Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae

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dc.contributor.author Gorman, Maureen J.
dc.contributor.author Sullivan, Lucinda I.
dc.contributor.author Nguyen, Thi D.T.
dc.contributor.author Dai, Huaien
dc.contributor.author Arakane, Yasuyuki
dc.contributor.author Dittmer, Neal T.
dc.contributor.author Syed, Lateef U.
dc.contributor.author Li, Jun
dc.contributor.author Hua, Duy H.
dc.contributor.author Kanost, Michael R.
dc.date.accessioned 2012-05-11T19:09:55Z
dc.date.available 2012-05-11T19:09:55Z
dc.date.issued 2012-03-01
dc.identifier.uri http://hdl.handle.net/2097/13817
dc.description Citation: Gorman, M.J., Sullivan, L.I., Nguyen, T.D.T., Dai,H., Arakane,Y., Dittmer, N.T.,…Kanost, M.R. (2012). Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae. Insect Biochemistry and Molecular Biology, 42(3), 193-202.
dc.description.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, Nacetyldopamine (NADA), N-b-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 (2,20-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. en_US
dc.relation.uri http://doi.org/10.1016/j.ibmb.2011.11.010 en_US
dc.subject Multicopper oxidase en_US
dc.subject Laccase en_US
dc.subject Substrate en_US
dc.subject Insect en_US
dc.subject Cuticle en_US
dc.title Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae en_US
dc.type Article (author version) en_US
dc.date.published 2012 en_US
dc.citation.doi 10.1016/j.ibmb.2011.11.010 en_US
dc.citation.epage 202 en_US
dc.citation.issue 3 en_US
dc.citation.jtitle Insect Biochemistry and Molecular Biology en_US
dc.citation.spage 193 en_US
dc.citation.volume 42 en_US
dc.citation Gorman, M.J., Sullivan, L.I., Nguyen, T.D.T., Dai,H., Arakane,Y., Dittmer, N.T.,…Kanost, M.R. (2012). Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae. Insect Biochemistry and Molecular Biology, 42(3), 193-202.
dc.contributor.authoreid duy en_US
dc.contributor.authoreid mgorman en_US
dc.contributor.authoreid huaien en_US
dc.contributor.authoreid ndittmer en_US
dc.contributor.authoreid lateefs en_US
dc.contributor.authoreid junli en_US
dc.contributor.authoreid kanost en_US


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