Biochemical and Structural Characterization of the Complex Agarolytic Enzyme System from the Marine Bacterium Zobellia galactanivorans
Biochemical and Structural Characterization of the Complex Agarolytic Enzyme System from the Marine Bacterium Zobellia galactanivorans
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DOI：
10.1074/jbc.m112.377184
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发表时间：
2012-08-31
影响因子：
4.8
通讯作者：
Czjzek, Mirjam
中科院分区：
文献类型：
--
作者：
Hehemann, Jan-Hendrik;Correc, Gaelle;Czjzek, Mirjam
半乳食藻是一种新兴的藻类生物量生物转化模式菌。值得注意的是，这种海洋类杆菌具有一个复杂的琼脂糖解系统，包括4个β-琼脂酶和5个β-卟啉酶，它们都属于糖苷水解酶家族16。虽然β-琼脂酶专属于中性琼脂糖基，但最近发现的β-卟啉酶能降解天然琼脂中不同数量的硫酸聚合物。在这里，我们报告了五种来自半乳糖酵母的β-卟啉酶和β-琼脂酶的生化和结构比较。通过对已定义的杂化低聚糖的作用，分析了两种β-卟啉酶和三种β-琼脂糖苷酶各自的降解模式。根据高分辨率的晶体结构，生化结果允许沿着酶的活性部位凹槽详细绘制底物特异性图。虽然PorA对-2和+1结合亚基中的C6-硫酸盐有严格的要求，但PorB对亚基-2中3-6-脱水-L-半乳糖的存在是容忍的。这两种酶都不接受-1亚基上半乳糖单位的甲基化。β-琼脂糖苷酶需要至少四个连续的琼脂糖基(DP8)，并且对修饰高度不耐受，而对于在-4、+1和+3位含有C6-硫酸盐基团的Agab寡糖，仍然被降解。结合对这些酶表达的转录分析，结构和生化结果允许提出半乳糖酵母琼脂糖解系统的模型方案。
Zobellia galactanivorans is an emerging model bacterium for the bioconversion of algal biomass. Notably, this marine Bacteroidetes possesses a complex agarolytic system comprising four beta-agarases and five beta-porphyranases, all belonging to the glycoside hydrolase family 16. Although beta-agarases are specific for the neutral agarobiose moieties, the recently discovered beta-porphyranases degrade the sulfated polymers found in various quantities in natural agars. Here, we report the biochemical and structural comparison of five beta-porphyranases and beta-agarases from Z. galactanivorans. The respective degradation patterns of two beta-porphyranases and three beta-agarases are analyzed by their action on defined hybrid oligosaccharides. In light of the high resolution crystal structures, the biochemical results allowed a detailed mapping of substrate specificities along the active site groove of the enzymes. Although PorA displays a strict requirement for C6-sulfate in the -2- and + 1-binding subsites, PorB tolerates the presence of 3-6-anhydro-L-galactose in subsite -2. Both enzymes do not accept methylation of the galactose unit in the -1 subsite. The beta-agarase AgaD requires at least four consecutive agarose units (DP8) and is highly intolerant to modifications, whereas for AgaB oligosaccharides containing C6-sulfate groups at the -4, +1, and +3 positions are still degraded. Together with a transcriptional analysis of the expression of these enzymes, the structural and biochemical results allow proposition of a model scheme for the agarolytic system of Z. galactanivorans.