Arylalkylamine N-acetyltransferase 1 gene (TcAANAT1) is required for cuticle morphology and pigmentation of the adult red flour beetle, Tribolium castaneum.
In the insect cuticle tanning pathway (sclerotization and pigmentation), the enzyme arylalkylamine N-acetyltransferase (AANAT) catalyzes the acetylation of dopamine to form N-acetyldopamine (NADA), which is one of the major precursors for quinone-mediated tanning. In this study we characterized and investigated the function of TcAANAT1 in cuticle pigmentation of the red flour beetle, Tribolium castaneum. We isolated a full length TcAANAT1 cDNA that encodes a protein of 256 amino acid residues with a predicted GCN5-related acetyltransferase domain containing an acetyl-CoA binding motif. TcAANAT1 transcripts were detected at all stages of development with lowest expressions at the embryonic and pharate pupal stages. We expressed and purified the encoded recombinant TcAANAT1 protein (rTcAANAT1) that exhibited highest activity at slightly basic pH values (for example, pH 7.5 to 8.5 using dopamine as the substrate). In addition, rTcAANAT1 acts on a wide range of substrates including tryptamine, octopamine and norepinephrine with similar substrate affinities with Kmvalues in the range of 0.05-0.11 mM except for tyramine (Km= 0.56 mM). Loss of function of TcAANAT1 caused by RNAi had no effect on larval and pupal development. The tanning of pupal setae, gin traps and urogomphi proceeded normally. However, the resulting adults (∼70%) exhibited a roughened exoskeletal surface, separated elytra and improperly folded hindwings. The body wall, elytra and veins of the hindwing of the mature adults were significantly darker than those of control insects probably due to the accumulation of dopamine melanin. A dark pigmentation surrounding the bristles located on the inter-veins of the elytron was evident primarily because of the underlying darkly pigmented trabeculae that partition the dorsal and ventral layers of the elytron. These results support the hypothesis that TcAANAT1 acetylates dopamine and plays a role in development of the morphology and pigmentation of T. castaneum adult cuticle.
Department of Applied Biology, Chonnam National University, Gwangju 500-757, South Korea.
Mi Young
MY
Noh
Department of Applied Biology, Chonnam National University, Gwangju 500-757, South Korea.
Bonwoo
B
Koo
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, USA.
Karl J
KJ
Kramer
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506, USA.
Subbaratnam
S
Muthukrishnan
Department of Applied Biology, Chonnam National University, Gwangju 500-757, South Korea. Electronic address: arakane@chonnam.ac.kr.
Yasuyuki
Y
Arakane
D000595:Amino Acid Sequence; D000818:Animals; D047068:Arylalkylamine N-Acetyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004298:Dopamine / Q000031:analogs & derivatives / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007814:Larva / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D010802:Phylogeny; D010858:Pigmentation; D011679:Pupa / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D034622:RNA Interference; D014231:Tribolium / Q000235:genetics* / Q000254:growth & development*
D018076:DNA, Complementary; D019476:Insect Proteins; C006374:N-acetyldopamine; D047068:Arylalkylamine N-Acetyltransferase; D004298:Dopamine
Cuticle pigmentation; Elytron; N-acetyltransferase; RNA interference (RNAi); Trabeculae; Tribolium castaneum