Ranocha P, McDougall G, Hawkins S, Sterjiades R, Borderies G, Stewart D, Cabanes-Macheteau M, Boudet A M, Goffner D
UMR CNRS/UPS 5546, Pôle de Biotechnologie Végetales, Castanet Tolosan, France.
Eur J Biochem. 1999 Jan;259(1-2):485-95. doi: 10.1046/j.1432-1327.1999.00061.x.
The nature of the enzyme(s) involved in the dehydrogenative polymerization of lignin monomers is still a matter of debate. Potential candidates include laccases which have recently received attention due to their capacity to oxidize lignin monomers and close spatial and temporal correlation with lignin deposition. We have characterized two H2O2-independent phenoloxidases with approximate molecular masses of 90 kDa and 110 kDa from cell walls of Populus euramericana xylem that are capable of oxidizing coniferyl alcohol. The 90-kDa protein was purified to apparent homogeneity and extensively characterized at the biochemical and structural levels. To our knowledge, this is the first report of a plant laccase purified to homogeneity from a lignifying tissue of an angiosperm. The cDNA clones corresponding to the 90-kDa and 110-kDa proteins, lac90 and lac110, were obtained by a PCR-based approach using specific oligonucleotides derived from peptide sequences. Sequence analysis indicated that lac90 and lac110 encoded two distinct laccases. In addition, heterologous screening using an Acer pseudoplatanus laccase cDNA enabled us to obtain three additional cDNAs (lac1, lac2, lac3) that did not correspond to lac90 and lac110. The five laccase cDNAs correspond to a highly divergent multigene family but Northern analysis with gene-specific probes indicated that all of the genes are exclusively and abundantly expressed in stems. These results highlight the polymorphism of plant laccases by an integrated biochemical and molecular approach, and provide the tools that will enable us to clearly determine the function of these enzymes in plants by molecular and genetic approaches.
参与木质素单体脱氢聚合反应的酶的性质仍存在争议。潜在的候选酶包括漆酶，由于其氧化木质素单体的能力以及与木质素沉积密切的时空相关性，漆酶最近受到了关注。我们从欧美杨木质部细胞壁中鉴定出两种不依赖H2O2的酚氧化酶，其分子量分别约为90 kDa和110 kDa，它们能够氧化松柏醇。90 kDa的蛋白质被纯化至表观均一，并在生化和结构水平上进行了广泛表征。据我们所知，这是首次从被子植物木质化组织中纯化出均一的植物漆酶的报道。通过基于PCR的方法，使用从肽序列衍生的特异性寡核苷酸，获得了与90 kDa和110 kDa蛋白质对应的cDNA克隆，即lac90和lac110。序列分析表明，lac90和lac110编码两种不同的漆酶。此外，使用假挪威槭漆酶cDNA进行的异源筛选使我们获得了另外三个与lac90和lac110不对应的cDNA（lac1、lac2、lac3）。这五个漆酶cDNA对应于一个高度分化的多基因家族，但用基因特异性探针进行的Northern分析表明，所有这些基因都仅在茎中大量表达。这些结果通过综合生化和分子方法突出了植物漆酶的多态性，并提供了工具，使我们能够通过分子和遗传方法明确确定这些酶在植物中的功能。
