Phenoloxidase (PO), which is classified as a type 3 copper protein, catalyzes the hydroxylation of monophenol to o-diphenol and subsequent oxidation to the corresponding o-quinone. The geometry and coordination environment of the active site of the arthropod PO are very similar to those of the arthropod hemocyanin (Hc). However, unlike the POs, Hc is an oxygen carrier in crustaceans, and does not possess PO activity in general. Recently, we identified a new type of proPO from a crustacean and designated it proPOβ. This enzyme has many characteristics that are rather similar to those of Hc, such as its maturation, localization, and oligomeric state. Here, we determined the crystal structure of proPOβ prepared from the hemolymph of kuruma prawns (Marsupenaeus japonicus) at 1.8-Å resolution. M. japonicus proPOβ forms a homohexamer rather similar to that of arthropod Hc. The geometry of the active copper site in proPOβ is nearly identical to that of arthropod Hc. Furthermore, the well-characterized ‘place-holder’ phenylalanine is present (Phe72). However, the accessibility to the active site differs in several ways. First, another phenylalanine, which shields the active site by interacting with a copper-coordinated histidine in crustacean Hc, is replaced by valine in the proPOβ structure. Second, two tyrosines, Tyr208 and Tyr209, both of which are absent in Hc, show the alternative conformations and form a pathway providing access to the reaction center. Thus, the present crystal structure clarifies the similarities and differences in the activity of two closely related proteins, PO and Hc.Database
Structural data are available in the RSCB protein data bank under the accession number 3WKY.Structured digital abstract
Arthropod phenoloxidase (PO) is classified as a type 3 copper protein. We solved the first crystal structure of crustacean PO at 1.8-Å resolution. Its active site structure closely resembled to that of another type 3 copper protein, hemocyanin. Thus, the PO activity of this type of protein depends on the substrate accessibility to its copper containing active site.