki, and the amino acid residues in chain B are shown in cyan.

Data collection and refinement statistics

Primers used for gene cloning.

Primers used for real-time PCR.