Functional expression and characterization of Aedes aegypti dopachrome conversion enzyme.
A full-length mosquito dopachrome conversion enzyme (DCE) and its truncated form lacking the last 54 carboxyl-terminal amino acid residues are expressed using a baculovirus/insect cell expression system. The full-length recombinant DCE displayed multiple bands during native PAGE with substrate staining, but only one active band was detected when the truncated recombinant DCE was analyzed under identical analysis conditions. Our data suggest that the last 50 some carboxyl-terminal residues are involved in the polymerization of the DCE molecules and that the proposed DCE isozymes likely reflect the presence of multimers of the same DCE molecules. The significance of the recombinant DCE in accelerating the melanization pathway is demonstrated by a rapid production of melanin in a dopa and tyrosinase reaction mixture in the presence of recombinant DCE. The DCE sequence data obtained in our previous study, together with results of functional expression and biochemical characterization achieved in this study, provide a necessary reference for the study of other insect DCEs.
Fang
Jianmin
J
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802, USA.
Han
Qian
Q
Johnson
Jody K
JK
Christensen
Bruce M
BM
Li
Jianyong
J
D000330:Aedes / Q000201:enzymology*; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D045563:Indolequinones; D007211:Indoles / Q000737:chemistry; D007313:Insecta; D019746:Intramolecular Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D008543:Melanins / Q000737:chemistry; D008956:Models, Chemical; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011809:Quinones / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D000596:Amino Acids; D018076:DNA, Complementary; D045563:Indolequinones; D007211:Indoles; D008543:Melanins; D020033:Protein Isoforms; D011809:Quinones; D011994:Recombinant Proteins; C001123:dopachrome; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase