Supplementary Materials

1

Mutation of active site serine residue with cysteine displays change in acyl-acceptor preference

of β-peptidyl aminopeptidase from Pseudomonas aeruginosa PAO1

Jiro Arima*, Ayumi Tanaka, Masazumi Morimoto, and Nobuhiro Mori

Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori

University, Tottori 680-8553, Japan

*Corresponding author: Jiro Arima

Tel.: +81-857-31-5363, Fax: +81-857-31-5363

E-mail: arima@muses.tottori-u.ac.jp

Contents

Fig. S1 Native-PAGE of purified wild-type BapF and S237C mutant enzyme

Fig. S2 Effect of βhGly-pNA concentration on p-nitroaniline releasing activity of BapF at pH 9.0 (a)

and pH 6.0 (b)

Fig. S3 pH Stability (a) and thermal stability (b) of BapF

Fig. S4 UPLC-ESI-TOF MS analysis of product synthesized with 2 mM βhGly-pNA as a substrate

Fig. S5 SDS-PAGE of crude wild-type BapF and S237T mutant enzyme

Fig. S6 Characterization of S237C mutant enzyme

Table S1 Purification of recombinant BapF

Table S2 Tested chemicals and peptides synthesized using BapF and S237C mutant enzyme

Supplementary Materials

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Fig. S1 Native-PAGE of purified wild-type BapF and S237C mutant enzyme. Samples (3 μg of enzymes) were loaded on a 7.5% gel.

Fig. S2 Effect of βhGly-pNA concentration on p-nitroaniline release activity of BapF at pH 9.0 (a) and pH 6.0 (b). The reaction was done at 25°C in 0.1 M borate-NaOH (pH 9.0) or 0.2 M Tris-malate (pH 6.0). Values are averages of four independent experiments ± standard deviation.

Supplementary Materials

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Fig. S3 pH Stability (a) and thermal stability (b) of BapF. For the pH stability test, enzyme (0.1 mg·ml-1) was treated in 0.1 M sodium acetate (pH 3.8–5.4), Tris-malate (pH 5.4–8.4), Tris-HCl (pH 7.4–9.0), and borate-NaOH (pH 9.0–9.9) for 24 h at 4°C. Residual activity was measured using β-Ala-pNA at pH 6.0. For the thermal stability test, the enzyme (0.1 mg·ml-1 protein) was incubated for 30 min at temperatures of 25–77°C. Residual activity was measured using βhGly-pNA at pH 6.0. Each value is the average of four independent experiments ± the standard deviation.

Fig. S4 UPLC-ESI-TOF MS analysis of product synthesized using wild type BapF with 2 mM βhGly-pNA as a substrate. The reaction was performed at pH 6.0 for 3 min. Lower panel, extracted ion chromatogram of 281.13. Upper panel, MS chart of peak top in the extracted ion chromatogram.

Supplementary Materials

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Fig. S5 SDS-PAGE of crude wild-type BapF and S237T mutant enzyme. Samples (10 μg of proteins) were loaded on a 15% gel.

Fig. S6 Characterization of S237C mutant enzyme. (a) Effect of βhGly-pNA concentration on p-nitroaniline release activity at pH 9.0. The reaction was conducted at 25°C in 0.2 M Tris-HCl (pH 9.0). (b) pH Stability. For the test, enzyme (0.1 mg·ml-1) was treated in 0.1 M sodium acetate (pH 3.8–5.4), Tris-malate (pH 5.4–8.4), Tris-HCl (pH 7.4–9.0), and borate-NaOH (pH 9.0–9.9) for 24 h at 4°C. Residual activity was measured using βhGly-pNA at pH 9.0. (c) Thermal stability. For the test, the enzyme (0.1 mg·ml-1 protein) was incubated for 30 min at temperatures of 25–77°C. Residual activity was measured using βhGly-pNA at pH 9.0. In all panels, each value is the average of four independent experiments ± standard deviation.

Supplementary Materials

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Table S1 Purification of recombinant BapF

Total protein Total activity Specific activity Purification Yield

(mg) (μ mol min-1) (μ mol min-1 mg-1) (fold) (%)

Crude extract 156 1360 8.72 1 100

15– 40% Ammonium sulfate fractionation

59.9 807 13.5 1.55 59.3

Butyl-Toyopearl 27.7 487 17.6 2.02 35.8

Vivapure Q spin 10.4 257 24.7 2.83 18.9

Supplementary Materials

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Table S2 Tested chemicals and peptides synthesized using BapF and S237C mutant enzyme. As an acyl donor for aminolysis reaction, 2 mM βhGly-pNA was added to the reaction mixture containing 50 mM acyl acceptor candidates (amino acids, aminoacyl derivatives, and other amine chemicals). The reaction was conducted for 2 h. Data of ESI-MS are presented later.

Acyl acceptor Product of aminolysis reaction Wild type BapF S237C mutant enzyme

α-Amino acids L-Leu bhGly-L-Leu n.d.a) L-Met bhGly-L-Met bhGly-L-Met L-Phe bhGly-L-Phe bhGly-L-Phe L-Trp bhGly-L-Trp n.d. L-Asp(β-OMe) n.d. bhGly-L-Asp(β-OMe) L-Glu(γ-OMe) n.d. bhGly-L-Glu(γ-OMe) L-α-Aminoacyl derivatives and peptides Gly-NH2 n.d. bhGly-Gly-NH2 Gly-OMe bhGly-Gly-OMe bhGly-Gly-OMe Gly-OBzl bhGly-Gly-OBzl bhGly-Gly-OBzl Gly-L-Phe bhGly-Gly-L-Phe bhGly-Gly-L-Phe L-Val-OMe bhGly-L-Val-OMe bhGly-L-Val-OMe L-Val-OBzl bhGly-L-Val-OBzl bhGly-L-Val-OBzl L-Leu-NH2 bhGly-L-Leu-NH2 n.d. L-Leu-OMe bhGly-L-Leu-OMe bhGly-L-Leu-OMe L-Leu-OEt bhGly-L-Leu-OEt bhGly-L-Leu-OEt L-Leu-OBzl bhGly-L-Leu-OBzl bhGly-L-Leu-OBzl L-Ile-OMe bhGly-L-Ile-OMe bhGly-L-Ile-OMe L-Ile-OBzl bhGly-L-Ile-OBzl bhGly-L-Ile-OBzl L-Met-OMe bhGly-L-Met-OMe bhGly-L-Met-OMe L-Phe-NH2 bhGly-L-Phe-NH2 bhGly-L-Phe-NH2 L-Phe-OMe bhGly-L-Phe-OMe bhGly-L-Phe-OMe L-Phe-OEt bhGly-L-Phe-OEt bhGly-L-Phe-OEt L-Phe-OBzl bhGly-L-Phe-OBzl bhGly-L-Phe-OBzl L-Phe-L-Ala bhGly-L-Phe-L-Ala n.d. L-Phe-L-Phe bhGly-L-Phe-L-Phe bhGly-L-Phe-L-Phe L-Trp-OMe bhGly-L-Trp-OMe bhGly-L-Trp-OMe L-Trp-OBzl bhGly-L-Trp-OBzl bhGly-L-Trp-OBzl L-Tyr-NH2 bhGly-L-Tyr-NH2 bhGly-L-Tyr-NH2 L-Tyr-OMe bhGly-L-Tyr-OMe bhGly-L-Tyr-OMe L-Tyr-OBzl bhGly-L-Tyr-OBzl bhGly-L-Tyr-OBzl L-Phg-OEtb) bhGly-L-Phg-OEt bhGly-L-Phg-OEt L-Ser-OMe bhGly-L-Ser-OMe n.d. L-Ser-OBzl bhGly-L-Ser-OBzl n.d. L-Ser(OBzl)-OMe bhGly-L-Ser(OBzl)-OMe bhGly-L-Ser(OBzl)-OMe L-Thr-OBzl bhGly-L-Thr-OBzl n.d. (L-Cys-OMe)2 bhGly-(L-Cys-OMe)2 bhGly-(L-Cys-OMe)2

Supplementary Materials

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L-Asn-OMe bhGly-L-Asn-OMe bhGly-L-Asn-OMe L-His-OMe bhGly-L-His-OMe bhGly-L-His-OMe L-Lys-OMe bhGly-L-Lys-OMe bhGly-L-Lys-OMe L-Arg-OMe bhGly-L-Arg-OMe n.d. L-Asp(β-OMe)-OMe bhGly-L-Asp(β-OMe)-OMe bhGly-L-Asp(β-OMe)-OMe L-Asp(β-OBzl)-OBzl bhGly-L-Asp(β-OBzl)-OBzl bhGly-L-Asp(β-OBzl)-OBzl L-Glu-OMe bhGly-L-Glu-OMe bhGly-L-Glu-OMe L-Glu-OBzl bhGly-L-Glu-OBzl n.d. L-Glu(γ-OMe)-OMe bhGly-L-Glu(γ-OMe)-OMe n.d. D-, and DL-α-Aminoacyl derivatives and peptides D-Val-OBzl bhGly-D-Val-OBzl n.d. D-Leu-OMe bhGly-D-Leu-OMe bhGly-D-Leu-OMe D-Leu-OBzl bhGly-D-Leu-OBzl bhGly-D-Leu-OBzl D-Ile-OBzl bhGly-L-Ile-OBzl n.d. D-Phe-OMe bhGly-D-Phe-OMe bhGly-D-Phe-OMe D-Trp-OMe bhGly-D-Trp-OMe bhGly-D-Trp-OMe D-Tyr-OMe n.d. bhGly-D-Tyr-OMe D-Phg-OEtb) bhGly-D-Phg-OEt bhGly-D-Phg-OEt D-His-OMe n.d. bhGly-D-His-OMe D-Lys-OMe bhGly-D-Lys-OMe bhGly-D-Lys-OMe D-Glu(γ-OMe)-OMe bhGly-D-Glu(γ-OMe)-OMe n.d. Other amine chemicals GABA-OBzl c) bhGly-GABA-OBzl bhGly-GABA-OBzl cis 4-ACHC-OMed) bhGly-cis 4-ACHC-OMe bhGly-cis 4-ACHC-OMe trans 4-ACHC-OMed) bhGly-trans 4-ACHC-OMe bhGly-trans 4-ACHC-OMe Cysteamine n.d. bhGly-cysteamine Cystamine bhGly-cystamine n.d. Dopamine bhGly-dopamine n.d. 1-Amino-2-butanol bhGly-1-amino-2-butanol bhGly-1-amino-2-butanol 6-Amino-1-hexanol bhGly-6-amino-1-hexanol bhGly-6-amino-1-hexanol Butylamine bhGly-butylamine bhGly-butylamine Hexylamine bhGly-hexylamine bhGly-hexylamine 1-Amino-decane bhGly-1-amino-decane bhGly-1-amino-decane Chemicals that showed no product of aminolysis reaction α-Amino acids Gly, L-Val, D-Val, L-tertLeu, D-Leu, DL-Nle, L-Ile, D-Ile, D-Phe, D-Trp, L-Tyr, D-Tyr, L-DOPA,

L-Pro, D-Pro, L-Ser, D-Ser, L-Thr, L-Cys, D-Cys, (L-Cys)2, L-Asn, D-Asn, L-Gln, L-His, D-His, L-Lys, D-Lys, L-Arg, D-Arg, L-homoArg, L-Asp, D-Asp, L-Glu, D-Glu, DL-2-aminobutylate

L-α-Aminoacyl derivatives and peptides Gly-Gly, Gly-L-Ala, Gly-L-Pro, L-tertLeu-OMe, L-Pro-OMe, L-Pro-OBzl, L-Thr-OMe, L-Cys-OMe,

L-Asp-OMe, L-Asp-L-Phe, L-Glu-OMe D-, and DL-α-aminoacyl derivatives and peptides D-Phe-OBzl, D-Trp-OBzl, D-Tyr-OBzl, D-Pro-OMe, D-Pro-OBzl, D-Ser-OMe, D-Ser-OBzl,

D-Arg-OMe, D-Glu-OBzl, 2-piperidine carboxylate-OEt

Supplementary Materials

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Other amine chemicals Taurine, histamine, 8-aminoquinoline, p-Amino diphenylamine, naphtylamine, D-glucosamine,

D-galactosamine, 2-aminoethanol, (R)-1-amino-2-propanol, (S)-1-amino-2-propanol, (R)-2-amino-1-propanol, (S)-2-amino-1-propanol, (R)-2-amino-1-butanol, (S)-2-amino-1-butanol, 4-amino-1-butanol, 10-amino-1-decanol, methylamine, ethylamine, propylamine, isopropylamine, 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane, 1,9-diaminononane, 1,10-diaminodecane, 1,12-diaminododecane, dimethylamine, diethylamine, allylamine, 3-amylamine, tert-amylamine

a) n.d., not detected (ion intensity [counts per second] less than 300)

b) Phg, phenylglycine

c) GABA, γ-amino butyric acid

d) 4-ACHC, 4-aminocyclohexane carboxylate

Mass spectra data of synthesized peptides and their analogues shown in Table 2 are as follows.

βhGly-L-Leu, calcd. for C9H18N2O3 [M+H]+: 203.1397, found: 203.1396. βhGly-L-Met, calcd. for

C8H16N2O3S1 [M+H]+: 221.0961, found: 221.0942 and 221.0952. βhGly-L-Phe, calcd. for C12H16N2O3

[M+H]+: 237.1240, found: 237.1239 and 237.1241. βhGly-L-Trp, calcd. for C14H17N3O3 [M+H]+:

276.1349, found: 276.1293. βhGly-L-Asp(β-OMe), calcd. for C8H14N2O5 [M+H]+: 219.0982, found:

219.0968. βhGly-L-Glu(γ-OMe), calcd. for C9H16N2O5 [M+H]+: 233.1138, found: 233.1129.

βhGly-Gly-NH2, calcd. for C5H11N3O2 [M+H]+: 146.0931, found: 146.1172. βhGly-Gly-OMe, calcd.

for C6H12N2O3 [M+H]+: 161.0927, found: 161.0897 and 161.0934. βhGly-Gly-OBzl, calcd. for

C12H16N2O3 [M+H]+: 237.1240, found: 237.1221 and 237.1215. βhGly-Gly-L-Phe, calcd. for

C14H19N3O4 [M+H]+: 294.1455, found: 294.1413 and 294.1420. βhGly-L-Val-OMe, calcd. for

C9H18N2O3 [M+H]+: 203.1397, found: 203.1391 and 203.1394. βhGly-L-Val-OBzl, calcd. for

C15H22N2O3 [M+H]+: 279.1710, found: 279.1709 and 279.1671. βhGly-L-Leu-NH2, calcd. for

C9H19N3O2 [M+H]+: 202.1557, found: 202.1545 and 202.1552. βhGly-L-Leu-OMe, calcd. for

C10H20N2O3 [M+H]+: 217.1553, found: 217.1542 and 217.1539. βhGly-L-Leu-OEt, calcd. for

C11H22N2O3 [M+H]+: 231.1710, found: 231.1708 and 231.1698. βhGly-L-Leu-OBzl, calcd. for

C16H24N2O3 [M+H]+: 293.1867, found: 293.1855 and 293.1866. βhGly-L-Ile-OMe, calcd. for

C10H20N2O3 [M+H]+: 217.1553, found: 217.1549 and 217.1536. βhGly-L-Ile-OBzl, calcd. for

Supplementary Materials

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C16H24N2O3 [M+H]+: 293.1867, found: 293.1868 and 293.1827. βhGly-L-Met-OMe, calcd. for

C9H18N2O3S1 [M+H]+: 235.1118, found: 235.1122 and 235.1114. βhGly-L-Phe-NH2, calcd. for

C12H17N3O2 [M+H]+: 236.1400, found: 236.1389 and 236.1380. βhGly-L-Phe-OMe, calcd. for

C13H18N2O3 [M+H]+: 251.1397, found: 251.1389 and 251.1377. βhGly-L-Phe-OEt, calcd. for

C14H20N2O3 [M+H]+: 265.1553, found: 265.1558 and 265.1534. βhGly-L-Phe-OBzl, calcd. for

C19H22N2O3 [M+H]+: 327.1710, found: 327.1719. βhGly-L-Phe-L-Ala, calcd. for C15H21N3O4

[M+H]+: 308.1612, found: 308.1587. βhGly-L-Phe-L-Phe, calcd. for C21H25N3O4 [M+H]+: 384.1925,

found: 384.1879 and 384.1921. βhGly-L-Trp-OMe, calcd. for C15H19N3O3 [M+H]+: 290.1506, found:

290.1505 and 290.1479. βhGly-L-Trp-OBzl, calcd. for C21H23N3O3 [M+H]+: 366.1819, found:

366.1839 and 366.1819. βhGly-L-Tyr-NH2, calcd. for C12H17N3O3 [M+H]+: 252.1349, found:

252.1342 and 252.1328. βhGly-L-Tyr-OMe, calcd. for C13H18N2O4 [M+H]+: 267.1346, found:

267.1342 and 267.1334. βhGly-L-Tyr-OBzl, calcd. for C19H22N2O4 [M+H]+: 343.1659, found:

343.1692 and 343.1651. βhGly-L-Phg-OEt, calcd. for C13H18N2O3 [M+H]+: 251.1397, found:

251.1398 and 251.1394. βhGly-L-Ser-OMe, calcd. for C7H14N2O4 [M+H]+: 191.1033, found:

191.1024. βhGly-L-Ser-OBzl, calcd. for C13H18N2O4 [M+H]+: 267.1346, found: 267.1348.

βhGly-L-Ser(OBzl)-OMe, calcd. for C14H20N2O4 [M+H]+: 281.1502, found: 281.1501 and 281.1484.

βhGly-L-Thr-OBzl, calcd. for C14H20N2O4 [M+H]+: 281.1502, found: 281.1499. βhGly-(L-Cys-OMe)2,

calcd. for C11H21N3O5S2[M+H]+: 340.1003, found: 340.0336 and 340.0968. βhGly-L-Asn-OMe, calcd.

for C8H15N3O4 [M+H]+: 218.1142, found: 218.1124 and 218.1132. βhGly-L-His-OMe, calcd. for

C10H16N4O3 [M+H]+: 241.1302, found: 241.1296 and 241.1290. βhGly-L-Lys-OMe, calcd. for

C10H21N3O3 [M+H]+: 232.1663, found: 232.1630 and 232.1683. βhGly-L-Arg-OMe, calcd. for

C10H21N5O3 [M+H]+: 260.1725, found: 260.1726. βhGly-L-Asp(β-OMe)-OMe, calcd. for C9H16N2O5

[M+H]+: 233.1138, found: 233.1139 and 233.1107. βhGly-L-Asp(β-OBzl)-OBzl, calcd. for

C21H24N2O5 [M+H]+: 385.1765, found: 385.1762 and 385.1737. βhGly-L-Glu-OMe, calcd. for

C9H16N2O5 [M+H]+: 233.1138, found: 233.1133 and 233.1140. βhGly-L-Glu-OBzl, calcd. for

Supplementary Materials

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C15H20N2O5 [M+H]+: 309.1451, found: 309.1450. βhGly-L-Glu(γ-OMe)-OMe, calcd. for C10H18N2O5

[M+H]+: 247.1295, found: 247.1284 and 247.1252. βhGly-D-Val-OBzl, calcd. for C15H22N2O3

[M+H]+: 279.1710, found: 279.1675. βhGly-D-Leu-OMe, calcd. for C10H20N2O3 [M+H]+: 217.1553,

found: 217.1531 and 217.1556. βhGly-D-Leu-OBzl, calcd. for C16H24N2O3 [M+H]+: 293.1867, found:

293.1827 and 293.1878. βhGly-D-Ile-OBzl, calcd. for C16H24N2O3 [M+H]+: 293.1867, found:

293.1831. βhGly-D-Phe-OMe, calcd. for C13H18N2O3 [M+H]+: 251.1397, found: 251.1394.

βhGly-D-Trp-OMe, calcd. for C15H19N3O3 [M+H]+: 290.1506, found: 290.1485 and 290.1501.

βhGly-D-Tyr-OMe, calcd. for C13H18N2O4 [M+H]+: 267.1346, found: 267.1340. βhGly-D-Phg-OEt,

calcd. for C13H18N2O3 [M+H]+: 251.1397, found: 251.1853 and 251.1441. βhGly-D-His-OMe, calcd.

for C10H16N4O3 [M+H]+: 241.1302, found: 241.1300. βhGly-D-Lys-OMe, calcd. for C10H21N3O3

[M+H]+: 232.1663, found: 232.1630 and 232.1661. βhGly-D-Glu(γ-OMe)-OMe, calcd. for

C10H18N2O5 [M+H]+: 247.1295, found: 247.1714. βhGly-GABA-OBzl, calcd. for C14H20N2O3

[M+H]+: 265.1553, found: 265.1528 and 265.1512. βhGly-cis 4-ACHC-OMe, calcd. for C11H20N2O3

[M+H]+: 229.1553, found: 229.1536 and 229.1543. βhGly-trans 4-ACHC-OMe, calcd. for

C11H20N2O3 [M+H]+: 229.1553, found: 229.1539 and 229.1536. βhGly-cysteamine, calcd. for

C5H12N2OS [M+H]+: 149.0750, found: 149.0744. βhGly-cystamine, calcd. for C7H17N3OS2 [M+H]+:

224.0893, found: 224.0881. βhGly-dopamine, calcd. for C11H16N2O3 [M+H]+: 225.1240, found:

225.1219. βhGly-1-amino-2-butanol, calcd. for C7H16N2O2 [M+H]+: 161.1291, found: 161.1284 and

161.1279. βhGly-6-amino-1-hexanol, calcd. for C9H20N2O2 [M+H]+: 189.1604, found: 189.1590 and

189.1615. βhGly-butylamine, calcd. for C7H16N2O [M+H]+: 145.1342, found: 145.1343 and 145.1327.

βhGly-hexylamine, calcd. for C9H20N2O [M+H]+: 173.1655, found: 173.1648 and 173.1655.

βhGly-1-aminodecane, calcd. for C13H28N2O [M+H]+: 229.2282, found: 229.2269 and 229.2270.