Amino acid sequences copper oxidases

Laccase, 36 318, 329, 40 122 see also Blue copper oxidases amino-acid sequences, 40 141 anaerobic reduction, 40 158-160 biological function, 40 124 electrochemistry, 36 360 fungal, 40 145-152 evolution, 40 153-154 inhibition, 40 162 kinetic properties, 40 157-162 molecular and spectroscopic properties, 40 125-126... 

The similarity matrix calculated in Messerschmidt and Huber (202) indicates clearly the six-domain structure of ceruloplasmin and three-domain structures for laccase and ascorbate oxidase. The internal triplication within the ceruloplasmin amino-acid sequence is reflected by values of about 60% difference. Comparison of both the N-terminal domains and the C-terminal domains of the blue oxidases indicates, respectively, a relationship that is closer and relevant values for percent difference that are significantly lower than those for other comparisons. This might reflect the requirements for the trinuclear copper site. The lowest values of about 70 to 73% difference are observed for both N-terminal and C-terminal domains of laccase and ascorbate oxidase, showing that the two oxidases are more closely related to ceruloplasmin than either of them. 

Redox potentials for the different copper centers in the blue oxidases have been determined for all members of the group but in each case only for a limited number of species. The available data are summarized in Table VI 120, 121). The redox potentials for the type-1 copper of tree laccase and ascorbate oxidase are in the range of 330-400 mV and comparable to the values determined for the small blue copper proteins plastocyanin, azurin, and cucumber basic protein (for redox potentials of small blue copper proteins, see the review of Sykes 122)). The high potential for the fungal Polyporus laccase is probably due to a leucine or phenylalanine residue at the fourth coordination position, which has been observed in the amino-acid sequences of fungal laccases from other species (see Table IV and Section V.B). Two different redox potentials for the type-1 copper were observed for human ceruloplasmin 105). The 490-mV potential can be assigned to the two type-1 copper sites with methionine ligand and the 580-mV potential to the type-1 center with the isosteric leucine at this position (see Section V.B). The... 

This progress is mainly due to the determination of the amino-acid sequences for all members of this group and the X-ray crystal structure of ascorbate oxidase. The three-dimensional structure of ascorbate oxidase showed the nature and spatial arrangement of the copper centers and the three-domain structure. However, modern spectroscopic techniques (e.g., low-temperature MCD and ENDOR) made invaluable contributions as well. 

A structurally based amino-acid sequence alignment strongly suggests a three-domain structure for laccase, closely related to ascorbate oxidase, and a six-domain structure for ceruloplasmin. These domains demonstrate homology with the small blue copper proteins. The relationship suggests that laccase, like ascorbate oxidase, has a mononuclear blue copper in domain 3 and a trinuclear copper between domain 1 and domain 3, and ceruloplasmin has mononuclear copper ions in domains 2, 4, and 6 and a trinuclear copper between domain 1 and domain 6. 

As shown in Sect. 3, the structure of type 2 copper centers is less uniform than in types 1 and 3. Type 2 copper proteins can, furthermore, neither be phylo-genetically grouped by comparison of amino acid sequences nor by a common tertiary structure. The class of type 2 copper proteins contains oxidases, mono-and di-oxygenases, as well as enzymes, which participate in metabolizing reactive species. 

Galactose oxidase has a unique tertiary structure for a copper protein, comparable with that of the non-copper protein methylamine dehydrogenase. Comparisons of the amino acid sequences [157] show, however, that the enzymes are not phylogenetically related. The tertiary structures developed separately [30]. 

A phylogenetic relationship may be assumed for amine oxidase and diamine oxidase which shows distinct sequence homologies in their C-terminal regions [128]. Lysyl oxidase is not related to the other amine oxidases as it does not share any homology in structure or amino acid sequence [128] with the other amine oxidases. Non-copper enzymes with similar structures or sequences have not yet been found for any of the three enzymes. 

The three-domain structure of ascorbate oxidase is the result of two duplications of the original domain, which was most similar to the small blue proteins and the C-terminal domain (domain 3) of the blue oxidases [71]. These duplications did not, however, lead to a protein with three type 1 copper centers arranged in a row. The original type 1 copper center has only been retained in domain 3, where it has still kept its function of transferring electrons to the active center [36]. The extensive alterations in the amino acid sequence of domain 2... 

Based on its amino acid sequence and three-dimensional structure, 2,3QP can be classified within the cupin superfamiliy. This superfamiliy includes functionally diverse proteins that are found in archaea, eubacteria, and eukaryota. Structural information shows that they contain a motif of six antiparallel /3-strands located within a conserved /3-barrel structure. The structure of gemin, which is a 16kDa Mn-containing oxalate oxidase, can be superimposed on the N-terminal domain of 2,3QD with an rms deviation of 1.8 A for 91 Ca atoms. One can see in the superposition that the Mn site of germin formed by three histidines, a glutamate, and two water molecules matches with the copper site of 2,3QD. 

The heme-copper oxidase superfamily is defined hy two criteria (1) a high degree of amino acid sequence similarity within the largest suhunit (suhunit I) and (2) a unique bimetallic active site, consisting of a heme and a closely associated copper atom (see Figure 8), where dioxygen is reduced to water. There are two main branches of the superfamily, which have distinct substrate specificities the mitochondrial respiratory oxidases use cytochrome r as a substrate and, hence, are called cytochrome c oxidases (COX). Bacteria, unlike most mitochondria, contain multiple respiratory oxidases. Many of the prokaryotic respiratory oxidases use membrane-bound quinol (ubiquinol or menaquinol) as a substrate rather than cytochrome c. A number of these quinol oxidases have been shown to be members of the heme-copper oxidase superfamily and to pump protons as efficiently as COXs. " ... 

With few exceptions [12,27], copper amine oxidases are homodimers in the native conformation. The molecular mass of the single subunit lies in the range 70-100 kDa. Most of the eukaryotic copper amine oxidases are glycoproteins. Their isoelectric point is usually slightly below pi 7.0, but some enzymes have pi > 7.0 such as pea seedling amine oxidase [12]. The amino acid composition is known for many amine oxidases and in the last decade, the complete amino acid sequence has been determined for a number of them. Some enzymes have been prepared in a crystalline form [12], four of them have been analyzed by X-ray diffraction with complete resolution of their three dimensional structure including detailed spatial conformation of the active site [28-31]. 

Fig. (I). Topa quinone as a part of the cofactor consensus amino acid sequence in the active site of copper amine oxidases [4],...

Table 1. Alignment of amino acid sequences of several copper amine oxidase around the position of topa quinone. The sequences were obtained by translation the corresponding cDNAs except for the enzymes from porcine kidney and porcine serum and the benzylamine oxidase from Hansenula polymorpha where they were determined by automated Edman degradation of peptides. Homologous consensus sequence around the cofactor is underlined, the tyrosyl precursor of topa quinone is shown as y.

Bilirubin oxidase, purified and characterized from the fungus Myrothecium verrucaria, has a strong amino acid sequence homology with the other blue copper oxidases and point mutations studies on the supposed copper binding residues have confirmed its identity as a member of the enzymatic family.In vitro, BO couples 02-reduction to the oxidation of bilirubin to biliverdin. This catalytic activity has found clinical application in the diagnosis and treatment of jaundice and hyperbilirubinemia. 

Ceruloplasmin is a blue a-2 glycoprotein of 132 kD. This plasma protein is responsible for the binding of 90 to 95% of the blood plasma copper in vertebrates. In addition to its primary role in copper transport and homeostasis, it possesses a number of additional enzymatic activities (see ref. 179 for a recent review). The complete amino acid sequence of human ceruloplasmin has been determined (42), establishing that this large multicopper oxidase is synthesized as a single chain polypeptide, containing 1046 amino acid residues. 

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