Mutation at a strictly conserved, active site tyrosine in the copper amine oxidase leads to uncontrolled oxygenase activity

Abstract

The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

Scheme 1

Proposed mechanism for the biogenesis of TPQ.

Scheme 2

A. Mechanism for the branching of a biogenesis intermediate to TPQ and TPO. B. Model to explain partial occupancy of a derivative at C-3 via formation of TPO-3,4. C. Model to explain partial occupancy of a derivative at C-2 via formation of TPO-2,4.

Figure 1

A. Mature HPAO active site, with TPQ in the reactive conformation (6). Acidic side chains are red, basic are cyan, aromatic are purple, neutral hydrophilic are lime and hydrophobic are green, the TPQ is red and the Cu(II) ion is crimson. A network of 6 active-site water molecules (W1-W6) and one axial copper-coordinating water (Wa) that forms hydrogen bonds to TTQ and side chain atoms (6) are shown in blue. A 7^th water, We, is also coordinated to the Cu(II) ion but is not shown. Hydrogen and metal coordination bonds are shown as black and red dashed lines, respectively. B. Precursor HPAO active-site (21), Zn(II) (gold) and unmodified Tyr405 (red). The color schemes for atoms and coordination or hydrogen bonds are as in Figure 1A (These figures are reprinted from (8)).

Figure 2

A stereo view of the Y305A mutant of HPAO. The (2Fo-Fc) electron density map (red contours at the 1σ level, dark blue at the 8 σ level) and the (Fo–Fc) electron density (green contours at the +3σ level) are shown (no contours are present at the -3 σ level). Also shown is the 6-fold averaged simulated annealing (Fo–Fc) omit map (cyan contours at the +3 σ level). Carbon atoms are green except for TPQ where carbon atoms are cyan; nitrogen atoms are blue, oxygen red, sulfur yellow, and copper magenta. Hydrogen bonds are shown as dashed black lines and coordination bonds as dashed crimson lines. All of the active-site water molecules present in the wild-type structure are shown (wa, w1-w6, shown in Figure 1A, plus an additional water, w7) are shown. Water we, present in the wild-type structure, is not visible in the Y305A mutant.

Figure 3

Simulated annealing omit maps of the Y305F mutants of HPAO computed using coefficients (Fo–Fc) where Fc were calculated from models in which the side chain of residue 405 was absent; in panels A-C Fo were the structure factors observed from crystals of the E-coli-expressed mutant and in panel D from the yeast-expressed mutant. The maps are contoured at sigma levels of 2.5 (cyan), 3.0 (crimson) and 10.0 (purple). Atoms are colored blue for nitrogen, red for oxygen, and magenta for copper; for the residue 405 cofactor, the carbon atoms are cyan. A. Stereo view of a model of TPQ is shown superimposed on the omit map. B. Stereo view of an initial model of TPO is superimposed on the map. C and D. Stereo views of the final model of TPO superimposed on the E. coli- and yeast-expressed simulated annealing omit maps, respectively, of the Y305F mutants. In addition, the modified methionine at position 634 (also omitted during the omit map calculation) is shown superimposed on its density.

Figure 3

Simulated annealing omit maps of the Y305F mutants of HPAO computed using coefficients (Fo–Fc) where Fc were calculated from models in which the side chain of residue 405 was absent; in panels A-C Fo were the structure factors observed from crystals of the E-coli-expressed mutant and in panel D from the yeast-expressed mutant. The maps are contoured at sigma levels of 2.5 (cyan), 3.0 (crimson) and 10.0 (purple). Atoms are colored blue for nitrogen, red for oxygen, and magenta for copper; for the residue 405 cofactor, the carbon atoms are cyan. A. Stereo view of a model of TPQ is shown superimposed on the omit map. B. Stereo view of an initial model of TPO is superimposed on the map. C and D. Stereo views of the final model of TPO superimposed on the E. coli- and yeast-expressed simulated annealing omit maps, respectively, of the Y305F mutants. In addition, the modified methionine at position 634 (also omitted during the omit map calculation) is shown superimposed on its density.

Figure 4

Final electron density maps of the Y305F mutants of HPAO computed using coefficients (2Fo–Fc) and (Fo–Fc) where Fc was calculated from the models refined with TPO and the modified Met634 present. The (2Fo–Fc) maps are contoured at the 0.8 (cyan), 1.0 (crimson) and 8.0 (dark blue) σ levels and the (Fo–Fc) maps are contoured at the -3.0 σ level (gold); no contours above the +3.0 σ level are present. Contours are drawn for the TPO and modified Met634 side chains, as well as for waters, w1-w4. The side chains of Asp319 and Asn430 are also shown without contours (for clarity). Hydrogen and coordination bonds are indicated by black dashed lines. The atom coloring and numbering scheme is the same as in Figure 3 with the additional numbering of four water molecules, w1-w4. These four waters correspond closely to the active site waters found in the native and Y305A structures, namely, waters w4, w7, w5 and w3, respectively. The residues, waters and cofactor oxygen atoms are labeled. A. The E. coli-expressed structure is shown. B. The yeast-expressed structure is shown.

Figure 5

Active-site configuration for the final model of the E. coli-expressed Y305F mutant of HPAO. The orientation of the molecule is approximately the same as shown in Figure 1A. The final TPO cofactor and modified Met634 side chain are shown along with four water molecules, w1-w4. Also included are the side chains of His456, His458, and His624 that form three of the four coordination bonds to copper. In addition, shown are the side chains of Phe305, Asp319, Ala402, and Asn430. Hydrogen and coordination bonds are indicated by black dashed lines. The atom coloring scheme is the same as described for Figure 3.