The high-resolution crystal structure of lobster hemocyanin shows its enzymatic capability as a phenoloxidase
Introduction
Hemocyanin (Hc) is a member of the family of type 3 copper proteins, which is characterized by binuclear copper atoms, each of which is coordinated by three histidine side chains [1]. In general, Hc is abundant in the hemolymph of arthropods and mollusks, and functions as a dioxygen-transporting protein. Although Hc from both arthropods and mollusks share common structural features, namely the copper center where dioxygen is reversibly bound as peroxide in side-on bridging coordination, the structural basis of the Hc molecule as a whole between arthropods and mollusks is far less conserved [2]. Molluscan Hc forms a huge cylindrical supermolecule composed of decameric or multidecameric 330 to 450-kDa subunits. Each subunit has a subset of approximately 50-kDa sequentially arranged paralogous functional units that contain a type 3 copper center [3]. In contrast, a subunit of arthropod Hc is approximately 75-kDa and is composed of three domains, among which the binuclear copper center is found in domain II. Each subunit assembles to form a hexamer, the basic unit of arthropod hemocyanin. These hexamers sometimes gather to form a larger structure, such as a dodecamer or higher aggregates [4].
The structure of the arthropod Hc subunit is rather similar to that of arthropod phenoloxidase (PO), another member of the family of type 3 copper proteins. Arthropod proPO catalyzes the key reactions of melanin formation, i.e. the hydroxylation of monophenols to o-diphenols (monophenolase activity; E.C. 1.14.18.1) and the subsequent oxidation to the corresponding quinone (diphenolase [catecholoxidase] activity; E.C. 1.10.3.1). Proteins with those enzymatic activities are ubiquitously distributed in bacteria, fungi, plants, and vertebrates, for example, tyrosinases and polyphenoloxidases (PPO) with both mono- and di-phenolase activities, and catechol oxidases (CO) with only diphenolase activity [5,6]. The overall structures of tyrosinases, PPO, and CO from bacteria, fungi, and plants are closely related to that of the functional unit of molluscan Hc, whereas these proteins are structurally distinct from Hc and PO from arthropods [6,7]. However, the structures of the copper-containing active site are comparable among these type 3 copper proteins.
The arthropod proPO system is a major component of the innate immunity of arthropods, as quinone species (the reactive intermediate of this enzymatic reaction) are harmful to pathogenic bacteria and fungi. PO also contributes to the physical encapsulation of pathogens and wound healing by producing melanin [8]. Molecular evolutionary studies and phylogenic analyses of the primary structures of arthropod Hc and PO suggest that they are derived from a common ancestral protein with enzymatic activity, and that Hc eventually lost its PO activity and became specialized as an oxygen-transporting protein [[9], [10], [11]]. However, significant or trace PO activity of some arthropod hemocyanins has been reported [[12], [13], [14], [15], [16]].
Due to the extreme abundance and biological significance of Hc, the structural analysis of arthropod Hc has a very long history. Crustacean Hc is one of the first multimeric proteins, the crystal structure of which was solved by X-ray crystallography [[17], [18], [19], [20]]. The first determined crystal structure of Hc is from a crustacean, the California spiny lobster (Panulirus interruptus), at 3.2 Å resolution (PDB ID: 1HC1 and 1HCY) [19]. The crystal structure of arthropodan Hc from the horseshoe crab (Limulus polyphemus), an ancient arthropod that belongs to the subphylum of chelicerates, was later solved at 2.18–2.4 Å resolution (PDB ID: 1LLA, 1NOL and 1OXY) [21,22].
Although these crystal structures contributed significantly to the establishment of the mechanisms underlying the oxygen binding and transition between the oxygenated and deoxygenated states, the high-resolution crystal structure of arthropodan Hc had not been obtained to date, partially due to the complexity and heterogeneity of the Hc subunits. In contrast, the structural information of the other type 3 copper protein, POs (e.g. tyrosinases from bacteria [[23], [24], [25], [26]], PPO from plants [[27], [28], [29], [30], [31]], proPO from arthropod [[32], [33], [34]], and catecholoxidases from plants and fungi [28,35]), was obtained in recent decades. These studies have shed light on the reaction mechanisms of mono- and di-PO reactions and provided an insight into the fundamental question concerning the functional diversity among these proteins despite the striking structural similarity of their binuclear copper centers.
The importance of the conserved asparagine and glutamate residues and water molecule around the copper site has been highlighted by several research groups, based on the structural analyses of bacterial tyrosinases [5,29,36,37]. These three elements are commonly observed in tyrosinases and POs with enzymatic activity, and are suggested to be crucial elements for the o-hydroxylation of mono-phenolic substrates. Although some exceptions have been reported in plant PPOs [38,39], the significance of these elements are further supported by the structural study of arthropod PO from a mosquito [34]. In the present study, we determined the high-resolution crystal structure of oxy-form Hc from the Japanese spiny lobster (Panulirus japonicus) (PjHc). PjHc had the conserved asparagine and glutamate residues and water molecule around the copper site at almost exactly the same positions as those in the proPO structures. However, there was a striking structural difference between PjHc and a proPO from a crustacean: the blocker residue phenylalanine (Phe371) stacked with a CuA-coordinated histidine (His198) side chain. This blocker phenylalanine seems to restrict the space of the cavity around the active site and hinder the access of substrates to the copper center. This crystal structure shows the significance of the blocker residue in the enzymatic activity of type 3 copper proteins, in addition to the elements highlighted earlier.
Section snippets
cDNA cloning
Total RNA was extracted from the hepatopancreas of P. japonicus (Japanese spiny lobsters) using Sepasol RNA extraction reagent (Nacalai Tesque, Kyoto, Japan), followed by mRNA purification using Oligotex dT-30 (Takara, Ohtsu, Japan). A reverse transcription polymerase chain reaction (PCR) was performed using an oligo dT adaptor primer containing the M13M4 or T7 promoter sequence and a PrimeScript RT-PCR Kit (Takara) according to the manufacturer's instruction.
We designed gene-specific primer
Structural analysis of PjHc
PjHc crystals were successfully obtained using purified PjHc (15 mg/ml). The initial phases of the diffraction data set were determined by molecular replacement using the coordinates of Hc from a California spiny lobster (1HCY). Subsequently, the amino acid sequence was replaced with the deduced amino acid sequence from the cDNA clone isolated from the hepatopancreas total RNA of a Japanese spiny lobster (GenBank accession no. LC509010). However, the sequence was partially mismatched to the
Acknowledgements
We are grateful to Dr. Takehiko Tosha at RIKEN SPring-8 for his helpful input and support with the crystallization. The synchrotron radiation experiments were performed at beamlines BL38B1 and BL26B1 of SPring-8, with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal numbers: 2017B6750, 2017A2533, 2017A2546, 2017B2547 and 2017B2546). The measurements of vacuum-ultraviolet circular-dichroism spectra were carried out with the approval of the Hiroshima
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2024, Fish and Shellfish ImmunologyImmune properties of invertebrate phenoloxidases
2021, Developmental and Comparative ImmunologyCitation Excerpt :It seems likely that these in vitro treatments serve to shift the position of an active site blocker (Li et al., 2009) residue such as the phenylalanine 371 in the crustacean Panulirus japonicus hemocyanin (Masuda et al., 2020) that is denying access to the copper to more bulkier substrates. This Phe residue is not present in crustacean POs; its absence and the presence of conserved glutamic acid and asparagine acid residues and a conserved water molecule around the Cu have been suggested to contribute to the enzyme activity of arthropodian PO (Masuda et al., 2020; Hu et al., 2016). The immunological relevance of hemocyanin-to-PO conversions remains to firmly establish in many cases.
The basicity of an active-site water molecule discriminates between tyrosinase and catechol oxidase activity
2021, International Journal of Biological MacromoleculesCitation Excerpt :Especially, the large movement of CuA seems to be necessary for the Ty reaction, which was also suggested by other research groups [22,23]. Until now, despite extensive studies based on the crystal structures of Ty or related proteins [5,6,10–17,21–32], its catalytic mechanism has not yet been clearly understood at an atomic level. For instance, researchers have been working for decades to elucidate the differences in substrate specificity between Ty and CO on a structural level.
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Current address: Bunzo Mikami, Laboratory of Metabolic Science of Forest Plants and Microorganisms, Research Institute for Sustainable Humanosphere and Laboratory of Structural Energy Bioscience, Institue of Advance Energy, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.