Table 7.
Summary of the mutations to the thioether bridge constituent, the seventh His and the disulfide bonds: effects on PPOs′ copper content and diphenolase/monophenolase activity. The modification of copper content and the activities′ rate are described compared to the wild type of the corresponding enzymes.
|
Multi‐mutations | |||||
|---|---|---|---|---|---|
|
Enzyme |
Mutant |
Diphenolase activity |
Monophenolase activity |
Copper % |
Ref. |
|
AoTYR[a] |
His63Asn‐His290Asn (HisA1‐HisB1) |
n.d. (l‐DOPA) |
n.d. (l‐tyrosine) |
0 |
|
|
|
His63Asn‐His294Asn (HisA1‐HisB2) |
n.d. (l‐DOPA) |
n.d. (l‐tyrosine) |
0 |
|
|
|
His63Asn‐His332Asn (HisA1‐7thHis) |
n.d. (l‐DOPA) |
n.d. (l‐tyrosine) |
0 |
|
|
|
His63Asn‐His333Asn (HisA1‐HisB3) |
n.d. (l‐DOPA) |
n.d. (l‐tyrosine) |
0 |
|
|
|
Cys82Ala‐His290Asn (Th. bridge‐HisB1) |
n.d. (l‐DOPA) |
n.d. (l‐tyrosine) |
0 |
|
|
|
His84Asn‐His290Asn (HisA2‐HisB1) |
n.d. (l‐DOPA) |
n.d. (l‐tyrosine) |
0 |
|
|
|
His93Asn‐His290Asn (HisA3‐HisB1) |
n.d. (l‐DOPA) |
n.d. (l‐tyrosine) |
0 |
|
|
JrPPO1 |
Asn240Lys‐Leu244Arg (HisB1+1‐HisB2+1) |
−3200‐fold (dopamine: 0.029 s−1) −1070‐fold (l‐DOPA: 0.104 s−1) |
n.d. (tyramine and l‐tyrosine) |
55 |
|
|
|
Asn240Thr‐Leu244Arg (HisB1+1‐HisB2+1) |
−117‐fold (dopamine: 0.789 s−1) −618‐fold (l‐DOPA: 0.180 s−1) |
n.d. (tyramine and l‐tyrosine) |
45 |
|
|
CgAUS |
Thr253Asp‐Arg257Asp (HisB1+1‐HisB2+1) |
−3.0‐fold (dopamine: 171 s−1) |
+generate (tyramine: 9.48 s−1) |
44 |
|
|
|
Thr253Asp‐Arg257Gly (HisB1+1‐HisB2+1) |
+1.2‐fold (dopamine: 662 s−1) |
+generate (tyramine: 1.91 s−1) |
33 |
|
|
|
Thr253Gly‐Arg257Leu (HisB1+1‐HisB2+1) |
∼same (dopamine: 535 s−1) |
+generate (tyramine: 0.05 s−1) |
34 |
|
|
|
Thr253Ser‐Arg257Gly (HisB1+1‐HisB2+1) |
−1.3‐fold (dopamine: 430 s−1) |
+generate (tyramine: 0.01 s−1) |
33 |
|
|
|
Thr253Gly‐Arg257Val (HisB1+1‐HisB2+1) |
−1.5‐fold (dopamine: 859 s−1) |
+generate (tyramine: 0.02 s−1) |
52 |
|
|
|
Thr253Gly‐Arg257Thr (HisB1+1‐HisB2+1) |
−3.0‐fold (dopamine: 191 s−1) |
+generate (tyramine: 0.56 s−1) |
11 |
|
|
|
Thr253Asp‐Phe273Asp (HisB1+1‐gatekeeper) |
−365‐fold (dopamine: 1.52 s−1) |
+generate (tyramine: 0.01 s−1) |
12 |
|
|
|
Thr253Asp‐Arg257Asp‐Phe273Asp (HisB1+1‐HisB2+1‐gatekeeper) |
−200‐fold (dopamine: 2.76 s−1) |
n.d. (tyramine) |
18 |
|
|
|
Glu248Ala‐Thr253Glu (waterkeeper‐HisB1+1) |
−567‐fold (dopamine: 0.98 s−1) |
n.d. (tyramine) |
1.2 |
|
|
|
Glu248Ala‐Phe273Glu (waterkeeper‐gatekeeper) |
−471‐fold (dopamine: 1.18 s−1) |
+generate (tyramine: 0.22 s−1) |
1.7 |
|
|
|
Cys97Ala‐Thr253Asp‐Arg257Asp (thioether bridge‐HisB1+1‐HisB2+1) |
−29‐fold (dopamine: 19 s−1) |
+generate (tyramine: 6.52 s−1) |
40 |
|
[a] Refers to AoTYR (mel0‐Q00234). n.d.: no detected activity, n.i.: no information from the particular study, ++: generation of monophenolase activity that was not present in the wild type, entries with s‐1 refer to the respective kcat values.