Table 2 Biochemical properties of the three enzymes Enzyme Temperature range(°C) Optimal temperature Thermal Stability① pH range Optimal pH Acid stability② Alkali Stability③ Specific activity Epigenetics Compound Library PdcDE 20-70 40°C 35% 3.0-10.0 6.0 20% 60% ND④ PdcG 20-70 50°C 65% 5.0-10.0 8.0 18% 75% 0.44 U/mg PdcF 20-70 40°C 10% 5.0-9.0 7.0 20% 58% 446.97 U/mg ①Relative activity of purified protein when it was treated in 60°C for 20 min; ②Relative activity of purified protein when it was treated in pH 3.0 for 30 min; ③Relative activity of purified protein when it was treated in pH 10.0 for 30 min; ④Not detectedEach value

represents the mean of at least three independent replicates. Autophagy Compound Library Table 3 Effect of various metal ions and chemical agent on the activity of the three enzymes Metal ion or chemical agent (5 mM)   Relative activity (%)     PdcDE PdcG PdcF No addition 100 100 100 K + (KCl) 113.04 ± 10.80 95.79 ± 16.49 129.00 ± 27.32 Na + (NaCl) 113.42 ± 2.27 88.22 ± 17.76 123.91 ± 25.82 Ba 2+ (BaCl 2 ) 99.19 ± 6.29 123.34 ± 7.79 129.02 ± 6.46 Mg 2+ (MgCl 2 ) 95.41 ± 5.96 138.06 ± 8.46 129.79 ± 18.11 Zn 2+ (ZnCl 2 ) 87.44 ± 8.68 145.95 ± 5.13 21.44 ± 3.71 Cu 2+ (CuCl 2 ) 22.46 ± 6.83 110.18 ± 11.17 59.23 ± 12.57 Ni 2+ (NiCl 2 ) 111.05 ± 2.61 183.93 ± 30.68 35.25 ± 16.67 Co 2+ (CoCl 2 ) 104.15 ± 6.79 147.08 ± 17.51 79.14 ± 13.21 Mn 2+ (MnCl 2 ) 77.45 ± 2.93

186.12 ± 9.99 136.59 ± 3.65 Cd 2 + (CdSO 4 ) 63.24 ± 3.61 58.93 ± 3.88 39.52 ± 7.01 Fe 2+ (FeCl 2 ) 82.13 ± 13.46 39.47 ± 9.49 118.90 ± 21.53 Fe 3+ (FeCl 3 ) 78.33 ±

10.74 187.37 ± 15.37 134.89 ± 28.19 EDTA 62.44 ± 3.90 83.17 ± 8.32 112.93 ± 40.43 SDS 97.47 ± 1.65 81.58 ± 24.05 136.59 ± 3.66 Each value represents the mean of at least three independent replicates. Enzymatic www.selleck.co.jp/products/cobimetinib-gdc-0973-rg7420.html assays of 4-HS dehydrogenase activity The catalysis of 4-HS to MA by 4-HS dehydrogenase (His6-PdcG) was determined by monitoring the spectral changes at 320 nm. His6-PdcG thus catalyzed the oxidation of 4-HS to MA, confirming that PdcG was the enzyme downstream of PdcDE in the PNP degradation pathway in strain 1-7. Figure 7 Enzyme activity assay of PdcG. (a) Absorbance from 270 nm to 320 nm in the absence of His6-PdcG; (b) Spectral changes during oxidation of 4-HS by His6-PdcDE. The spectra were recorded a total of five times over a five minute period (marked 1-5). The arrow indicates the direction of spectral changes. (c) Spectral changes at 320 nm during metabolism of HQ by purified His6-PdcDE and oxidation of 4-HS by purified His6-PdcG. The arrow indicates when NAD+ was added. The specific activity of PdcG was calculated to be 0.44 Umg-1 (Table 2).