Hemocyanin molluscan

Table 5.2 contains data about selected copper enzymes from the references noted. It should be understood that enzymes from different sources—that is, azurin from Alcaligenes denitrificans versus Pseudomonas aeruginosa, fungal versus tree laccase, or arthropodan versus molluscan hemocyanin—will differ from each other to various degrees. Azurins have similar tertiary structures—in contrast to arthropodan and molluscan hemocyanins, whose tertiary and quaternary structures show large deviations. Most copper enzymes contain one type of copper center, but laccase, ascorbate oxidase, and ceruloplasmin contain Type I, Type II, and Type III centers. For a more complete and specific listing of copper enzyme properties, see, for instance, the review article by Solomon et al.4... 

Figure 7 of reference 11 compares similarities between Limulus and Octopus hemocyanins by illustrating the overlap of 30 amino acids about the CuB region. The authors find that all residues within 5.0 A of the Cu-Cu midpoint are conserved between arthropodan and molluscan hemocyanins and occupy analogous positions except for one (see the following paragraph). The three histidines about CuB overlap almost exactly in orientation and are less than 1 A apart. The histidines... 

The tropomyosins of mite and insect species show some sequence identity (63-65%) with snail tropomyosin and share similar epitopes (EFSA, 2006 Fig. 4.1). Still, tropomyosin appears to play a minor role in the crossreactivity of dust mites and snails (Asturias et ah, 2002 Guilloux et ah, 1998 Van Ree et ah, 1996a). Other non-tropomyosin allergens are likely to be involved including Der p 4 (amylase), Der p 5, Der p 7, and hemocyanin (Martins et ah, 2005 Mistrello et ah, 1992 Van Ree et ah, 1996). While snail is the main molluscan shellfish species involved in cross-reactions with dust mites, some patients allergic to dust mites and snails are also sensitized to mussels (DeMaat-Bleeker et ah, 1995 Van Ree et ah, 1996b). In their study of 70 patients sensitized to molluscan shellfish, Wu and Williams (2004) noted that 90% were also sensitized to dust mites. However, the clinical significance of this sensitization was not documented. 

Molluscan hemocyanins. Two FUs from moUuscan hemocyanins were resolved, the oxy-form of O. dofleini He FU g (Figure 5b) and the deoxy-form of R. thomasiana He (Figure 5c ). Each FU consists of two domains. The N-terminal domain II carries the active site with a four alpha-helix bundle folding motif with two copper atoms. The C-terminal domain III replaces topologically the domain I in arthropod subunits and looks like a squeezed beta-barrel. Although the Rapana structure is not resolved as well as the Octopus FU, two different conformations can be deduced. In the oxy FU of Octopus hemocyanin, domain III covers the entrance to the active site completely while in the deoxy-form this domain is shifted a few degrees so that the channel to the active site becomes completely uncovered. 

Comparing the most conserved parts, the active site, reveals two different types of type 3 copper proteins, an arthropod hemocyanin-like one and a molluscan hemocyanin-hke one. While the Cn-B site is highly conserved in both types, the Cu-A site differs. In the case of molluscan hemocyanins, one helix is too short for stabihzing the important histidine complexing Cu-A so that it has to be tied down by an unusual His ys bond as shown in Figure 2 (d-f). 

A similar substrate-binding pocket was identified for molluscan hemocyanins, which was not surprising since the active site of mollusc hemocyanins and that of arthropod hemocyanins and catecholoxidases are very similar. Therefore, we proposed the following mechanism for the activation of the molluscan hemocyanin based on the recently solved X-ray structure of a fimctional unit of the hemocyanin from the molluscan O. dofleini The C-terminal domain covers the entrance to the active site located on the N-terminal site by sticking Leu2830 into the entrance door. Detergents... 

Evolution of two phenoloxidases, an arthropod and molluscan type. A close relationship between phenoloxidase and hemocyantn was deduced based on their similar sequences, physico-chemical properties and similar functions. But sequence comparisons also revealed that there is not a common phenoloxidase type the enzymes found in animals, plants, and fungi are different with respect to their sequences, size, glycosylation, and activation. Two different types of tyrosinases can be distinguished based on their sequences, structure, and function. One type (m-phenoloxidase) is more related to molluscan hemocyanin with respect to the active site. The other type (a-phenoloxidase), which is very similar to arthropod hemocyanins, is found in arthropods together with hemocyanins (Figure 9). ... 

Comparison of the arrangements of the domains within an arthropod hemocyanin subunit and the functional unit of a molluscan hemocyanin also suggests two different types of phenoloxidases, the a-phenoloxidase and the m-phenoloxidase. Cleavages of the N-terminal domain of a-phenoloxidase and the C-terminal domain of m-phenoloxidase open the entrances to the active sites for bulky substrates. The key amino acids are indicated in Figure 9. 

Figure 9 The orientation of the domains from an arthropod and a functional unit from a molluscan hemocyanin are compared. In the first case (a) F49 in the N-terminal domain blocks the entrance to the active site, in the latter one (b) L2830 located in the C-terminal domain ...

The second type of oxidized hemocyanin is that produced by the action of H2 02 on deoxyhemocyanin, as carried out recently by Mason and co-workers83 , in this regard there are substantial differences between arthropod and molluscan hemocyanins. The oxy forms of either hemocyanin do not react with H2 02 the deoxy forms both do react, but differently. Molluscan hemocyanin reacts with small amounts of H2 02 to produce an inactive Cu(II) form but additional H2 02 regenerates the active oxyhemocyanin similar to the behavior of aged hemocyanin65). Arthropod deoxyhemocyanin reacts irreversibly with H2 0283 . That product is a methemocyanin which (aside from that due to small amounts of isolated Cu(II)... 

Contrary to the results for molluscan hemocyanin, arthropod hemocyanin does not undergo rapid aging upon fluoride treatment as evidenced by the lack of change in the optical spectrum84. However, an interaction of F- and arthropod oxyhemocyanin was observed by NMR studies82 (vide ante) and the maintenance of the 340 nm absorption is due to the retention of 02 by the oxyhemocyanin in spite of the presence ofF". 

Light-absorption and CD studies of hemocyanins from two different molluscan species showed several bands in the visible region characteristic also of other copper proteins. The greater resolving power of CD spectra yielded more accurate information than previous ORD measurements. d-d Transitions with distorted coordination sites could account for the absorption intensities observed without invoking charge transfer. By comparison of the CD data with those of peptide-Cu(II) complexes, the... 

Figure 3 Hierarchies in the structures of hemocyanins from arthropods and molluscs. A kidney shaped arthropod subunit (Mr 72 kDa) binds one molecule of dioxygen. Depending on the species, the subunits associate to 1 x 6, 2 x 6, 4 x 6, 6 x 6 and 8 X 6-meric hemocyanins found freely dissolved in the hemolymph. A molluscan hemocyanin subunit (Mr 400 kDa) folds into eight functional units each carrying one active site (two cannot be seen being hidden in the interior of the cylinder). Ten of these subunits form cylinders. Depending on the species, decamers or didecamers are found. Thus, hemocyanins can bind up to 160 dioxygen molecules with a cooperativity h of over 11...

Figures Comparison of the tertiary structures of a catecholoxidase from a sweet potato Ipomea sp. (a), functional units of molluscan hemocyanins O. dofleini (b) and R. thomasiana (c)) and arthropod hemocyanin subunits from L. poIyphemus (d) and P. inteiruptus (e). All structures are centered with respect to domain II (red) carrying the active site, domain I is colored green, domain III blue. The histidines are colored gray. In the Limulus structure (d) the alpha 1,3 helix is missing compared to the Panulirus (e) structure (ellipse)...

Hemocyanins are large, multisubunit dioxygen transporting proteins found in the hemolymph of many invertebrate species of the phyla of molluscs and arthropods 14). The subunits of molluscan hemocyanins contain functional units with a molecular weight of about 50,000 Da, each of which contains a dioxygen binding dicopper center. Arthropodal hemocyanins occur as hexamers, or multihexamers of subunits with a molecular weight of about 75,000 Da. As shown by the comparison of various X-ray crystal structures of the proteins from Panulirus interruptus (IS), Limulus polyphemus... 

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