Activation of prophenoloxidase with 2-propanol and other organic compounds in Drosophilamelanogaster

Asada, Nobuhiko; Fukumitsu, Takashi; Fujimoto, Kengo; Masuda, Ken-Ichiro

doi:10.1016/0965-1748(93)90060-6

Cited by 62 publications

(10 citation statements)

References 23 publications

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“…It was then confirmed that the partially purified tyrosinases and catechol oxidases were activated to show the catalytic activity by a variety of treatments, such as pH control, 20,21 addition of detergents, 22,23 organic solvents, 24,25 or protein denaturants. 26,27 To date, such phenomena have been discussed for more than 70 years, and nowadays most tyrosinases and catechol oxidases are thought to be expressed as inactive forms (pro-tyrosinase and pro-catechol oxidase) in the cell in order to avoid undesirable intracellular degradation.…”

Section: Introductionmentioning

confidence: 88%

Controlling Dicopper Protein Functions

Fujieda¹,

Itoh²

2016

Bulletin of the Chemical Society of Japan

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Maturation processes of dinuclear copper proteins such as tyrosinase, catechol oxidase, and hemocyanin have been a long-standing mystery in copper protein chemistry. Until now, several crystal structures have revealed that these copper proteins share a similar dinuclear copper active site, where each copper ion is ligated by three histidine imidazoles, and binds molecular oxygen in a side-on fashion to form a (¯-η 2 :η 2 -peroxido)dicopper(II) species not only as the dioxygen-adduct in oxy-hemocyanins but also as the key reactive intermediate for the hydroxylation of phenols to catechols (phenolase reaction) and the oxidation of catechols to o-quinones (catecholase reaction) in tyrosinases and catechol oxidases. Recently, we have succeeded in determining the high-resolution crystal structures of the recombinant pro-form of yellow koji mold tyrosinase to find the existence of a distinct C-terminal domain containing a CXXC unit, that is the common sequence motif of the copper chaperons. Thus, the C-terminal domain apparently acts as a copper chaperon, helping construction of the dinuclear copper active site of tyrosinase. Furthermore, we have found that the proteolytic cleavage of the C-terminal domain from the pro-form (inactive-form) of tyrosinase greatly enhances the tyrosinase activity, thus suggesting that the Cterminal domain also acts as a shielding domain to regulate the enzymatic activity. In fact, overall structure of the pro-form resembles the structure of one of the functional units of octopus hemocyanin (oxygen carrier protein), which also has a similar C-terminal domain prohibiting the monooxygenase activity. On the basis of these results together with the detailed kinetic and spectroscopic analyses, the maturation process of the dinuclear copper proteins is discussed to provide new insights into the regulation mechanism of the dicopper protein functions; dioxygen binding and activation. We have also succeeded in evolving phenolase activity from molluscan and arthropod hemocyanins by treating them with a hydrolytic enzyme or an acid, and demonstrated that the reaction mechanism of their phenolase activity is the same to that of tyrosinase itself, that is the electrophilic aromatic substitution mechanism. Furthermore, we have developed an artificial dicopper protein exhibiting catecholase activity using metallo-β-lactamase, a dinuclear zinc enzyme, as a metal binding platform.

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Section: Introductionmentioning

confidence: 88%

Controlling Dicopper Protein Functions

Fujieda¹,

Itoh²

2016

Bulletin of the Chemical Society of Japan

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“…Based on their crystal structures, MsPPO1 and MsPPO2 differ in their surface electric charge and structure (Figure 2), which may complicate PPO activation since the electrostatic interactions of MsPPO, SPH, and PAPs are involved in the activation process (Li et al, 2009). In addition to serine protease, detergents such as sodium dodecyl sulfate (SDS), CPC, and ethanol can activate insect PPO (Asada et al, 1993; Ashida and Brey, 1997). Cationic detergents are thought to interact with PPO through electrostatic interaction, causing conformational changes and dislodging the place holder (Li et al, 2009).…”

Section: Ppo Activationmentioning

confidence: 99%

Insect prophenoloxidase: the view beyond immunity

et al. 2014

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Insect prophenoloxidase (PPO) is an important innate immunity protein due to its involvement in cellular and humoral defense. It belongs to a group of type-3 copper-containing proteins that occurs in almost all organisms. Insect PPO has been studied for over a century, and the PPO activation cascade is becoming clearer. The insect PPO activation pathway incorporates several important proteins, including pattern-recognition receptors (PGRP, β GRP, and C-type lectins), serine proteases, and serine protease inhibitors (serpins). Due to their complexity, PPO activation mechanisms vary among insect species. Activated phenoloxidase (PO) oxidizes phenolic molecules to produce melanin around invading pathogens and wounds. The crystal structure of Manduca sexta PPO shows that a conserved amino acid, phenylalanine (F), can block the active site pocket. During activation, this blocker must be dislodged or even cleaved at the N-terminal sequence to expose the active site pockets and allow substrates to enter. Thanks to the crystal structure of M. sexta PPO, some domains and specific amino acids that affect PPO activities have been identified. Further studies of the relationship between PPO structure and enzyme activities will provide an opportunity to examine other type-3 copper proteins, and trace when and why their various physiological functions evolved. Recent researches show that insect PPO has a relationship with neuron activity, longevity, feces melanization (phytophagous insects) and development, which suggests that it is time for us to look back on insect PPO beyond the view of immunity in this review.

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“…The cleavage site in PPO has been experimentally confirmed in several insect species, including 51 Arg-Phe 52 in B. mori PPO [26], 51 Arg-Phe 52 in M. sexta PPO [27, 28], and 50 Arg-Phe 51 followed by the second cleavage at 162 Arg-Ala 163 in H. diomphalia PPO [23, 24]. In vitro, PPO can also be activated without proteolytic cleavage by certain chemicals such as ethanol or detergents (e.g., sodium dodecyl sulfate (SDS), cetylpyridinium chloride (CPC)) [29, 30]. …”

Section: Introductionmentioning

confidence: 99%

Cloning, Expression, and Characterization of Prophenoloxidases from Asian Corn Borer,Ostrinia furnacalis(Gunée)

Zhang

Fang

et al. 2016

Journal of Immunology Research

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Insect phenoloxidase (PO) belongs to the type 3 copper protein family and possesses oxidoreductase activities. PO is typically synthesized as a zymogen called prophenoloxidase (PPO) and requires the proteolytic activation to function. We here cloned full-length cDNA for 3 previously unidentified PPOs, which we named OfPPO1a, OfPPO1b, and OfPPO3, from Asian corn borer, Ostrinia furnacalis (Gunée), in addition to the previously known OfPPO2. These conceptual PPOs and OfPPO2 all contain two common copper-binding regions, two potential proteolytic activation sites, a plausible thiol-ester site, and a conserved C-terminal region but lack a secretion signal peptide sequence at the N-terminus. O. furnacalis PPOs were highly similar to other insect PPOs (42% to 79% identity) and clustered well with other lepidopteran PPOs. RT-PCR assay showed the transcripts of the 4 OfPPOs were all detected at the highest level in hemocytes and at the increased amounts after exposure to infection by bacteria and fungi. Additionally, we established an Escherichia coli (E. coli) expression system to produce recombinant O. furnacalis PPO proteins for future use in investigating their functions. These insights could provide valuable information for better understanding the activation and functioning mechanisms of O. furnacalis PPOs.

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Activation of prophenoloxidase with 2-propanol and other organic compounds in Drosophilamelanogaster

Cited by 62 publications

References 23 publications

Controlling Dicopper Protein Functions

Controlling Dicopper Protein Functions

Insect prophenoloxidase: the view beyond immunity

Cloning, Expression, and Characterization of Prophenoloxidases from Asian Corn Borer,Ostrinia furnacalis(Gunée)

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