Additional file 1 of Efficient production of salvianic acid A from l-dihydroxyphenylalanine through a tri-enzyme cascade
Creators: name: Yang, Jiahui
name: Wei, Wanqing
name: Gao, Changzheng
name: Song, Wei
name: Gao, Cong
name: Chen, Xiulai
name: Liu, Jia
name: Guo, Liang
name: Liu, Liming
name: Wu, Jing
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Information Systems not elsewhere classified

description: Additional file 1: Table S1. The primers used in this study. Table S2. Data collection and refinement statistics of apo-LaPPR crystal structure. Table S3. Specific enzyme activities of L-AAD from different organisms. Table S4. Specific enzyme activities of α-keto acid reductase from different organisms. Table S5. Specific enzyme activities of PmLAAD, LaPPR and CbFDH in E. coli. Table S6. Types and sequences of different RBS. Figure S1. SDS-PAGE analysis. a The crude PmLAAD enzyme, purified LaPPR and purified CbFDH enzyme; b The purified PmLAAD enzyme; c Three enzymes PmLAAD, LaPPR and CbFDH co-expressed in E. coli YJH01. Figure S2. Detection of SAA product in vitro biosynthesis pathway. a Analysis of the standard sample and the retention time of SAA was 18.17 min; b Analysis of the reaction mixture in vitro, and SAA product was detected at 18.17 min. Figure S3. Characterization of pure SAA by LC-MS.m/z: C9H10O5 [M-H]-, 197. Figure S4. Detection of DHPPA intermediate in the reaction mixture by HPLC. a Standard sample of DHPPA, the retention time was 10.66 min; b Analysis of the remaining DHPPA intermediate in reaction mixture, and DHPPA was detected at 10.67 min. Figure S5. The asymmetric unit of LaPPR contains two essentially identical units. Figure S6. Multiple sequence alignment of LaPPR with homologous PGDH sequences from superfamily. Figure S7. a Structural alignment of LaPPRand LaPPRMu2; b Ramachandran plot of LaPPRMu2 structure. The initial structure of LaPPRMu2 was predicted from AlphaFold. Figure S8. a Root-mean-square-deviationcalculated from MD simulationsof LaPPR-NADH-DHPPA and LaPPRMu2-NADH-DHPPA complex. b B-factor calculated from MD simulations of LaPPR-NADH-DHPPA and LaPPRMu2-NADH-DHPPA complex. c Distances of hydride transfer and proton transfercalculated from 50 ns MD simulations on the LaPPR-NADH-DHPPA complex. Distances of hydride transfer and proton transfercalculated from 50 ns MD simulations on the LaPPRMu2-NADH-DHPPA complex. Mean ± standard deviation is shown for 25000 snapshots of the 50 ns MD simulations. The initial structure of LaPPRMu2 was predicted from AlphaFold. Figure S9. a The wild-type LaPPR pocket, shown in yellow, has a volume size of 989 Å3; b The mutant LaPPRMu2 pocket, shown in yellow, has a volume size of 1359 Å3. The pocket volume for each of the structures has been computed with the POCASA server. The initial structure of LaPPRMu2 was predicted from AlphaFold. Figure S10. a Effect of conversion temperature on SAA concentration. The conversion reactions were performed in a 10 mL volume of 50 mM Tris-HCl buffer, pH 7.0, containing 3 mM NAD+, with 20 g L-1 wet whole-cell biocatalysts, 50 g L-1 L-DOPA and 60 g L-1 sodium formate, in 20–40 °C for 12 h. b Effect of conversion pH on SAA concentration. The conversion reactions were performed in a 10 mL volume of 50 mM Tris-HCl buffer, pH 6.5-9.0 containing 3 mM NAD+, with 20 g L-1 wet whole-cell biocatalysts, 50 g L-1 L-DOPA and 60 g L-1 sodium formate, in 30 °C for 12 h. c Effect of concentration of NAD+ on SAA concentration. The conversion reactions were performed in a 10 mL volume of 50 mM Tris-HCl buffer, pH 7.0, containing 0.2-1.6 mM NAD+, with 20 g L-1 wet whole-cell biocatalysts, 50 g L-1 L-DOPA and 60 g L-1 sodium formate, in 30 °C for 12 h.Effect of concentration of whole-cell catalyst on SAA concentration. The conversion reactions were performed in a 10 mL volume of 50 mM Tris-HCl buffer, pH 7.0, containing 0.6 mM NAD+, with 10-40 g L-1 wet whole-cell biocatalysts, 50 g L-1 L-DOPA and 60 g L-1 sodium formate, in 30 °C for 12 h. The data represent mean ± SD, as determined from three independent experiments.