Crystal structure of Manduca sexta prophenoloxidase provides insights into the mechanism of type 3 copper enzymes

Abstract

Arthropod phenoloxidase (PO) generates quinones and other toxic compounds to sequester and kill pathogens during innate immune responses. It is also involved in wound healing and other physiological processes. Insect PO is activated from its inactive precursor, prophenoloxidase (PPO), by specific proteolysis via a serine protease cascade. Here, we report the crystal structure of PPO from a lepidopteran insect at a resolution of 1.97 Å, which is the initial structure for a PPO from the type 3 copper protein family. Manduca sexta PPO is a heterodimer consisting of 2 homologous polypeptide chains, PPO1 and PPO2. The active site of each subunit contains a canonical type 3 di-nuclear copper center, with each copper ion coordinated with 3 structurally conserved histidines. The acidic residue Glu-395 located at the active site of PPO2 may serve as a general base for deprotonation of monophenolic substrates, which is key to the ortho-hydroxylase activity of PO. The structure provides unique insights into the mechanism by which type 3 copper proteins differ in their enzymatic activities, albeit sharing a common active center. A drastic change in electrostatic surface induced on cleavage at Arg-51 allows us to propose a model for localized PPO activation in insects.innate immune ͉ tyrosinase ͉ melanization ͉ zymogen activation ͉ hemocyanin P henoloxidase (PO), a critical component of the innate immune system in insects and crustaceans, is present as a zymogen [prophenoloxidase (PPO)] in hemolymph and becomes activated on wounding or infection (1, 2). Possessing ohydroxylase (EC 1.14.18.1) and o-di-PO (EC 1.10.3.1) activities, PO converts a variety of monophenolic and o-diphenolic substrates to o-quinones (3). Quinones can act as cross-linkers for wound healing, and they also polymerize to form melanin capsules around parasites and parasitoids (4-6). Quinones and other reactive intermediates (e.g., 5,6-dihydroxyindole) directly kill microbial pathogens (7). Although PO-generated compounds are powerful weapons against pathogens, they could also cause damage to host tissues and cells. Consequently, the activation of PPO is mediated by a cascade of highly specific serine proteases and regulated as a local transient reaction against invading organisms (8).The proteolytic activation of PPO requires a trypsin-like serine protease, known as PPO activating protease (PAP) or PPO activating enzyme, which cuts the protein substrate next to an Arg residue near its amino-terminus (9-13). PAP contains regulatory clip domain(s) followed by a catalytic domain that hydrolyzes synthetic substrates at various ionic strengths and cleaves PPO in low-salt buffers preferably. In some insects, PAP generates active PO in the presence of an auxiliary factor consisting of clip-domain serine protease homolog (SPH), which lacks catalytic activity because of the substitution of the active site Ser by Gly (14,15). Unlike its precursor, active PO selfassociates into oligomers, binds to other proteins, and sticks to column matrices; consequently, it has n...

Peptide toxin glacontryphan-M is present in the wings of the butterfly Hebomoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)

“…Some plants use peroxidases as defensive mechanisms, possibly explaining why oxidative modifications are observed on the Lepidoptera feeding on them (18). Furthermore, Lepidoptera and other arthropods are known to contain phenol oxidases that may be responsible for quinone modification of GT of HG (19).…”

Hemolymph Melanization in the Silkmoth Bombyx mori Involves Formation of a High Molecular Mass Complex That Metabolizes Tyrosine

“…It is generally assumed that processing of PPO to PO confers the enzymatic activity needed to cause hemolymph to melanize (1,(12)(13)(14)(15). Two lines of investigation, however, raise questions as to whether this is fully correct.…”

Determination of tyrosinase substrate-binding modes reveals mechanistic differences between type-3 copper proteins

“…The catalytic mechanism of type-3 copper proteins has been studied extensively for the past two decades, recently assisted by the availability of three-dimensional structures [4][5][6][7][8] . Based on these structures, it is evident that the differences in function are due to variations in the residues surrounding the substrate-binding pocket.…”