Oxygen Activation Switch in the Copper Amine Oxidase of Escherichia coli.

Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu2+ in the active site, two peripheral calcium sites, ∼32 Šfrom the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca2+ (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Ca2+ (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Q mutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (∼60% WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity (∼4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet-visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu2+-substrate reduced TPQ (TPQAMQ) Cu+-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu2+-TPQAMQ or destabilizes Cu+-TPQSQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu2+-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu+-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.

Probing the molecular mechanisms in copper amine oxidases by generating heterodimers.

For some homodimeric copper amine oxidases (CuAO), there is suggestive evidence of differential activity at the two active sites implying potential cooperativity between the two monomers. To examine this phenomenon for the Arthrobacter globiformis CuAO (AGAO), we purified a heterodimeric form of the enzyme for comparison with the homodimer. The heterodimer comprises an active wild-type monomer and an inactive monomer in which an active-site tyrosine is mutated to phenylalanine (Y382F). This mutation prevents the formation of the trihydroxyphenylalanine quinone (TPQ) cofactor. A pETDuet vector and a dual fusion tag strategy was used to purify heterodimers (WT/Y382F) from homodimers. Purity was confirmed by western blot and native PAGE analyses. Spectral and kinetic studies support the view that whether there are one or two functional monomers in the dimer, the properties of each functional monomer are the same, thus indicating no communication between the active sites in this bacterial enzyme.

Tyrosine 381 in E. coli copper amine oxidase influences substrate specificity.

Copper amine oxidases are important for the metabolism of a range of biogenic amines. Here, we focus on substrate specificity in the E. coli copper amine oxidase (ECAO) and specifically the role of Tyr 381. This residue, and its equivalent, in other copper amine oxidases has been referred to as a "gating" residue able to move position depending upon the presence or absence of amine substrate. The position of this residue suggests a role in substrate selectivity. We have compared the properties of two variant forms of ECAO, Y381F and Y381A, with wild-type enzyme by steady-state kinetics of oxidation of a number of amine substrates, modes of inhibitor interactions and X-ray structure determination. Y381F displays a similar catalytic efficiency to wild type against the preferred substrate ß-phenylethylamine. In both cases oxidation of the alternative aromatic amine substrate benzylamine is relatively poor, although Y381F represents an efficient benzylamine oxidase. By contrast, Y381A performed poorly against both aromatic substrates predominantly due to an increased K (M) which we propose is due to the lack of an aromatic residue to orient substrate towards the TPQ and active site base. These results are supported by different behaviour of Y381A to inhibition with 2-hydrazinopyridine. We also report on methylamine turnover by the three enzymes. We propose that Y381, together with another residue Y387, may be considered of critical importance for the substrate selectivity of ECAO, through stacking or hydrophobic interactions with substrate.

Exploring the roles of the metal ions in Escherichia coli copper amine oxidase.

To investigate the role of the active site copper in Escherichia coli copper amine oxidase (ECAO), we initiated a metal-substitution study. Copper reconstitution of ECAO (Cu-ECAO) restored only approximately 12% wild-type activity as measured by k(cat(amine)). Treatment with EDTA, to remove exogenous divalent metals, increased Cu-ECAO activity but reduced the activity of wild-type ECAO. Subsequent addition of calcium restored wild-type ECAO and further enhanced Cu-ECAO activities. Cobalt-reconstituted ECAO (Co-ECAO) showed lower but significant activity. These initial results are consistent with a direct electron transfer from TPQ to oxygen stabilized by the metal. If a Cu(I)-TPQ semiquinone mechanism operates, then an alternative outer-sphere electron transfer must also exist to account for the catalytic activity of Co-ECAO. The positive effect of calcium on ECAO activity led us to investigate the peripheral calcium binding sites of ECAO. Crystallographic analysis of wild-type ECAO structures, determined in the presence and absence of EDTA, confirmed that calcium is the normal ligand of these peripheral sites. The more solvent exposed calcium can be easily displaced by mono- and divalent cations with no effect on activity, whereas removal of the more buried calcium ion with EDTA resulted in a 60-90% reduction in ECAO activity and the presence of a lag phase, which could be overcome under oxygen saturation or by reoccupying the buried site with various divalent cations. Our studies indicate that binding of metal ions in the peripheral sites, while not essential, is important for maximal enzymatic activity in the mature enzyme.

Cross-link formation of the cysteine 228-tyrosine 272 catalytic cofactor of galactose oxidase does not require dioxygen.

Galactose oxidase (GO) belongs to a class of proteins that self-catalyze assembly of their redox-active cofactors from active site amino acids. Generation of enzymatically active GO appears to require at least four sequential post-translational modifications: cleavage of a secretion signal sequence, copper-dependent cleavage of an N-terminal pro sequence, copper-dependent formation of a C228-Y272 thioether bond, and generation of the Y272 radical. The last two processes were investigated using a truncated protein (termed premat-GO) lacking the pro sequence and purified under copper-free conditions. Reactions of premat-GO with Cu(II) were investigated using optical, EPR, and resonance Raman spectroscopy, SDS-PAGE, and X-ray crystallography. Premat-GO reacted anaerobically with excess Cu(II) to efficiently form the thioether bond but not the Y272 radical. A potential C228-copper coordinated intermediate (lambda max = 406 nm) in the processing reaction, which had not yet formed the C228-Y272 cross-link, was identified from the absorption spectrum. A copper-thiolate protein complex, with copper coordinated to C228, H496, and H581, was also observed in a 3 min anaerobic soak by X-ray crystallography, whereas a 24 h soak revealed the C228-Y272 thioether bond. In solution, addition of oxygenated buffer to premat-GO preincubated with excess Cu(II) generated the Y272 radical state. On the basis of these data, a mechanism for the formation of the C228-Y272 bond and tyrosyl radical generation is proposed. The 406 nm complex is demonstrated to be a catalytically competent processing intermediate under anaerobic conditions. We propose a potential mechanism which is in common with aerobic processing by Cu(II) until the step at which the second electron acceptor is required.

Medical implications from the crystal structure of a copper-containing amine oxidase complexed with the antidepressant drug tranylcypromine.

The X-ray crystal structure of the copper-containing quinoprotein amine oxidase from E. coli has been determined in complex with the antidepressant drug tranylcypromine to 2.4 A resolution. The drug is a racemic mix of two enantiomers, but only one is seen bound to the enzyme. The other enantiomer is not acting as a substrate for the enzyme as no catalytic activity was detected when the enzyme was initially exposed to the drug. The inhibition of human copper amine oxidases could be a source of side-effects in its use as an antidepressant to inhibit the flavin-containing monoamine oxidases in the brain.

Cofactor processing in galactose oxidase.

GO (galactose oxidase; E.C. 1.1.3.9) is a monomeric 68 kDa enzyme that contains a single copper ion and an amino acid-derived cofactor. The enzyme is produced by the filamentous fungus Fusarium graminearum as an extracellular enzyme. The enzyme has been extensively studied by structural, spectroscopic, kinetic and mutational approaches that have provided insight into the catalytic mechanism of this radical enzyme. One of the most intriguing features of the enzyme is the post-translational generation of an organic cofactor from active-site amino acid residues. Biogenesis of this cofactor involves the autocatalytic formation of a thioether bond between Cys-228 and Tyr-272, the latter being one of the copper ligands. Formation of this active-site feature is closely linked to the loss of an N-terminal 17 amino acid prosequence. When copper and oxygen are added to this pro-form of GO (pro GO), purified in copper-free conditions from the heterologous host Aspergillus nidulans, mature GO is formed by an autocatalytic process. Structural comparison of pro GO with mature GO reveals overall structural similarity, but with some regions showing significant local differences in main-chain position. Some side chains of the active-site residues differ significantly from their positions in the mature enzyme. These structural effects of the prosequence suggest that it may act as an intramolecular chaperone to provide an open active-site structure conducive to copper binding and chemistry associated with cofactor formation. The prosequence is not mandatory for processing, as a recombinant form of GO lacking this region and purified under copper-free conditions can also be processed in an autocatalytic copper- and oxygen-dependent manner.

Dissecting the mechanism of oxygen trafficking in a metalloenzyme.

A key question in the biological activation of oxygen is how the protein matrix regulates the delivery of oxygen to its site of activation. We are using Escherichia coli copper amine oxidase as a model system to investigate the roles played by both local active site residues as well as long range interactions in this process. We have generated active site mutants, as well as mutants in the putative oxygen delivery channel and characterised their affects on enzyme structure and catalysis.

The stacking tryptophan of galactose oxidase: a second-coordination sphere residue that has profound effects on tyrosyl radical behavior and enzyme catalysis.

The function of the stacking tryptophan, W290, a second-coordination sphere residue in galactose oxidase, has been investigated via steady-state kinetics measurements, absorption, CD and EPR spectroscopy, and X-ray crystallography of the W290F, W290G, and W290H variants. Enzymatic turnover is significantly slower in the W290 variants. The Km for D-galactose for W290H is similar to that of the wild type, whereas the Km is greatly elevated in W290G and W290F, suggesting a role for W290 in substrate binding and/or positioning via the NH group of the indole ring. Hydrogen bonding between W290 and azide in the wild type-azide crystal structure are consistent with this function. W290 modulates the properties and reactivity of the redox-active tyrosine radical; the Y272 tyrosyl radicals in both the W290G and W290H variants have elevated redox potentials and are highly unstable compared to the radical in W290F, which has properties similar to those of the wild-type tyrosyl radical. W290 restricts the accessibility of the Y272 radical site to solvent. Crystal structures show that Y272 is significantly more solvent exposed in the W290G variant but that W290F limits solvent access comparable to the wild-type indole side chain. Spectroscopic studies indicate that the Cu(II) ground states in the semireduced W290 variants are very similar to that of the wild-type protein. In addition, the electronic structures of W290X-azide complexes are also closely similar to the wild-type electronic structure. Azide binding and azide-mediated proton uptake by Y495 are perturbed in the variants, indicating that tryptophan also modulates the function of the catalytic base (Y495) in the wild-type enzyme. Thus, W290 plays multiple critical roles in enzyme catalysis, affecting substrate binding, the tyrosyl radical redox potential and stability, and the axial tyrosine function.

Role of the interactions between the active site base and the substrate Schiff base in amine oxidase catalysis. Evidence from structural and spectroscopic studies of the 2-hydrazinopyridine adduct of Escherichia coli amine oxidase.

2-Hydrazinopyridine (2HP) is an irreversible inhibitor of copper amine oxidases (CAOs). 2HP reacts directly at the C5 position of the TPQ cofactor, yielding an intense chromophore with lambda(max) approximately 430 nm (adduct I) in Escherichia coli amine oxidase (ECAO). The adduct I form of wild type (WT-ECAO) was assigned as a hydrazone on the basis of the X-ray crystal structure. The hydrazone adduct appears to be stabilized by two key hydrogen-bonding interactions between the TPQ-2HP moiety and two active site residues: the catalytic base (D383) and the conserved tyrosine residue (Y369). In this work, we have synthesized a model compound (2) for adduct I from the reaction of a TPQ model compound (1) and 2HP. NMR spectroscopy and X-ray crystallography show that 2 exists predominantly as the azo form (lambda(max) at 414 nm). Comparison of the UV-vis and resonance Raman spectra of 2 with adduct I in WT, D383E, D383N, and Y369F forms of ECAO revealed that adduct I in WT and D383N is a tautomeric mixture where the hydrazone form is favored. In D383E adduct I, the equilibrium is further shifted in favor of the hydrazone form. UV-vis spectroscopic pH titrations of adduct I in WT, D383N, D383E, and 2 confirmed that D383 in WT adduct I is protonated at pH 7 and stabilizes the hydrazone tautomer by a short hydrogen-bonding interaction. The deprotonation of D383 (pKa approximately 9.7) in adduct I resulted in conversion of adduct I to the azo tautomer with a blue shift of the lambda(max) to 420 nm, close to that of 2. In contrast, adduct I in D383N and D383E is stable and did not show any pH-dependent spectral changes. In Y369F, adduct I was not stable and gradually converted into a new species with lambda(max) at approximately 530 nm (adduct II). A detailed mechanism for the adduct I formation in WT has been proposed that is consistent with the mechanism proposed for the oxidation of substrate by CAOs but addresses some key differences in the active site chemistry of hydrazine inhibitors and substrate amines.

Active site rearrangement of the 2-hydrazinopyridine adduct in Escherichia coli amine oxidase to an azo copper(II) chelate form: a key role for tyrosine 369 in controlling the mobility of the TPQ-2HP adduct.

Adduct I (lambda(max) at approximately 430 nm) formed in the reaction of 2-hydrazinopyridine (2HP) and the TPQ cofactor of wild-type Escherichia coli copper amine oxidase (WT-ECAO) is stable at neutral pH, 25 degrees C, but slowly converts to another spectroscopically distinct species with a lambda(max) at approximately 530 nm (adduct II) at pH 9.1. The conversion was accelerated either by incubation of the reaction mixture at 60 degrees C or by increasing the pH (>13). The active site base mutant forms of ECAO (D383N and D383E) showed spectral changes similar to WT when incubated at 60 degrees C. By contrast, in the Y369F mutant adduct I was not stable at pH 7, 25 degrees C, and gradually converted to adduct II, and this rate of conversion was faster at pH 9. To identify the nature of adduct II, we have studied the effects of pH and divalent cations on the UV-vis and resonance Raman spectroscopic properties of the model compound of adduct I (2). Strikingly, it was found that addition of Cu2+ to 2 at pH 7 gave a product (3) that exhibited almost identical spectroscopic signatures to adduct II. The X-ray crystal structure of 3 shows that it is the copper-coordinated form of 2, where the +2 charge of copper is neutralized by a double deprotonation of 2. These results led to the proposal that adduct II in the enzyme is TPQ-2HP that has migrated onto the active site Cu2+. The X-ray crystal structure of Y369F adduct II confirmed this assignment. Resonance Raman and EPR spectroscopy showed that adduct II in WT-ECAO is identical to that seen in Y369F. This study clearly demonstrates that the hydrogen-bonding interaction between O4 of TPQ and the conserved Tyr (Y369) is important in controlling the position and orientation of TPQ in the catalytic cycle, including optimal orientation for reactivity with substrate amines.

Probing the catalytic mechanism of Escherichia coli amine oxidase using mutational variants and a reversible inhibitor as a substrate analogue.

Copper amine oxidases are homodimeric enzymes containing one Cu(2+) ion and one 2,4,5-trihydroxyphenylalanine quinone (TPQ) per monomer. Previous studies with the copper amine oxidase from Escherichia coli (ECAO) have elucidated the structure of the active site and established the importance in catalysis of an active-site base, Asp-383. To explore the early interactions of substrate with enzyme, we have used tranylcypromine (TCP), a fully reversible competitive inhibitor, with wild-type ECAO and with the active-site base variants D383E and D383N. The formation of an adduct, analogous to the substrate Schiff base, between TCP and the TPQ cofactor in the active site of wild-type ECAO and in the D383E and D383N variants has been investigated over the pH range 5.5-9.4. For the wild-type enzyme, the plot of the binding constant for adduct formation (K(b)) against pH is bell-shaped, indicating two pK(a)s of 5.8 and approximately 8, consistent with the preferred reaction partners being the unprotonated active-site base and the protonated TCP. For the D383N variant, the reaction pathway involving unprotonated base and protonated TCP cannot occur, and binding must follow a less favoured pathway with unprotonated TCP as reactant. Surprisingly, for the D383E variant, the K(b) versus pH behaviour is qualitatively similar to that of D383N, supporting a reaction pathway involving unprotonated TCP. The TCP binding data are consistent with substrate binding data for the wild type and the D383E variant using steady-state kinetics. The results provide strong support for a protonated amine being the preferred substrate for the wild-type enzyme, and emphasize the importance of the active-site base, Asp-383, in the primary binding event.

Enhanced fructose oxidase activity in a galactose oxidase variant.

Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previously, computer-aided docking experiments have been used to develop a plausible model for interactions between GO and the D-galactose substrate. Residues implicated in such interactions include Arg330, Gln406, Phe464, Phe194 and Trp290. In the present study we describe an improved expression system for recombinant GO in the methylotrophic yeast Pichia pastoris. We use this system to express variant proteins mutated at Arg330 and Phe464 to explore the substrate binding model. We also demonstrate that the Arg330 variants display greater fructose oxidase activity than does wild-type GO.