3-1. The Construction of Mutants of AGHO and Expression of Wild Type and Mutants of AGHO
In this study, A156D, A156D/L158W, A156S/P157G,
A156S/P157G/L158D, L158D mutants of AGHO were generated using QuickChangeTM Site-Directed Mutagenesis with pGEM-T easy/AGHO as a template. Then the pET-based bacterial expression system was chosen to express the wild-type and mutant of AGHO in E. coli. These proteins were cultivated in a copper-depleted medium and purified to homogeneity following protocol described by Yang in our laboratory [27]. As shown in Figure 2, the purity of wild type and mutants of AGHO was >99% as verified by SDS-PAGE.
3-2. Biogenesis of TPQ in AGHO Mutants 3-2-1. TPQ of Wild Type AGHO
The production of an active quinone-containing form of CAOs is dependent on the presence of Cu2+ ions. In previous study, we had
demonstrated that the conversion of the precursor tyrosine to TPQ could be done in the presence of 50 µM CuSO4 [27]. To reduce the oxidative damage of expressed recombinant protein by Cu(II), the enzyme was purified as an apo-form. Upon incubation with 50 µM CuSO4 for
overnight, the purified AGHO may exhibit a pale pink color suggesting the formation of TPQ; whereas the untreated, inactive enzyme
(presumably an apo form) is colorless. To verify that TPQ does form in
recombinant AGHO after Cu2+ treatment, the redox-cycling stanining was employed. The cofactor TPQ has been shown to catalyze redox cycling under an alkaline pH with excess glycine as a reducting agent. In the presence of nitroblue tetrazolium and oxygen, tetrazolium is reduced to formazan and deposited onto the nitrocellulose membrane that contains recombinant AGHO (Figure 3). This result ensures that the TPQ is really formed in AGHO following the treatment of Cu2+. After treatment, the unbound Cu(II) is removed by dialysis to ensure the maintenance of high enzyme activity for a long time.
3-2-2. TPQ formation of Mutants of AGHO
Similarly, all the mutants of AGHO used in this study were also purified as an apo form and then were converted to the holo form prior to the experiment. The formation of TPQ in the mutants of AGHO was verified by NBT/Glycine staining (Figure 4). This result reveals no obvious differences in the TPQ contents were observed among mutants and wild type AGHO, indicating that the formation of TPQ in these mutants is unaltered by the mutagenesis.
3-3. Study of Substrate Preference of Wild Type AGHO
Although histamine is reported to be the primary substrate for AGHO, other amines are also suggested to be catalyzed by this AGHO [38]. Therefore, the reactivity of active AGHO to various, natural, or xenobiotic amines (Appendix 4) were studied. The relative activities of AGHO to various amines (compared with histamine) at the
concentration of 0.2 mM were calculated and listed in Table 4.
Accordingly, compared with histamine (100%), wild type AGHO
exhibited higher reactivity to phenylethylamine (156.0 %) and tyramine (134.6 %). However, it exhibited 41.9 % and 23.9 % activity to
phenylbutylamine and phenylpropylamine, respectively. AGHO shows a little or no activity to benzylamine (1.5%), cadverine (1.4 %),
putrescine (0.7 %), and all the aliphatic amines studied.
Although CAOs exhibit common structural features, the substrate specificities of these enzymes appear to be different. Table 5 shows the kinetic constants (Km, Kcat, Kcat/Km) of wild type AGHO to various amines. Initial rate of AGHO at each concentration of corresponding amine substrate was determined at 30ºC within a time range of 0~5 minutes. The non-linear curve of initial rate vs. substrate concentration was fitted by an appropriate Michaelis-Menten equation (Eq.1 or Eq.2).
The results show that almost all amines (except histamine and benzylamine) exhibit substrate inhibition to wild-type AGHO.
As shown in Table 5, the Km value of wild type AGHO decreases when the alkyl carbon chain length that connects aromatic ring and the amino group of the aromatic amines increases from 1 to 4 carbons. The Km value of AGHO for tyramine, which has one hydroxyl group on the aromatic ring, is slightly higher than that of phenylethylamine (Table 5).
The values of kcat/Km, a representative of substrate specificity, are 0.054, 0.0013, 0.658, 0.385, 0.974, and 0.586 for histamine, benzylamine,
phenylethylamine, phenylpropylamine, phenylbutylamine, and tyramine, respectively. The result indicates that phenylethylamine and its
deverivatives are good substrates for wild type AGHO.
3-4. Studies of Substrate Preference of Mutants AGHO
3-4-1. Selection of Residues in AGHO for Site-Directed Mutagenesis In our previous study, we have shown that AGHO prefer
hydrophobic amines as its substrates [27]. The hydrophobicity is probably the determinant in substrate to affect its binding to AGHO, implying the presence of a lipophilic binding pocket at the active site of AGHO [3]. Thus, the hydrophobicity was used as an important factor to generate QSAR model of AGHO [30]. Based on the generated AGHO QSAR model, some amino acids residues, including F126, A156, P157, Y316, D318, Y322, V399, N401, and F427 are selected (Table 2). They are the consensus residues used in this study to evaluate the effect for QSAR modeling. Most of the selected residues are non-polar except Y316, D317, Y322, and N400. The multiple sequences alignment of CAOs (Table. 3) reveals that the residues Y316, D317, Y322, and N400 are either identical or highly conserved cross all five kingdoms.
Interestingly, residues A156, P157, and L158 of AGHO are highly conserved within each kingdom; whereas the homology of these residues is low cross the kingdoms (Table 3). Thus, in this study, several single, double or triple mutants of AGHO was generated to mimic the active site residues of Hansenula Polymorpha amine oxidase (HPAO) (A156D and A156D/L158W) or bovine serum amine oxidase (BSAO) (L158D, A156S/P157G and A156S/P157G/L158D). Further kinetic studies will be performed.
3-4-2. Relative Activity and Kinetic studies of A156D mutant with
Various Substrates
The A156D mutant of AGHO was generated, overexpressed, and purified as demonstrated in Figure 2. The catalytic activity of A156D mutant toward various amines was much lower than those of wild type (Table. 4). The mutant exhibits a moderate to low reactivity to
phenylethylamine (40.5 %), tyramine (23.5 %), histamine (18.9 %), phenylbutylamine (11.9 %), and phenylpropylamine (7.9 %). It shows nearly no activity to benzylamine (1.0%) and aliphatic amines.
Table 6 shows the kinetic parameters of A156D mutant. Compared with wild type AGHO, the Km values of A156D to histamine,
benzylamine, phenylethylamine and tyramine increased; whereas the kcat
values for the above amine were decreased (Table 10). Hence, the kcat/Km
values for histamine, benzylamine, phenylethylamine and tyramine, were 0.004, 0.0002, 0.012 and 0.005, respectively, which were much lower than those of wild type AGHO (kcat/Km(histamine) = 0.054;
kcat/Km(benzylamine) = 0.0013; kcat/Km(phenylethylamine) = 0.658;
kcat/Km(tyramine) = 0.586). Different from wild type AGHO, no substrate inhibition was observed in A156D mutant, even the concentration of amines reached 1 mM. 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.
3-4-3. Relative Activity and Kinetic studies of A156D/L158W Mutant with Various Substrates
The double mutant of AGHO (A156D/L158W) was also generated,
A156D/L158W mutant shows no activity toward the amines studied.
This result suggests that Leu 158 may be important in controlling the access of amines toward active site pocket. The molecular modeling of AGHO shows that Leu 158 lies in the proximity of substrate with its side chain closes to the ring of aromatic amines (Figure 5). When Leu was changed to Trp, a steric effect may be exerted by its bulk side chain.
The bulk side chain of Trp may either hinder the access of substrate to the TPQ or cause the improper orientation of substrate directing to TPQ (Figure 6). In conclusion, A156 and L158 in AGHO may play important roles in controlling the binding and/or catalysis of substrates.
3-4-4. Relative Activity and Kinetic studies of A156S/P157G/L158D with Various Substrates
The A156S/P157G/L158D triple mutant of AGHO, which mimics the active site residues of BSAO (bovine serum amine oxidase), was
generated, overexpressed, and purified (Figure 2). The substrate
specificity of BSAO has been shown to prefer aliphatic amines, including putrescine, spermine, and spermidine [39-41]. In contrast, these aliphatic amines are neither substrates nor inhibitors of wild type AGHO.
Therefore, the reactivity of this triple mutant toward putrescine and spermine will be studied. Different from wild type AGHO,
A156S/P157G/L158D mutant has a low activity to aromatic amines, including tyramine (18.7 %), phenylbutylamine (16.1 %),
phenylpropylamine (15.2 %), histamine (12.2 %), and phenylpropylamine (2.8 %) (Table 4). Interestingly, this triple mutant shows about
4.4~30-fold higher relative activity to aliphatic amines, including
putrescine (8.3 %), cadcerine (6.2 %), spermine (6.1 %), spermidine (7.2
%), and norspermidine (7.5 %), than that of wild type AGHO (Table 4).
The Km values of A156S/P157G/L158D triple mutant to histamine, phenylbutylamine, phenylpropylamine, phenylpropylamine and tyramine are similar; whereas the kcat values to above aromatic amine reduced 50~95% (Table 11). Surprisingly, A156S/P157/L158D mutant exhibits a high affinity to spermine and putrescine with Km values of 23.81 µM and 25.35 µM, respectively (Table 7). The kcat values of the
A156S/P157G/L158D mutant to spermine and putrescine are 0.32 s-1 and 0.29 s-1, respectively. More interestingly, the Km of BSAO to spermine is 20 µM [40]. This result suggests that the residues A156, P157, and L158 in AGHO are truly involved in the substrate recognition. 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. Compared with wild-type AGHO, the kcat/Km values of A156S/P156G/L158D mutant reduced 5~11-fold to histamine (0.009), benzylamine (0.006), phenylethylamine (0.074), phenylpropylamine (0.012), phenylbutylamine (0.352), and tyramine (0.044). Interestingly, the kinetic parameters for phenylbutylamine were not affected much by this mutagenesis with Km and kcat of 2.56 µM and 0.9 s-1, respectively. A long butyl carbon chain may partially mimic the aliphatic amine that can be fitted into the active site of
A156S/P157G/L158D mutant. A low substrate inhibition was observed on the A156S/P157G/L158D mutant with the Ki values of 344, 2248, 2402 and 2865 µM for phenylbutylamine, phenylethylamine, tyramine,
3-4-5. Relative Activity and Kinetic studies of A156S/P157G with Various Substrates
A156S/P157G double mutant, partially mimic the active site residues of BSAO (bovine serum amine oxidase), was generated, overexpressed and purified (Figure 2). Compared with wild type AGHO, A156S/P157G exhibiteds normal reactivity to phenylethylamine (103.2 %) and tyramine (90.6 %), and moderate activity to histamine (64.4 %) and
phenylbutylamine (60.4 %) (Table 4). Additionally, A156S/P157G shows a little or no activity to benzylamine (~1.0 %) and spermine. Interesting, the activity of A156S/P157G mutant toward diamine, such as putrescine, is higher than that of wild-type.
Table 8 displays the kinetic parameters of A156S/P157G mutant of AGHO to various substrates. Interestingly, substrate inhibition was not observed in A156S/P157G mutant. The Km values for histamine,
benzylamine, phenylethylamine and tyramine were higher than those of wild type AGHO (Table 12). However, the kinetic parameters of
A156S/P157G mutant to phenylpropylamine, phenylbutylamine and spermine could not be determined in this study. 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 0.020, 0.0004, 0.158, 0.181 and 0.0001, respectively. These results reveal that replacement of A156 and P157 to Ser and Gly,
respectively, can induce its activity to putrescine, but may not be enough to turn the substrate specificity of AGHO from aromatic amines to
aliphatic amines. Apparently, 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.
3-4-6. Relative Activity and Kinetic studies of L158D with Various Substrates
To understand the role of L158 in the substrate specificity of AGHO, a mutant L158D was generated, overexpressed and purified. As shown in Table 3, L158D mutant exhibited low activity to amines tested, including phenylethylamine (15.4 %), tyramine (11.8 %), phenylbutylamine (11.3
%), and histamine (5.0 %). L156D mutant shows little or no activity to phenylpropylamine (1.9 %), benzylamine (1.7 %), and aliphatic amines, including putrescine (4.0 %), cadcerine (3.1 %), spermine (1.1 %), spermidine (0.6 %), and norspermidine (0.5 %).
The Km for histamine, phenylethylamine and tyramine was slight increased or decreased due to the mutation; whereas the Km for
benzylamine was largely decreased compared with that of wild-type AGHO (Table 14). For the aliphatic amines, including putrescine and spermine, the Km values were 124.2 and 1830 µM, respectively, which were about 5- and 77-fold higher than that of A156S/P157G/L158D mutant (Table 15). However, the kcat values of L156D mutant to
putrescine and spermine were similar to that of A156S/P157G/L158D mutant. Several amines also exhibit substrate inhibition to L158D mutant, including benzylamine (574 µM), tyramine (744 µM), and
phenylethylamine (1575 µM) (Table 9). Therefore, the single mutation of L158D also contributes to mediate the substrate binding and orientation
Although L158D, A156S/P157G and A156S/P157G/L158D mutants can utilize putrescine and sperimine as substraes, only
A156S/P157G/L158D mutant of AGHO mimic the substrate specificity of BSAO. Maybe when Pro was changed to Gly, the lack of β-carbon atom permits 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 there is a suitable
circumstance driving the long chains and positively charged amino group of the substrate to the active site, in a correct orientation for the catalytic reaction (Figure 7). This result suggests that A156, P157 and L158 are essential in the active site of AGHO to mediate the substrate recognition and binding. The incomplete replacement of these three amino acid residues may lead to incomplete conversion of AGHO to BSAO in term of substrate specificity.
3-5. Future Application of AGHO
Although the reductive half-reaction and the reaction intermediates of the catalysis of AGHO have been well understoodl, the factors that
influence the recognition and interaction between the substrate and the active site of CAOs are still unknown.
Since the substrates of AGHO are involved in numerous cellular functions, such as regulation of the synthesis of protein and nucleic acid, regulation of cell proliferation, differentiation and development, and involvement in detoxification and cell signaling processes. Based on the clinical investigation, SSAO have been detected under several
pathophysiological conditions, particularly in diabetes mellitus,
congestive heart failure and cirrhotic liver inflammation [42]. It has been suggested that some of the complications associated with diabetes, such as retinopathy, rephropathy, neuropathy, atherosclerosis and
cardiovascular complications, may be caused by toxic products of SSAO-catalyzed reactions [43]. Therefore, CAOs have shown to be a potential target for anti-inflammatory drug screening. The inhibitors of CAOs may be used clinically to alleviate with diabetes.
Hence, knowledge of the factors controlling enzyme and substrate interactions can facilitate the design and optimization of selective
agonist/antagonist. The results presented in this study may be helpful for the future application of AHGO mutants in fabrication of biosensors and drug screening.
Reference
1. Binda, C., A. Mattevi, and D.E. Edmondson, Structure-function relationships in flavoenzyme-dependent amine oxidations: a comparison of polyamine oxidase and monoamine oxidase. J Biol Chem, 2002. 277(27): p. 23973-6.
2. Mure, M., S.A. Mills, and J.P. Klinman, Catalytic mechanism of the topa quinone containing copper amine oxidases.
Biochemistry, 2002. 41(30): p. 9269-78.
3. Wilce, M.C., et al., Crystal structures of the copper-containing amine oxidase from Arthrobacter globiformis in the holo and apo forms: implications for the biogenesis of topaquinone.
Biochemistry, 1997. 36(51): p. 16116-33.
4. Carter, S.R., et al., Purification and active-site characterization of equine plasma amine oxidase. J Inorg Biochem, 1994. 56(2): p.
127-41.
5. Elmore, B.O., J.A. Bollinger, and D.M. Dooley, Human kidney diamine oxidase: heterologous expression, purification, and characterization. J Biol Inorg Chem, 2002. 7(6): p. 565-79.
6. McGuirl, M.A., et al., Purification and characterization of pea seedling amine oxidase for crystallization studies. Plant Physiol, 1994. 106(3): p. 1205-11.
7. McIntire, W., and Hartmann, C., Copper-containing amine oxidases. In Principles and Applications of Quinoproteins.
Davidson, VL, Ed.; Marcel Dekker: New York, 1993: p. 97-172.
8. Sebela, M.F., I.; Petrivalsky, M.; Pec, P., Copper/topa quinone-containing amine oxidases - recent research developments. 2002. 26: p. 1259-99.
9. Salmi, M., J. Hellman, and S. Jalkanen, The role of two distinct endothelial molecules, vascular adhesion protein-1 and
peripheral lymph node addressin, in the binding of lymphocyte subsets to human lymph nodes. J Immunol, 1998. 160(11): p.
5629-36.
10. Yu, P.H., et al., Physiological and pathological implications of semicarbazide-sensitive amine oxidase. Biochim Biophys Acta, 2003. 1647(1-2): p. 193-9.
11. Ruggiero, C.E., et al., Mechanistic studies of topa quinone biogenesis in phenylethylamine oxidase. Biochemistry, 1997.
36(8): p. 1953-9.
12. Ruggiero, C.E. and D.M. Dooley, Stoichiometry of the topa quinone biogenesis reaction in copper amine oxidases.
Biochemistry, 1999. 38(10): p. 2892-8.
13. Prabhakar, R. and P.E. Siegbahn, A theoretical study of the mechanism for the biogenesis of cofactor topaquinone in copper amine oxidases. J Am Chem Soc, 2004. 126(12): p. 3996-4006.
14. Wang, S.X., et al., A crosslinked cofactor in lysyl oxidase: redox function for amino acid side chains. Science, 1996. 273(5278): p.
1078-84.
15. Wilmot, C.M., et al., Visualization of dioxygen bound to copper during enzyme catalysis. Science, 1999. 286(5445): p. 1724-8.
16. Murray, J.M., et al., Conserved tyrosine-369 in the active site of Escherichia coli copper amine oxidase is not essential.
Biochemistry, 2001. 40(43): p. 12808-18.
17. O'Connell, K.M., et al., Differential Inhibition of Six Copper Amine Oxidases by a Family of 4-(Aryloxy)-2-butynamines:
Evidence for a New Mode of Inactivation. Biochemistry, 2004.
43(34): p. 10965-78.
18. Parsons, M.R., et al., Crystal structure of a quinoenzyme: copper amine oxidase of Escherichia coli at 2 A resolution. Structure, 1995. 3(11): p. 1171-84.
19. Li, R., et al., Crystallographic study of yeast copper amine oxidase. Acta Crystallogr D Biol Crystallogr, 1997. 53(Pt 4): p.
364-70.
20. Duff, A.P., et al., The crystal structure of Pichia pastoris lysyl oxidase. Biochemistry, 2003. 42(51): p. 15148-57.
21. Kumar, V., et al., Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A resolution. Structure, 1996. 4(8): p. 943-55.
22. Lunelli, M., et al., Crystal structure of amine oxidase from bovine serum. J Mol Biol, 2005. 346(4): p. 991-1004.
23. Airenne, T.T., et al., Crystal structure of the human vascular adhesion protein-1: unique structural features with functional implications. Protein Sci, 2005. 14(8): p. 1964-74.
24. Li, R., J.P. Klinman, and F.S. Mathews, Copper amine oxidase from Hansenula polymorpha: the crystal structure determined at
6(3): p. 293-307.
25. Shepard, E.M., et al., Towards the development of selective amine oxidase inhibitors. Mechanism-based inhibition of six copper containing amine oxidases. Eur J Biochem, 2002. 269(15): p.
3645-58.
26. Mure, M., Tyrosine-derived quinone cofactors. Acc Chem Res, 2004. 37(2): p. 131-9.
27. Yang, J.-G., Research of Substrate Specificity of Arthrobacter globiformis Histamine Oxidase., in institute of Biological Science and Technology 2004, National Chiao Tung University.
28. Yoshida, S., et al., Structure of rice-straw arabinoglucuronoxylan and specificity of Streptomyces xylanase toward the xylan. Agric Biol Chem, 1990. 54(2): p. 449-57.
29. Choi, Y.H., et al., Copper/topa quinone-containing histamine oxidase from Arthrobacter globiformis. Molecular cloning and sequencing, overproduction of precursor enzyme, and generation of topa quinone cofactor. J Biol Chem, 1995. 270(9): p. 4712-20.
30. Chang, L.-J., Integrating GEMDOCK with GEMPLS and
GEMkNN for QSAR model of huAChE and AGHO. , in Institute of Bioinformatics 2005, National Chiao Tung University.
31. Choi, Y.H., et al., Role of conserved Asn-Tyr-Asp-Tyr sequence in bacterial copper/2,4, 5-trihydroxyphenylalanyl
quinone-containing histamine oxidase. J Biol Chem, 1996.
271(37): p. 22598-603.
32. Kishishita, S., et al., Role of copper ion in bacterial copper amine oxidase: spectroscopic and crystallographic studies of
metal-substituted enzymes. J Am Chem Soc, 2003. 125(4): p.
1041-55.
33. Contakes, S.M., et al., Reversible inhibition of copper amine
oxidase activity by channel-blocking ruthenium(II) and rhenium(I) molecular wires. Proc Natl Acad Sci U S A, 2005. 102(38): p.
13451-6.
34. Chang, S.-P., Characterization of Arthrobacter globiformis histamine oxidase by mutagenesis., in institute of Biological Science and Technology. 2003, National Chiao Tung University.
35. Lin, Y.-H., Expression, mutagenesis and characterization of Arthrobacter globiformis amine oxidase I (histamine oxidase), in institute of Biological Science and Technology. 2002, National
Chiao Tung University.
36. Stoner, P., An improved spectrophotometric assay for histamine and diamine oxidase (DAO) activity. Agents Actions, 1985. 17(1):
p. 5-9.
37. Paz, M.A., et al., Specific detection of quinoproteins by
redox-cycling staining. J Biol Chem, 1991. 266(2): p. 689-92.
38. Shimizu, E., Odawara, T., Tanizawa, K., and Yorifuji, T., Histamine oxidase, a quinoprotein enzyme of Arthrobacter globiformis. Biosci. Biotech. Biochem., 1994. 58: p. 2118-120.
39. Houen, G., et al., Substrate specificity of the bovine serum amine oxidase and in situ characterisation of aminoaldehydes by NMR spectroscopy. Bioorg Med Chem, 2005. 13(11): p. 3783-96.
40. Di Paolo, M.L., et al., Electrostatic compared with hydrophobic interactions between bovine serum amine oxidase and its
substrates. Biochem J, 2003. 371(Pt 2): p. 549-56.
41. Lee, Y. and L.M. Sayre, Reaffirmation that metabolism of
polyamines by bovine plasma amine oxidase occurs strictly at the primary amino termini. J Biol Chem, 1998. 273(31): p. 19490-4.
42. Boomsma, F., et al., Plasma semicarbazide-sensitive amine oxidase in human (patho)physiology. Biochim Biophys Acta, 2003. 1647(1-2): p. 48-54.
43. Gokturk, C., et al., Overexpression of semicarbazide-sensitive amine oxidase in smooth muscle cells leads to an abnormal
structure of the aortic elastic laminas. Am J Pathol, 2003. 163(5):
p. 1921-8.
44. Cai, D. and J.P. Klinman, Copper amine oxidase: heterologous expression, purification, and characterization of an active enzyme in Saccharomyces cerevisiae. Biochemistry, 1994. 33(24): p.
7647-53.
45. Tipping, A.J. and M.J. McPherson, Cloning and molecular analysis of the pea seedling copper amine oxidase. J Biol Chem, 1995. 270(28): p. 16939-46.
MBTH/DMAB standard curve
y = 0.0449x - 0.0031 R2 = 0.9996
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
0 2 4 6 8 10 12 14 16
H2O2(n mole) O.D 595nm
Figure 1. H2O2 standard curve.
The H2O2 standard curve was determined within the range between 0.1 to 15 nmole.
M
1 2 3 4 5 6 7 8 9
10
11
12
Figure 2. 10% SDS-PAGE of crude extract and purified wild type and AGHO mutants.
The crude extracts, purified wild-type AGHO, and AGHO mutants (all 3 µg) were separated on a 10 % SDS-PAGE and stained with Coomassie Blue. M: molecular weight standards (MBI Marker): 116.0, 66.2, 45.0, 35.0, and 25.0 kDa. Lane 1, crude extract containing Cu2+-free apo form of AGHO; Lane 2, purified wild type AGHO; Lane 3, crude extract containing Cu2+-free apo form of A156D mutant; Lane 4, purified A156D mutant; Lane 5, crude extract containing Cu2+-free apo form of
The crude extracts, purified wild-type AGHO, and AGHO mutants (all 3 µg) were separated on a 10 % SDS-PAGE and stained with Coomassie Blue. M: molecular weight standards (MBI Marker): 116.0, 66.2, 45.0, 35.0, and 25.0 kDa. Lane 1, crude extract containing Cu2+-free apo form of AGHO; Lane 2, purified wild type AGHO; Lane 3, crude extract containing Cu2+-free apo form of A156D mutant; Lane 4, purified A156D mutant; Lane 5, crude extract containing Cu2+-free apo form of