Ascorbate oxidase amino-acid sequences

This chapter will concentrate mainly on structural and functional aspects of these enzymes with the major emphasis on ascorbate oxidase and laccase. Significant progress has been achieved in the last 10 years the determination of amino acid sequences of all three enzymes, each from several sources, and the X-ray structure of ascorbate oxidase. The new information forms the basis of a much deeper understanding of the function of the enzymes as will be demonstrated in this chapter. 

Knowledge of the three-dimensional structure of ascorbate oxidase and of amino-acid sequences of all members of the blue oxidases made it possible to carry out a structurally based amino-acid sequence alignment 101) involving N. crassa laccase, cucumber ascorbate oxidase, and human ceruloplasmin. The structural basis of this alignment was... 

Structurally Based Amino-Acid Sequence Alignment of Fungal Laccases, Ascorbate Oxidases, and Related Proteins "... 

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. 

Comparison of amino acid sequences shows that small blue proteins and blue oxidases have developed from a common precursor [71]. Whereas the former are found in all three kingdoms, blue oxidases are only found in fungi, plants, and animals. In contrast to the small blue proteins, which have chain lengths of approximately 100 amino acid residues, blue oxidases have chain lengths of ca. 540 (laccase, ascorbate oxidase) and 1046 amino acid residues (ceruloplasmin) [71]. The following mechanisms were applied to increase the size of these proteins [71] ... 

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... 

The amino acid sequence predicted from pTOM13 shows a significant similarity to flavanone-3-hydroxylase, which requires Fe and 2-oxoglutarate [96]. Ververidis and John [98] applied the conditions to stabilise flavanone-3-hydroxylase to extract EFE from melon fruits and found that authentic EFE activity was recovered in a soluble fraction in the presence of Fe and ascorbate. This finding allowed other researchers to extract authentic EFE from apple [100,136,137] and avocado [99]. The molecular size of extracted ACC oxidase was about 35 kDa in both SDS-PAGE and gel filtration. This result indicates that ACC oxidase is a monomeric enzyme, and isolated cDNA clones represented by pTOM13 encode the entire ACC oxidase. 

Laccase and ascorbate oxidase consist of three, ceruloplasmin of six, homologous domains containing approximately 170 amino acids each. Sequence comparisons were conducted for the individual domains of blue oxidases to clarify the development of the domain structure. The phylogenetic relationship of the individual domains of laccase and ascorbate oxidase on the one hand,... 

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