Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases
Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases
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DOI：
10.1016/s0969-2126(97)00222-0
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发表时间：
1997-05-15
期刊：
影响因子：
5.7
通讯作者：
Jenkins, J
中科院分区：
文献类型：
--
作者：
Mayans, O;Scott, M;Jenkins, J
背景：微生物果胶和果胶酸裂解酶是降解植物细胞壁果胶组分的毒力因子。果胶的同型半乳糖主链在其甲基化程度上从被称为果胶的高度甲基化和相对疏水的形式变化到被称为果胶酸盐的完全去甲基化和高度带电的形式。果胶的甲基化和去甲基化区域分别被果胶裂解酶和钙依赖性果胶酸裂解酶裂解。结果：在pH6.5（2.4埃分辨率）和pH8.5（1.93埃分辨率）条件下，测定了黑曲霉（Aspergillusniger）菌株N400和4 M-147果胶裂解酶A的晶体结构。通过分子置换、多晶型置换和晶间平均相结合的方法确定了结构。果胶裂解酶A折叠成平行的β螺旋，并且共享果胶酸裂解酶的许多结构特征，尽管在基于成对结构的比对后不超过17%的序列同一性。这些共有的结构特征包括氨基酸堆叠和天冬酰胺梯。然而，这两种酶的底物结合裂缝的差异是惊人的。在果胶裂解酶A中，裂缝由芳香残基主导，并被负静电势包围。在果胶酸裂解酶中，该裂缝富含带电残基，并且当Ca 2+结合时包含正电位的细长带。来自两种菌株的两种果胶裂解酶A结构之间的主要差异在于由残基182-187形成的环的构象。这些观察到的差异是由于不同的pH值的crystallization.Conclusions：底物结合裂缝和催化机制的果胶和果胶酸裂解酶有分歧显着。特异性由蛋白质-碳水化合物相互作用的性质和长程静电力决定。在果胶裂解酶中已经鉴定出三种潜在的催化残基，其中两种是果胶酸裂解酶所共有的。果胶裂解酶A不结合Ca 2+，但在果胶酸裂解酶中的Ca 2+离子的等同位置发现精氨酸残基，表明在催化中具有类似的作用。果胶裂解酶A的活性是pH依赖性的，最佳活性在pH 5.5。活性下降到pH 7.0以上，这是由于结合裂缝处的构象变化，这是由两个掩埋的天冬氨酸残基的接近触发的。
Background: Microbial pectin and pectate lyases are virulence factors that degrade the pectic components of the plant cell wall. The homogalacturan backbone of pectin varies in its degree of methylation from the highly methylated and relatively hydrophobic form known as pectin, to the fully demethylated and highly charged form known as pectate. Methylated and demethylated regions of pectin are cleaved by pectin lyase and calcium-dependent pectate lyases, respectively. Protein engineering of lyases specific far particular patterns of methylation, will yield modified pectins of high value to the food and pharmaceutical industries.Results: The crystal structures of pectin lyase A from two strains of Aspergillus niger, N400 and 4M-147, have been determined at pH 6.5 (2.4 Angstrom resolution) and pH 8.5 (1.93 Angstrom resolution), respectively. The structures were determined by a combination of molecular replacement, multiple isomorphous replacement and intercrystal averaging. Pectin lyase A folds into a parallel beta helix and shares many of the structural features of pectate lyases, despite no more than 17% sequence identity after pairwise structure-based alignment. These shared structural features include amino acid stacks and the asparagine ladder. However, the differences in the substrate-binding clefts of these two enzymes are striking. In pectin lyase A, the cleft is dominated by aromatic residues and is enveloped by negative electrostatic potential. In pectate lyases, this cleft is rich in charged residues and contains an elongated ribbon of positive potential when Ca2+ is bound. The major difference between the two pectin lyase A structures from the two strains is in the conformation of the loop formed by residues 182-187. These observed differences are due to the different pH values of crystallization.Conclusions: The substrate-binding clefts and catalytic machinery of pectin and pectate lyases have diverged significantly. Specificity is dictated by both the nature of the protein-carbohydrate interaction and long-range electrostatic forces. Three potential catalytic residues have been identified in pectin lyase, two of these are common to pectate lyases. Pectin lyase A does not bind Ca2+ but an arginine residue is found in an equivalent position to the Ca2+ ion in pectate lyase, suggesting a similar role in catalysis. The activity of pectin lyase A is pH-dependent with an optimum activity at pH 5.5. The activity drops above pH 7.0 due to a conformational change at the binding cleft, triggered by the proximity of two buried aspartate residues.