title: BmPAH catalyzes the initial melanin biosynthetic step in Bombyx mori.
abstract: Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4(th) instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.
authors.affiliation: State Key Laboratory of Silkworm Genome Biology and College of Biotechnology, Southwest University, Chongqing, China ; College of Biotechnology, Southwest University, Chongqing, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Jiying
authors.initials: J
authors.lastname: Wang
authors.forename: Haiyin
authors.initials: H
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Yin
authors.initials: Y
authors.lastname: Lv
authors.forename: Cheng
authors.initials: C
authors.lastname: Lu
mesh_terms: D000818:Animals; D053898:Biosynthetic Pathways / Q000187:drug effects; D012831:Bombyx / Q000196:embryology / Q000201:enzymology* / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D007267:Injections; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007814:Larva / Q000235:genetics / Q000378:metabolism; D008543:Melanins / Q000096:biosynthesis*; D018978:Molting / Q000235:genetics; D010063:Ovum / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000008:administration & dosage / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D061987:Sf9 Cells; D014468:Umbelliferones / Q000494:pharmacology
chemical_list: D019476:Insect Proteins; D008543:Melanins; D012330:RNA, Double-Stranded; D014468:Umbelliferones; D010651:Phenylalanine Hydroxylase; C007628:esculetin