TABLE 1.
Substrate specificity of ht-PhnII in the NADH oxidation assay and GC-MS analysis of the products formed
| Substratea | Enzyme activityb
|
Product detected by GC-MSc
|
|||
|---|---|---|---|---|---|
| μmol NADH· min−1·mg−1 | % | Retention time (min) | Molecular mass (Da) | Identity | |
| Salicylate | 0.578 | 100 | 8.87 | 254 | Catechol |
| 3-Methylsalicylate | 0.44 | 76 | 9.86 | 268 | 3-Methylcatechol |
| 4-Methylsalicylate | 0.625 | 108 | 9.74 | 268 | 4-Methylcatechol |
| 5-Methylsalicylate | 0.396 | 68.5 | 9.74 | 268 | 4-Methylcatechol |
| 5-Chlorosalicylate | 0.267 | 46 | 10.79 | 288 | 4-Chlorocatechol |
| 5-Nitrosalicylate | 0.184 | 32 | 13.39 | 299 | 4-Nitrocatechol |
| Anthranilate | 0.39 | 67 | 10.21 | 253 | 2-Aminophenol |
| 3,5-Dichlorosalicylate | NDd | ND | 12.14 | 322 | 3,5-Dichlorocatechol |
| 2,4-Dihydroxybenzoate | 0.425 | 91 | 11.98 | 342 | Trihydroxybenzene |
| 2,6-Dihydroxybenzoate | 0.327 | 70 | 11.34 | 342 | Trihydroxybenzene |
| Gentisate | 0.462 | 99 | No product | ||
| 1-Hydroxy-2-naphthoate | 0.64 | 137 | No product | ||
| 2-Hydroxy-1-naphthoate | 0.75 | 160 | No product | ||
a
The initial substrate concentration was 0.5 mM except for 1-hydroxy-2-naphthoate(0.25 mM), 2-hydroxy-1-naphthoate (0.25 mM), and 5-nitrosalicylate (0.125 mM).
b
The values are means calculated from duplicate assays. The standard deviations are less than 5%.
c
Products were extracted with ethyl acetate after 2 min of reaction and analyzed as trimethylsilyl derivatives.
d
ND, not determined. The absorbance of the oxidation product at 340 nm precluded accurate estimation of the activity.