title: Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae.
abstract: Laccase-2 is a highly conserved multicopper oxidase that functions in insect cuticle pigmentation and tanning. In many species, alternative splicing gives rise to two laccase-2 isoforms. A comparison of laccase-2 sequences from three orders of insects revealed eleven positions at which there are conserved differences between the A and B isoforms. Homology modeling suggested that these eleven residues are not part of the substrate binding pocket. To determine whether the isoforms have different kinetic properties, we compared the activity of laccase-2 isoforms from Tribolium castaneum and Anopheles gambiae. We partially purified the four laccases as recombinant enzymes and analyzed their ability to oxidize a range of laccase substrates. The predicted endogenous substrates tested were dopamine, N-acetyldopamine (NADA), N-β-alanyldopamine (NBAD) and dopa, which were detected in T. castaneum previously and in A. gambiae as part of this study. Two additional diphenols (catechol and hydroquinone) and one non-phenolic substrate (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) were also tested. We observed no major differences in substrate specificity between the A and B isoforms. Dopamine, NADA and NBAD were oxidized with catalytic efficiencies ranging from 51 to 550 min⁻¹ mM⁻¹. These results support the hypothesis that dopamine, NADA and NBAD are endogenous substrates for both isoforms of laccase-2. Catalytic efficiencies associated with dopa oxidation were low, ranging from 8 to 30 min⁻¹ mM⁻¹; in comparison, insect tyrosinase oxidized dopa with a catalytic efficiency of 201 min⁻¹ mM⁻¹. We found that dopa had the highest redox potential of the four endogenous substrates, and this property of dopa may explain its poor oxidation by laccase-2. We conclude that laccase-2 splice isoforms are likely to oxidize the same substrates in vivo, and additional experiments will be required to discover any isoform-specific functions.
authors.affiliation: Department of Biochemistry, Kansas State University, 141 Chalmers, Manhattan, KS 66506, USA. mgorman@ksu.edu
authors.forename: Maureen J
authors.initials: MJ
authors.lastname: Gorman
authors.forename: Lucinda I
authors.initials: LI
authors.lastname: Sullivan
authors.forename: Thi D T
authors.initials: TD
authors.lastname: Nguyen
authors.forename: Huaien
authors.initials: H
authors.lastname: Dai
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Arakane
authors.forename: Neal T
authors.initials: NT
authors.lastname: Dittmer
authors.forename: Lateef U
authors.initials: LU
authors.lastname: Syed
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Duy H
authors.initials: DH
authors.lastname: Hua
authors.forename: Michael R
authors.initials: MR
authors.lastname: Kanost
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles / Q000737:chemistry / Q000201:enzymology*; D002395:Catecholamines / Q000032:analysis; D005260:Female; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D042845:Laccase / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014231:Tribolium / Q000201:enzymology*
chemical_list: D002395:Catecholamines; D019476:Insect Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D042845:Laccase