Supplementary Information

Genetic incorporation of D-Lysine into diketoreductase in Escherichia coli cells

Zhizhi Liu · XinYang · Denghuan Yi · Shuzhen Wang · Yijun Chen

Z. Liu · X. Yang · D. Yi · S. Wang · Y. Chen

Laboratory of Chemical Biology and State Key Laboratory of Natural Medicines,

China Pharmaceutical University, 24 Tongjia Street, Nanjing, Jiangsu Province,

210009, People’s Republic of China.

Tel: 86-25-83271045, Fax: 86-25-83271249

E-mail: ;

1. Supplementary figures

a)

b)

Supplementary Figure. 1 Schematic views of the interactions between PhLysRS and D-Lys and L-Lys. Lys (in red) and active site residues of PhLysRS are indicated as sticks.The carbon, nitrogen, oxygen and hydrogen atoms are shown in gray, blue, red and white respectively. Hydrogen bondings between Lys and active site residues are displayed with green dashed lines. a) Interactions between D-Lys and active site residues of PhLysRS. The carboxyl group of D-Lys forms two hydrogen bondings with the guanido group of Arg225, and α-amino group of D-Lys produces two hydrogen bondings with the carboxyl groups of Asp66 and Gly27 respectively. The ε-amino group of D-Lys forms two hydrogen bondings with the carboxyl group of Glu41 and the amino group of Gly237. The binding energy between PhLysRS and D-Lys was calculated to be -13.629 kJ mol-1. b) Interactions between L-Lys and active site residues of PhLysRS. The α-amino group of L-Lys forms a hydrogen bonding with the carboxyl group of Asp66, and the ε-amino group of L-Lys produces two hydrogen bondings with the carboxyl group of Glu41. The binding energy between PhLysRS and D-Lys was calculated to be -14.027 kJ mol-1.

a)

b)

Supplementary Fig. 2 Constructed plasmid map. (a) pAC-ph△-AK3. Plasmid pAC-ph△-AK3 was constructed to express the Ph tRNA/LysRS pair. “cat” (in blue) represents the gene of chloramphenicol acetyltransferase. “TAG” represents the amber codon at the 112th site of chloramphenicol acetyltransferase. Ph Lysyl-tRNA synthetase (in yellow) represents the gene of Ph Lysyl-tRNA synthetase. GlnRS promoter and terminator (in green) were encoded to control the expression of Ph Lysyl-tRNA synthetase gene. Tandem tRNA gene cassettes were consisted of three copies of suppressor tRNA genes (shown in blue) with lpp promoter and rrnC terminator (shown in green). (b) pETDuet-T2DK-dkr. Plasmid pETDuet-T2DK-dkr was constructed to encode an N-terminal His tagged mutant DKR gene with an amber codon at 2nd site (in blue) and a Ph Lysyl-tRNA synthetase gene (in blue). The two genes were expressed under the control of T7 promoter and terminator (in brown).

Supplementary Fig. 3 SDS-PAGE for protein expression of T2DK-DKR mutant and wild type DKR in recombinant E. coli cells after induction. Lane 1, Soluble fraction of T2DK-DKR mutant; Lane 2, Soluble fraction of wild type DKR; Lane 3, Whole cells of T2DK-DKR mutant; Lane 4, Whole cells of wild type DKR; Lane 5, Markers for protein molecular weight. Arrow indicates the target protein.

Supplementary Fig. 4 SDS-PAGE for the purification of T2DK-DKR mutant. Lane 1, Purified T2DK-DKR protein after DEAE- Sepharose column; Lane 2, T2DK-DKR protein after Ni-NTA column; Lane 3, Crude extract of T2DK-DKR mutant; Lane 4, Markers for protein molecular weight.

a)

b)

Supplementary Fig. 5 Mass spectra of trypsin digested peptide fragments. (a) WT-DKR; (b) T2DK-DKR.

2. Supplementary tables

Supplementary Table 1 Primers used in this experiment

Primer 5' to 3' Restriction site
PHKRSU: 5'-CGGGGTACCATGGTTCATTGGGCCGATTATATTG-3' KpnI
PHKRSL: 5'-ATACTGCAGTTATGAAAGCTCGTAAGTCCTCCTT-3' PstI
AKxbaU: 5'-ATATCTAGAGGTAAGATCTCGAACGATCAAAAATAAGTGC-3' XbaI
AKBamL:5'-ATAGGATCCGGTTGCATGCAAAAAAAATCCTTAGCTTTCG-3' BamHI
AKBamU:5'-ATAGGATCCGGTAAGATCTCGAACGATCAAAAATAAGTGC-3' BamHI
AKEcoRL:5'-CCGGAATTCGGTTGCATGCAAAAAAAATCCTTAGCTTTCG-3' EcoRI
AKEcoRU:5'-CCGGAATTCGGTAAGATCTCGAACGATCAAAAATAAGTGC-3' EcoRI
AKHindL:5'-CCCAAGCTTGGTTGCATGCAAAAAAAATCCTTAGCTTTCG-3' HindIII
DKRBamHI: 5'-CGCGGATCCATGTAGGGCATCACGAATGTCACCGTTCT-3' BamHI
DKRHindIII5'-CCCAAGCTTTCAGTACCGGTAGAAGCCCTCGCCGCTCGC-3' HindIII

Underlines represent the sequences of restriction sites.

Supplementary Table 2 Enzyme activity of T2DK-DKR mutant and WT-DKR

Enzyme Specific activity (µmol/mg·min)
T2DK-DKR 4.85±0.14 (n=3)
WT-DKR 6.78±0.31 (n=3)

Supplementary Table 3 Mass analysis of trypsin digested peptide fragments from WT-DKR and T2DK-DKR mutanta

Peptide fragment Theoretical mass (Da) Observed mass (Da)
LGLATGEGFYRY 1347.5 1346.8 (T2DK-DKR)
1346.6 (WT-DKR)
HHSQDPMTGITNVTV 1637.8 NDb (T2DK-DKR)
1637.7 (WT-DKR)
TIFATNSSTLLPSDLVGYTGR 2214.5 2214.1 (T2DK-DKR)
2214.1 (WT-DKR)
GSSHHHHHHSQDPMDKGITN 2614.9 2616.3(T2DK-DKR)
VTVLG NDb (WT-DKR)
IFATNSSTLLPSDLVGYTGRG 2745.1 2744.3(T2DK-DKR)
DKFLAGRGDKFLA 2744.4(WT-DKR)

aMALDI-TOF mass spectrometry was performed with a positive ionization mode.

bNot detected.

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