Michele A. McGuirl

Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A resolution.

BACKGROUND Copper-containing amine oxidases catalyze the oxidative deamination of primary amines to aldehydes, in a reaction that requires free radicals. These enzymes are important in many biological processes, including cell differentiation and growth, would healing, detoxification and signalling. The catalytic reaction requires a redox cofactor, topa quinone (TPQ), which is derived by post-translational modification of an invariant tyrosine residue. Both the biogenesis of the TPQ cofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site. The crystal structure of a prokaryotic copper amine oxidase from E. coli (ECAO) has recently been reported. RESULTS The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 A resolution. The crystallographic phases were derived from a single phosphotungstic acid derivative. The positions of the tungsten atoms in the W12 clusters were obtained by molecular replacement using E. coli amine oxidase as a search model. The methodology avoided bias from the search model, and provides an essentially independent view of a eukaryotic amine oxidase. The PSAO molecule is a homodimer; each subunit has three domains. The active site of each subunit lies near an edge of the beta-sandwich of the largest domain, but is not accessible from the solvent. The essential active-site copper atom is coordinated by three histidine side chains and two water molecules in an approximately square-pyramidal arrangement. All the atoms of the TPQ cofactor are unambiguously defined, the shortest distance to the copper atom being approximately 6 A. CONCLUSIONS There is considerable structural homology between PSAO and ECAO. A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic grouf to rotate about the Cbeta-Cgamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom. Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.

Copper-containing oxidases.

Major advances have been made during 1997 and 1998 toward understanding the structure/function relationships of the active sites in copper-containing oxidases. Central to this progress has been the elucidation of crystal structures for many of these enzymes. For example, studies of the mechanisms of biogenesis and/or catalysis of amine oxidase and galactose oxidase have been both stimulated and directed by the availability of structures for these proteins. Similarly, it is anticipated that the recently published crystal structures of peptidylglycine alpha-hydroxylating monooxygenase and laccase will contribute greatly toward understanding the roles of copper in these two proteins.

Purification and characterization of pea seedling amine oxidase for crystallization studies.

Pea (Pisum sativum L.) seedling amine oxidase (EC 1.4.3.6) is the first amine oxidase to be crystallized that diffracts to atomic resolution (2.5 A). Extensive modifications of a published purification procedure were necessary to obtain protein that would give diffraction-quality crystals. Here we report the improved purification and also use this high-purity protein to reexamine some fundamental characteristics of pea seedling amine oxidase. The extinction coefficient at 280 nm ([epsilon]1%280) and the molecular mass of the protein are investigated by a variety of techniques, yielding [epsilon]1%280 = 20 cm-1 and a mass of 150 [plus or minus] 6 kD. In addition, the stoichiometry of the metal and organic cofactors, Cu(II) and 6-hydroxy dopa (Topa) quinone, respectively, is examined. The ratio of Cu(II):Topa:protein monomer is found to be 1:1:1.

The generation of an organic free radical in substrate-reduced pig kidney diamine oxidase-cyanide

When the cyanide complex of the copper protein, pig kidney diamine oxidase, is reduced anaerobically by cadaverine (1,5‐diaminopentane), the broad, 480 nm absorption band characteristic of the resting enzyme is bleached and a new absorption spectrum with features at 457, 429, 403 (shoulder), 360 (shoulder) and 332 nm appears. Concomitantly, the EPR spectrum of the enzyme Cu(II)‐CN complex decreases in intensity and a new signal is observed that is attributable to an organic free radical. The g values and hyperfine splittings are similar to those previously assigned to a free radical observed when the cyanide complex of lentil seedling diamine oxidase is reacted with the substrate p‐dimethylaminomethylbenzylamine [(1984) FEBS Lett. 176, 378–380]. The optical absorption and EPR spectra of the organic radical observed in both proteins are consistent with the same semiquinone‐type structure, as expected if pyrroloquinolinequinone (PQQ) is the bound cofactor found in both enzymes.

The nos (nitrous oxide reductase) gene cluster from the soil bacterium Achromobacter cycloclastes: Cloning, sequence analysis, and expression

The nitrous oxide (N2O) reductase (nos) gene cluster from Achromobacter cycloclastes has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ (structural N2O reductase gene), nosD, nosF, nosY, nosL, and nosX are detected, indicating a genetic organization similar to that of Rhizobium meliloti. To aid homology studies, nosR from R. meliloti has also been sequenced. Comparison of the deduced amino acid sequences with corresponding sequences from other organisms has also allowed structural and functional inferences to be made. The heterologous expression of NosD, NosZ (N2O reductase), and NosL is also reported. A model of the CuA site in N2O reductase, based on the crystal structure of this site in bovine heart cytochrome c oxidase, is presented. The model suggests that a His residue of the CuA domain may be a ligand to the catalytic CuZ site. In addition, the origin of the spectroscopically-observed Cys coordination to CuZ is discussed in terms of the sequence alignment of seven N2O reductases.

Use of Microcalorimetry To Determine the Costs and Benefits to Pseudomonas putida Strain KT2440 of Harboring Cadmium Efflux Genes

ABSTRACT A novel microcalorimetric approach was used to analyze the responses of a metal-tolerant soil bacterium (Pseudomonas putida strain KT2440) to metal resistance gene deletions in cadmium-amended media. As hypothesized, under cadmium stress, the wild-type strain benefited from the resistance genes by entering the exponential growth phase earlier than two knockout strains. In the absence of cadmium, strain KT1, carrying a deletion in the main component (czcA1) of a Cd/Zn chemiosmotic efflux transporter (CzcCBA1), grew more efficiently than the wild type and released ∼700 kJ (per mole of biomass carbon) less heat than the wild-type strain, showing the energetic cost of maintaining CzcCBA1 in the absence of cadmium. A second mutant strain (KT4) carrying a different gene deletion, ΔcadA2, which encodes the main Cd/Pb efflux transporter (a P-type ATPase), did not survive beyond moderate cadmium concentrations and exhibited a decreased growth yield in the absence of cadmium. Therefore, CadA2 plays an essential role in cadmium resistance and perhaps serves an additional function. The results of this study provide direct evidence that heavy metal cation efflux mechanisms facilitate shorter lag phases in the presence of metals and that the maintenance and expression of tolerance genes carry quantifiable energetic costs and benefits.

Hydroxylase Activity of Met471Cys Tyramine β-Monooxygenase

A series of mutations was targeted at the methionine residue, Met471, coordinating the Cu(M) site of tyramine beta-monooxygenase (TbetaM). The methionine ligand at Cu(M) is believed to be key to dioxygen activation and the hydroxylation chemistry of the copper monooxygenases. The reactivity and copper binding properties of three TbetaM mutants, Met471Asp, Met471Cys, and Met471His, were examined. All three mutants show similar metal binding affinities to wild type TbetaM in the oxidized enzyme forms. EPR spectroscopy suggests that the Cu(II) coordination geometry is identical to that of the WT enzyme. However, substrate hydroxylation was observed for the reaction of tyramine solely with Met471Cys TbetaM. Met471Cys TbetaM provides the first example of an active mutant directed at the Cu(M) site of this class of hydroxylases. The reactivity and altered kinetics of the Met471Cys mutant further highlight the central role of the methionine residue in the enzyme mechanism. The sole ability of the cysteine residue to support activity among the series of alternate amino acids investigated is relevant to theoretical and biomimetic investigations of dioxygen activation at mononuclear copper centers.

Purification and active-site characterization of equine plasma amine oxidase.

An improved purification scheme for an amine oxidase from equine plasma (EPAO), a nonruminant source, is described and the proteins active-site is characterized. EPAO is dimeric and contains one Type-2 Cu(II) ion per monomer. The EPAO Cu(II) site is spectroscopically very similar to the Cu(II) sites in other amine oxidases. Unlike the extensively investigated nonruminant amine oxidase from porcine plasma, EPAO does not display half-of-the-sites reactivity; titrations with p-nitrophenylhydrazine and phenylhydrazine indicate two active cofactors per dimer. This cofactor is determined to be the same as that of other copper-containing amine oxidases, 6-hydroxydopa quinone (topa quinone). Upon anaerobic reduction with substrate at ambient temperature, the EPR spectrum of EPAO exhibits a sharp signal at g congruent to 2, attributable to the topa semiquinone. Equine plasma amine oxidase possesses novel in vitro substrate specificity; while other mammalian amine oxidases oxidize norepinephrine only slowly or not at all, EPAO displays significant activity toward this biogenic amine.

Cyanide as a copper-directed inhibitor of amine oxidases: implications for the mechanism of amine oxidation

Abstract The interactions of five copper-containing amine oxidases with substrates and substrate analogues in the presence of the copper ligands cyanide, azide, chloride, and 1,10-phenanthroline have been investigated. While cyanide inhibits, to varying degrees, the reaction of phenylhydrazine with porcine kidney amine oxidase (PKAO), porcine plasma amine oxidase (PPAO), bovine plasma amine oxidase (BPAO), and pea seedling amine oxidase (PSAO), it enhances the reaction of Arthrobacter P1 amine oxidase (APAO) with this substrate analogue. This indicates that cyanide exerts an indirect effect on topa quinone (TPQ) reactivity via coordination to Cu(II) rather than through cyanohydrin formation at the TPQ organic cofactor. Moreover, cyanide binding to the mechanistically relevant TPQ• semiquinone form of substrate-reduced APAO and PSAO was not observable by EPR or resonance Raman spectroscopy. Hence, cyanide most likely inhibits enzyme reoxidation by binding to Cu(I) and trapping the Cu(I)-TPQ• form of amine oxidases, and thus preventing the reaction of O2 with Cu(I). In contrast, ligands such as azide, chloride, and 1,10-phenanthroline, which preferentially bind to Cu(II), inhibit by stabilizing the aminoquinol Cu(II)-TPQred redox state, which is in equilibrium with Cu(I)-TPQ•.

Spectroscopic studies of pig kidney diamine oxidase-anion complexes

Abstract Complexes of pig kidney diamine oxidase with azide, thiocyanate, and cyanide have been characterized by EPR and circular dichroism spectroscopy. Cu(II) d-d bands are observed in the CD spectrum of the resting enzyme at ≈800 nm (12 500 cm −1 ) and 575 nm (17 400 cm −1 ). Anion binding produces characteristic changes in the CD spectra. N 3 − /SCN − → Cu(II) ligand-to-metal charge-transfer transitions are located at 390 nm (25 600 cm −1 ) and 365 nm (27 400 cm −1 ), respectively. In addition, an intense new band grew in at ≈500 nm (20 000 cm −1 ) when azide or thiocyanate were added, which may be assigned as a Cu(II) electronic transition that gains rotational strength in the anion complex. EPR spectra established that the Cu(II)-anion complexes are tetragonal; however, the magnitudes of the anion-induced shifts in the EPR parameters were consistent with substantial perturbations of the Cu(II) electronic ground state in the thiocyanate and cyanide complexes. Prominent superhyperfine splitting was apparent in the EPR spectra of the diamine oxidase complexes with thiocyanate and cyanide. The superhyperfine structure is (at least) partially attributable to endogenous Cu(II) ligands, probably from imidazole.