E. coli Expression of Recombinant AGHO WT and AGHO mutants
4. Studies of Substrate Preference of Mutants AGHO
4-1. Selection of Residues in AGHO for Site-Directed Mutagenesis
We have found that AGHO prefer hydrophobic amines as its substrates in our previous study [27]. The hydrophobicity of substrates is probably the determinant that affects the binding to AGHO, implying the presence of a lipophilic binding pocket at the active site of AGHO [8]. Thus, the
hydrophobicity was set to be a crucial factor to build the QSAR model of AGHO [21]. Based on the built AGHO QSAR model, amino acids residues including F126, A156, P157, Y316, D318, Y322, V399, N401, and F427 were selected (Table 4). Most of the selected residues are non-polar except Y316, D317, Y322, and N400. The multiple sequences alignment of CAOs (Table 5) reveals that the residues Y316, D317, Y322, and N400 are either identical or highly conserved cross all five kingdoms. We discovered that residues A156, P157, L158 and F427 of AGHO are highly conserved within each kingdom; whereas the
homology of these residues is low cross the kingdoms (Table 5). Hence, in this
study, several sets of single, double, triple and quadruple mutants of AGHO was generated to mimic the active site residues of a fungi amine oxidase, Hansenula Polymorpha amine oxidase (HPAO) (A156D, F427N, A156D/F427N), and an animal amine oxidase, bovine serum amine oxidase (BSAO) (L158D,
F427S,A156S/P157G, A156S/P157G/L158D and A156S/P157G/L158D/F427S).
Further kinetic studies were performed.
4-2. A156D, F427N and A156D/F427N mutants
4-2-1. Relative activities of A156D, F427N and A156D/F427N mutants with various substrates
In table 6, all the relative activities were compared to the activity of wild type AGHO toward histamine (100%). A156D exhibits lower relative activities to histamine (from 100.0% to 7.4%), benzylamine (from 1.8% to 0.8%),
phenylethylamine (from 145.6% to 21.2%), phenylpropylamine (from 38.1% to 3.1%), phenylbutylamine (from 54.2% to 5.2%) and tyramine (from 112.7% to 14.6%). This set of mutants shows no reactivities to the HPAO substrates including methylamine and ethylamine. In the result, the mutation intends to mimic HPAO did not increase the reactivities to the preferred substrates of
HPAO. F427N also shows lower relative activities to histamine (from 100.0% to 63.9%), benzylamine (from 1.8% to 1.5%), phenylethylamine (from 145.6% to 82.1%), phenylpropylamine (from 38.1% to 32.7%), phenylbutylamine (from 54.2% to 44.5%) and tyramine (from 112.7% to 73.5%), but mutation on F427
made a less obvious decrease than on A156 (figure 5). Both A156D and F427N show nearly no activity to putrescine and spermine.
Relative activities of A156D/F427N toward histamine, phenylethylamine and tyramine are 6.4 %, 12.8% and 9.4%, respectively. And it shows no detected activity to benzylamine, phenylpropylamine, phenylbutylamine, methylamine and ethylamine , putrescine and spermine. In figure 5, it shows a similar pattern on the activity change from mutation on A156D, F247N, A156D/F427N. Hence, Mutation of A156D and F427N may affect the catalytic activity of AGHO.
When A156 was mutated to polar aspartic acid and F427 was mutated to asparagine at the same time, it may alter the shape of the active site, and aromatic amines with longer chains could not fit into the active site.
4-2-2. Kinetic studies of A156D, F427N and A156D/F427N mutants with various substrates
Table 7 shows the kinetic parameters of A156D, F427N and A156D/F427N mutant. Compared with wild type AGHO, the Km values of A156D to histamine, benzylamine, phenylethylamine and tyramine increased, but the reactivities of methylamine, ethylamine, phenylpropylamine, phenylbutylamine, putrescine and spermine were too low for the kinetic constants determination.
The kcat values for the above amine were decreased in A156D. Hence, the kcat/Km values for histamine, benzylamine, phenylethylamine and tyramine, were 0.0045, 0.0007, 0.0153 and 0.0112 (µM-1S-1), respectively, which were much
lower than those values of the wild type AGHO. Different from wild type AGHO, no substrate inhibition was observed in this set of mutants including A156D, F427N and A156D/F427N. It was shown that the biogenesis of TPQ of this mutant was unaltered. Thus, A156 of AGHO may play a key role in
mediating the catalysis of amines.
The Km values of F427N to histamine decreased about 50%. And the Km
values of F427N to benzylamine, phenylethylamine, phenylpropylamine and tyramine slightly decreased or remained close. The kcat values for the above amine were decreased.
The Km value of A156D/F427N towards histamine is 3 times more than it of the wild type AGHO, to phenylethylamine it is 24 times, and to tyramine it is 10 times. The mutations did change the specificity of these amines. A156D/F427N shows higher reactivities to histamine, phenylethylamine and tyramine but shows no activity to benzylamine. The result shows that the mutation on A156 to aspartic acid and on F427 to asparagine may change the shape or size of the active channel of AGHO, and improve the selectivity on the chain length of these substrates.
4-3. A156S/P157G and L158D mutants
4-3-1. Relative activities of A156S/P157G, and L158D mutants with various substrates
Compared with the wild type AGHO, A156S/P157G exhibits lower
activities to histamine (from 100.0 % to 60.6 %), phenylethylamine (from 145.6
% to 97.6 %) and tyramine (from 112.7 % to 84.8 %), and a slightly increased activity to phenylbutylamine (from 54.2 % to 60.5 %) (Table 8) .Additionally, A156S/P157G shows a relatively low activity to benzylamine (1.1 %).
Interesting, the activity of A156S/P157G mutant toward putrescine is higher than that of wild-type (from 0.9% to 1.3%).
L158D mutant exhibited low activity to most of the amines we tested, including histamine (6.3%), benzylamine (1.4 %), phenylethylamine (12.3 %), phenylpropylamine (1.6 %), phenylbutylamine (14.8%) and tyramine (11.9 %).
We noticed that the activities toward histamine, phenylethylamine and tyramine decreased more than 10 times of the wild type AGHO. But the activities to aliphatic amines, including putrescine (3.4 %) and spermine (0.9 %) are increased. The result shows that the mutation of L158 to aspartic acid did increase the AGHO activities to the substrates of BSAO and it confirms the result of the previous study made by Chen [16].
4-3-2. Kinetic studies of A156S/P157G and L158D mutant with various substrates
Table 9 displays the calculated kinetic constants of A156S/P157G and L158D mutants of AGHO to various substrates.
The previously observed substrate inhibition in wild type by
phenylethylamine, phenylpropylamine, phenylbutylamine and tyramine was not
observed in A156S/P157G mutant. The Km values for histamine (98.58 ± 1.66 µM), benzylamine (91.02 ± 15.69 µM) and phenylethylamine (14.04 ± 0.13 µM) were higher than those of wild type AGHO. The kcat/Km values of A156S/P157G mutant for histamine, benzylamine, phenylethylamine, tyramine and putrescine were lower than that of wild type AGHO and calculated as 0.0499, 0.0010, 0.3255, 0.3541 and 0.0606 (µM-1S-1), respectively. These results reveal that replacement of A156 to Serine and P157 to Glycine could induce the activity to putrescine. Hence, L158 may be important in determining the substrate
selectivity of CAOs. However, A156 and P157 in AGHO may somewhat play roles mediating the substrate selectivity of the enzyme.
For the L158D mutant, the Km for histamine (148.15 ± 0.62 µM), phenylethylamine (4.64 ± 0.63 µM) and tyramine (12.08 ± 1.16 µM) was slightly increased or decreased due to the mutation; whereas the Km for benzylamine (3.6 ± 2.24 µM) decreased to about 1/10 of wild-type AGHO (Table 9). For the aliphatic amines, including putrescine and spermine, the Km values were 12.86 ± 1.23 µM and 9.78 ± 1.92 µM, which were to low to be determined in the wild type AGHO. Several amines also exhibit substrate inhibition to L158D mutant, including benzylamine (Ki = 606 ± 270 µM), phenylethylamine (Ki =1401 ± 250 µM), and tyramine (Ki =1199 ± 201 µM).
Hence, the single mutation of L158D also contributes to mediate the substrate binding and orientation in the active pocket.
L158D and A156S/P157G mutants utilize putrescine and sperimine as
substrates. Due to the replacement from Proline to Glycine, the lack of β-carbon atom may permit a substantially greater degree of conformational flexibility and attainable conformational space to admit long chains of amine. As for L158D, it’s probably consistent with electrostatic attraction of positively charged substrates into the channel. Therefore it drives the long chains and positively charged amino groups of the substrate to the active site in a correct orientation for the catalytic reaction (Figure 6). This result suggests that A156, P157 and L158 are essential in the active site of AGHO to mediate the substrate
recognition and binding.
4-4. A156S/P157G/L158D, F427S and A156S/P157G/L158D/F427S mutants 4-4-1. Relative activities of A156S/P157G/L158D, F427S and
A156S/P157G/L158D/F427S mutants with various substrates
Table 10 shows that A156S/P157G/L158D, F427S andA156S/P157G/L158D/F427S mutants all made increased activities to benzylamine, putrescine, and spermine. And they exhibit lower activities to histamine, phenylethylamine, phenylpropylamine, phenylbutylamine and
tyramine. The quadruple A156S/P157G/L158D/F427S mutant shows the lowest activities to theses amines.
The A156S/P157G/L158D triple mutant shows decreased activities toward histamine (from 100.0% to 9.8%) and phenyletheylamine (from 145.6% to 13.3%) to about 1/10 of the wild type AGHO. The A156S/P157G/L158D/F427S
quadruple mutant shows a relatively lower activity toward phenylpropylamine (1.4 ± 0.1%) which is 1/27 of the wild type AGHO (38.1 ± 1.9%).
In the F427S mutant the activities toward benzylamine (from 1.8% to 4.0%), putrescine (from 0.9% to 1.9%), and spermine (from 0.4% to 0.8%) were about 2 times for it of the wild type AGHO .In the A156S/P157G/L158D triple mutant the activities toward putrescine (6.3 ± 1.2%) is 7 times for it of the wild type AGHO (0.9 ± 0.1%) and for spermine (5.6 ± 0.2%) it is 14 times for it of the wild type AGHO (0.4 ± 0.1%). In the A156S/P157G/L158D/F427S quadruple mutant, the activities were even higher. It shows that the mutation on F427 to Serine could enhance the activities toward these aliphatic amines which were typical substrate of BSAO.
4-4-2 Kinetic studies of A156S/P157G/L158D, F427S and
A156S/P157G/L158D/F427S mutants with various substrates
The calculated kinetic constants of A156S/P157G/L158D, F427S and A156S/P157G/L158D/F427S mutants of AGHO to various substrates are listed in table 11.A156S/P157G/L158D, F427S and A156S/P157G/L158D/F427S mutant shows similar Km values to phenylethylamine, phenylpropylamine,
phenylbutylamine and tyramine with the wild type AGHO. Km values to
histamine and benzylamine were reduced. Interestingly, A156S/P157G/L158D and A156S/P157G/L158D/F427S exhibit higher affinity to putrescine and
spermine with Km values larger than 20 µM. And the values are close to the Km of BSAO to spermine (20 µM) [19]. Substrate inhibition was also observed in A156S/P157G/L158D , F427S and A156S/P157G/L158D/F427S mutants. The replacement of these residues with the active site sequence in BASO alters the substrate preference of AGHO from aromatic amine to aliphatic diamines, including putrescine and spermine.