Table 1. Kinetic parameters of P. trichocarpa PtAAS1, PtAAS2, PtAADC1, PtAADC2, and PtAADC3.
Enzymes were heterologously expressed in E. coli, purified, and incubated with Phe, Tyr, or Trp. Kinetic parameters are shown as means ± se (n = 3). Volatile phenylacetaldehyde was analyzed via TDU-GC-MS; all other compounds were measured using LC-MS/MS. PHA, phenylacetaldehyde; PEA, 2-phenylethylamine; 4-OH-PHA, 4-hydroxy phenylacetaldehyde; IAAld, indole-3-acetaldehyde; TyrA, tyramine; TrpA, tryptamine.
| Enzyme | Substrate | Product | Km (mm) | Vmax (nmola µg−1 a min−1) | kcat (s−1) | kcat /Km (s−1 mm−1) |
|---|---|---|---|---|---|---|
| PtAAS1 | Phe | PHA | 0.46 ± 0.02 | — | — | — |
| Phe | PEA | — | — | — | — | |
| Tyr | 4-OH-PHA | — | — | — | — | |
| Trp | IAAld | — | — | — | — | |
| PtAAS2 | Phe | PHA | 0.69 ± 0.39 | — | — | — |
| Phe | PEA | — | — | — | — | |
| Tyr | 4-OH-PHAa | detected | — | — | — | |
| Trp | IAAlda | detected | — | — | — | |
| PtAADC1 | Phe | PHA | — | — | — | — |
| Phe | PEA | 0.17 ± 0.03 | 430 ± 12 | 0.39 | 2.3 | |
| Tyr | TyrA | 2.0 ± 0.2 | 85.4 ± 3.9 | 0.08 | 0.04 | |
| Trp | TrpA | 3.0 ± 0.4 | 7.7 ± 0.5 | 0.007 | 0.002 | |
| PtAADC2 | Phe | PHA | — | — | — | — |
| Phe | PEA | 0.19 ± 0.01 | 0.89 ± 0.01 | 0.81 | 4.2 | |
| Tyr | TyrA | 1.2 ± 0.2 | 148 ± 7 | 0.14 | 0.11 | |
| Trp | TrpA | — | — | — | — | |
| PtAADC3 | Phe | PHA | — | — | — | — |
| Phe | PEA | 2.3 ± 0.2 | 6.7 ± 0.2 | 0.14 | 0.003 | |
| Tyr | TyrA | 0.043 ± 0.004 | 3.61 ± 0.06 | 0.003 | 0.08 | |
| Trp | TrpA | — | — | — | — |
a
Compounds were chemically converted into the corresponding aldoximes before LC-MS/MS analysis (see “Materials and Methods”).