Department of Biochemistry, Kansas State University, 141 Chalmers, Manhattan, KS 66506, USA.
Insect Biochem Mol Biol. 2012 Mar;42(3):193-202. doi: 10.1016/j.ibmb.2011.11.010. Epub 2011 Dec 16.
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.
漆酶-2 是一种高度保守的多铜氧化酶，在昆虫表皮色素沉着和鞣制中发挥作用。在许多物种中，选择性剪接产生了两种漆酶-2 同工型。对来自三个昆虫目的漆酶-2 序列的比较揭示了 A 和 B 同工型之间有 11 个保守差异的位置。同源建模表明，这 11 个残基不是底物结合口袋的一部分。为了确定同工型是否具有不同的动力学特性，我们比较了来自赤拟谷盗和冈比亚按蚊的漆酶-2 同工型的活性。我们将四个漆酶作为重组酶部分纯化，并分析了它们氧化一系列漆酶底物的能力。测试了先前在赤拟谷盗中检测到的预测内源性底物多巴胺、N-乙酰多巴胺（NADA）、N-β-丙氨酸多巴胺（NBAD）和多巴，以及作为本研究一部分在冈比亚按蚊中检测到的这些底物。还测试了另外两种二酚（儿茶酚和对苯二酚）和一种非酚类底物（2,2'-联氮双（3-乙基苯并噻唑啉-6-磺酸））。我们没有观察到 A 和 B 同工型之间在底物特异性上有重大差异。多巴胺、NADA 和 NBAD 的氧化催化效率范围为 51 至 550 min⁻¹ mM⁻¹。这些结果支持多巴胺、NADA 和 NBAD 是两种同工型漆酶-2 的内源性底物的假设。多巴氧化的催化效率较低，范围为 8 至 30 min⁻¹ mM⁻¹；相比之下，昆虫酪氨酸酶氧化多巴的催化效率为 201 min⁻¹ mM⁻¹。我们发现多巴是这四种内源性底物中具有最高氧化还原电位的，多巴的这一特性可能解释了其被漆酶-2 氧化的能力较差。我们得出结论，漆酶-2 剪接同工型可能在体内氧化相同的底物，需要进行额外的实验才能发现任何同工型特异性的功能。
