The copper-topaquinone-phenylhydrazine-adduct geometry in Escherichia coli amine oxidase derivatized with phenylhydrazines substituted with 19F-NMR relaxation measurements.

The copper quinoprotein amine oxidase from Escherichia coli was derivatized with phenylhydrazine, substituted with a F3C group at the ortho, meta, or para position. The derivatization of the topaquinone cofactor was verified by ultraviolet/visible spectroscopy. The reduction (with dithionite) of Cu(II) to Cu(I), which was required to obtain reference samples, was verified by EPR spectroscopy. 19F-NMR spectroscopy was carried out on the derivatized enzyme forms, and the spectra showed the line-broadening effect due to the paramagnetic Cu(II). The distance between the Cu and the mean of the three F positions in the F3C groups was calculated by means of the Solomon-Bloembergen equation for the distance-dependent contribution of CU(II) to the transversal-relaxation time of the F resonance. Assuming that the F3C-phenylhydrazines in the enzyme are always aligned towards the Cu in the same way, four configurations can be envisaged that should be taken into account to determine the topology of the two cofactors. Based on these configurations, two spatial positions were found where the calculated distances triangulated, each of these positions having a symmetry-related counterpart above or below the topaquinone-phenylhydrazine plane. If it is assumed that the geometric positions of the phenylhydrazine and topaquinone moieties in the adduct remain the same in the derivatized enzymes, a number of minimum distances between the Cu and certain atoms in the topaquinone moiety of the adduct can be calculated (1.52 +/- 0.06 nm from the C2-O, 1.30 +/- 0.04 nm from the C4-O, and 1.26 +/- 0.04 nm from the C5-N). However, one of the configurations yields very similar distances between the Cu and the C2-O and C4-O. Therefore, no conclusions can be made with regard to which OH group is closest to the Cu. By application of the same approach to the 19F-NMR data obtained for porcine-plasma marine oxidase [Williams, T J. & Falk, M.C.(1986) J. Biol. Chem. 261, 15949- 15954] we observed substantial differences between the topologies of the cofactors in the two enzymes. Possible reasons for this are discussed.

Cloning of the maoA gene that encodes aromatic amine oxidase of Escherichia coli W3350 and characterization of the overexpressed enzyme.

The mao operon of Escherichia coli W3350, which comprises the genes maoC and maoA, was cloned and appeared to be similar to that of Klebsiella aerogenes [Sugino, H., Sasaki, M., Azakami, H., Yamashita, M. & Murooka, Y. (1992) J. Bacteriol. 174, 2485-2492]. The gene that encodes aromatic amine oxidase (maoA) was isolated, sequenced, and expressed in E. coli TG2. The purified enzyme exhibited properties characteristic of a copper/topaquinone(TPQ)-containing amine oxidase with respect to the optical absorption and EPR spectra, the size of the subunits, and the optical absorption spectra obtained upon derivatization with hydrazines. However, high-resolution anion-exchange chromatography revealed that the preparation was heterogeneous. The enzyme preparation appeared to consist of at least four enzyme species with different specific activities, A474nm/A340nm ratios and TPQ/subunit ratios. Since the overall properties of the overexpressed enzyme and the authentic enzyme were similar and the separated enzyme species had identical N-terminal amino acid sequences, the heterogeneity does not seem to be caused by improper expression of the gene in the recombinant strain but by factors that interfere with the processing of the specific tyrosine in the precursor enzyme to functional TPQ. Although other causes cannot be excluded, the spectral data and TPQ/subunit ratios reported in the literature for other amine oxidases suggest that suboptimal synthesis of functional TPQ also occurs in other organisms.

Intermediates in the catalytic cycle of copper-quinoprotein amine oxidase from Escherichia coli.

Investigations on the reduction of copper quinoprotein amine oxidases (EC 1.4.3.6) by substrate indicate that the nature of the reduced enzyme species formed varies, as judged from the spectroscopic data reported in the literature for different enzymes and substrates. The availability of substantial amounts of overproduced, homogeneous Escherichia coli amine oxidase (ECAO) enabled us to investigate this aspect with a number of different approaches: quantitative titration of enzyme with substrate, stopped-flow kinetic spectrophotometry (anaerobic and semianaerobic), EPR spectroscopy of stable intermediates in the catalytic cycle, and conversions with H2O2 as the oxidant. Reduction of ECAO by a variety of substrates led to spectra (UV/Vis, EPR) identical to those that have been ascribed to the semiquinone form of the topaquinone cofactor. The extent of semiquinone formation was enhanced in the presence of KCN, but the properties of the artificially induced semiquinone were different from those of the spontaneously induced one, as shown by the spectroscopic data and the reactivity toward O2 and H2O2. On titrating ECAO at high concentrations with substrate, evidence was obtained that disproportionation takes place of the semiquinone formed, the reaction most probably proceeding via intermolecular electron transfer, leading to a topaquinone- and Cu1+-containing enzyme species that is able to perform substrate conversion. The latter, as well as OH*, is probably also formed when H2O2 replaces O2 as oxidant, explaining why substrate conversion with concomitant enzyme inactivation occurs under this condition. Formation of the semiquinone was always preceded by that of a hitherto unknown species with an absorbance maximum at 400 nm. The structure proposed for this species is a protonated form of the aminoquinol cofactor, the Zwitter ionic structure being stabilized by amino acid residues in the active site having opposite charges. Based on the properties observed and the moment of appearance during conversions, a proposal is made for the sequence in which the three reduced enzyme species convert into each other.

Identification of topaquinone, as illustrated for pig kidney diamine oxidase and Escherichia coli amine oxidase.

Pig kidney diamine oxidase was purified to homogeneity. The reaction product of the cofactor with p-nitrophenylhydrazine (pNPH) was liberated with pronase treatment and purified. 1H NMR, uv/vis, and electrospray tandem mass spectroscopy revealed it to be a dipeptide with the sequence topaquinone-pNPH and aspartate. No heterogeneity was observed, indicating that no intramolecular cyclization of the quinone moiety occurs in the time span of the isolation and of the measurements. Similar results were obtained with the more widely applicable reagent, phenylhydrazine, and using the aromatic amine oxidase from Escherichia coli. From the amount and ease with which the dipeptide could be isolated, the procedure used here is more convenient than the existing one for the identification of protein-integrated quinone cofactors.

Characterization of the topa quinone cofactor in amine oxidase from Escherichia coli by resonance Raman spectroscopy.

The aromatic amine oxidase from Escherichia coli (ECAO) utilizes Cu(II) and 2,4,5-trihydroxyphenylalanine quinone (TPQ) as cofactors in enzymatic catalysis. The TPQ cofactor is clearly identified by a set of characteristic vibrational modes between 1200 and 1700 cm-1 in the resonance Raman (RR) spectrum of the native enzyme. This is the first report of a RR spectrum for an underivatized TPQ cofactor in an enzyme, showing that it is possible to study changes in the cofactor during the natural reaction cycle. The RR spectrum of ECAO closely matches that of a 2-hydroxy-1,4-benzoquinone model compound, particularly in the deprotonated state in aqueous solution. The principal in-phase C = O symmetric stretching mode of the quinone occurs at 1681 cm-1 in ECAO and at 1666 cm-1 in the model compound and, in both cases, undergoes a downshift of approximately 25 cm-1 upon substitution of one of the carbonyl oxygens with 18O. The overall similarity of the 18O and D shifts in their RR spectra shows that the TPQ cofactor and model compound have the same structure and reactivity, with oxygen exchange occurring at the carbonyl adjacent to the hydroxyl group. Substrate reduction of ECAO under anaerobic conditions leads to a stable semiquinone (lambda max at 442 and 468 nm) with a RR spectrum characteristic of an amine-substituted semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)