Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
J Biol Chem. 2010 Aug 6;285(32):24519-28. doi: 10.1074/jbc.M110.125450. Epub 2010 May 27.
Alginate, a major component of the cell wall matrix in brown seaweeds, is degraded by alginate lyases through a beta-elimination reaction. Almost all alginate lyases act endolytically on substrate, thereby yielding unsaturated oligouronic acids having 4-deoxy-l-erythro-hex-4-enepyranosyluronic acid at the nonreducing end. In contrast, Agrobacterium tumefaciens alginate lyase Atu3025, a member of polysaccharide lyase family 15, acts on alginate polysaccharides and oligosaccharides exolytically and releases unsaturated monosaccharides from the substrate terminal. The crystal structures of Atu3025 and its inactive mutant in complex with alginate trisaccharide (H531A/DeltaGGG) were determined at 2.10- and 2.99-A resolutions with final R-factors of 18.3 and 19.9%, respectively, by x-ray crystallography. The enzyme is comprised of an alpha/alpha-barrel + anti-parallel beta-sheet as a basic scaffold, and its structural fold has not been seen in alginate lyases analyzed thus far. The structural analysis of H531A/DeltaGGG and subsequent site-directed mutagenesis studies proposed the enzyme reaction mechanism, with His(311) and Tyr(365) as the catalytic base and acid, respectively. Two structural determinants, i.e. a short alpha-helix in the central alpha/alpha-barrel domain and a conformational change at the interface between the central and C-terminal domains, are essential for the exolytic mode of action. This is, to our knowledge, the first report on the structure of the family 15 enzyme.
褐藻细胞壁基质的主要成分——褐藻胶，可被褐藻胶裂解酶通过 β-消除反应降解。几乎所有的褐藻胶裂解酶都以内切方式作用于底物，从而生成非还原端具有 4-脱氧-L-赤-己-4-烯糖醛酸的不饱和低聚褐藻酸。相比之下，土壤农杆菌褐藻胶裂解酶 Atu3025 是多糖裂解酶家族 15 的一个成员，以外切方式作用于褐藻胶多糖和寡糖，从底物末端释放不饱和单糖。通过 X 射线晶体学，Atu3025 及其无活性突变体与褐藻三糖（H531A/DeltaGGG）复合物的晶体结构在 2.10 和 2.99Å分辨率下分别被解析，最终 R 因子分别为 18.3%和 19.9%。该酶由一个α/α-桶和反平行β-折叠组成基本支架，其结构折叠在迄今分析的褐藻胶裂解酶中尚未出现。H531A/DeltaGGG 的结构分析和随后的定点突变研究提出了酶反应机制，其中 His（311）和 Tyr（365）分别作为催化碱和酸。两个结构决定因素，即中央α/α-桶域中的短α-螺旋和中央与 C-末端域之间界面的构象变化，对于外切作用模式至关重要。这是，据我们所知，家族 15 酶的第一个结构报告。
