Mode of action and subsite studies of the guluronan block-forming mannuronan C-5 epimerases AlgE1 and AlgE6
Mode of action and subsite studies of the guluronan block-forming mannuronan C-5 epimerases AlgE1 and AlgE6
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DOI：
10.1042/bj20051804
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发表时间：
2006-04-15
影响因子：
4.1
通讯作者：
Skjåk-Bræk, G
中科院分区：
文献类型：
--
作者：
Holtan, S;Bruheim, P;Skjåk-Bræk, G
AlgE 1、AlgE 5和AlgF.6是由棕色固氮菌（Azotobacter vinelandii）编码的甘露糖醛酸C-5差向异构酶家族的成员，并且在藻酸盐的生物合成中具有活性，其中它们催化β-D-甘露糖醛酸（M）残基聚合后转化为α-L-葡糖醛酸残基（G）。所有的酶都显示出在预先存在的G附近引入G-残基的偏好。它们也有能力转化侧翼为G的单个M残基，从而“缩合”G-嵌段以形成几乎均聚的古洛糖醛酸。基于特异性酶降解结合尺寸排阻色谱、电喷雾电离质谱、HPAEC-PAD分析G G-块的长度和分布（高效阴离子交换色谱和脉冲安培检测）（基质辅助激光解吸电离）-MS和NMR揭示了AlgE 1和AlgE 6产生的嵌段长度和分布的巨大差异，这可能反映了它们的持续合成能力的不同程度。当作用于作为底物的polyMG时。AlgE 1最初仅形成> 50的长均聚G-嵌段，而AlgE 6给出具有更宽嵌段尺寸分布的较短嵌段。AlgE 1和AlgE 6亚位点特异性的分析表明，通过相同的方法，甘露糖醛酸八聚体和lieptamer分别是最小的底物链长度需要适应酶的活动。从非还原端起的第四个M残基首先被两种酶差向异构化。当作用于MG-寡聚体时，AlgE 1需要十聚体，而AlgE 6需要八聚体来调节活性。通过对差向异构化和标准MG-寡聚物的裂解酶底物进行FIA（流动注射分析）-MS，两种酶优先攻击非还原末端的位置5中的M残基，在barG-序列下产生MGMG（G）（下划线和粗体表示差向异构化残基）。
AlgE1, AlgE5 and AlgF.6 are members of a family of mannuronan C-5 epimerases encoded by the bacterium Azotobacter vinelandii, and are active in the biosynthesis of alginate, where they catalyse the post-polymerization conversion of ss-D-mannuronic acid (M) residues into alpha-L-uluronic acid residues (G). All enzymes show preference for introducing G-residues neighbouring a pre-existing G. They also have the capacity to convert single M residues flanked by G, thus 'condensing' G-blocks to form almost homopolymeric guluronan. Analysis of the length and distribution of G G-blocks based on specific enzyme degradation combined with size-exclusion chromatography, electrospray ionization MS, HPAEC-PAD (high-performance anion-exchange chromatography and pulsed amperometric detection), MALDI (matrix-assisted laser-desorption ionization)-MS and NMR revealed large differences in block length and distribution generated by AlgE1 and AlgE6, probably reflecting their different degree of processivity. When acting on polyMG as substrates. AlgE1 initially forms only long homopolymeric G-blocks > 50, while AlgE6 gives shorter blocks with a broader block size distribution. Analyses of the AlgE1 and AlgE6 subsite specificities by the same methodology showed that a mannuronan octamer and lieptamer respectively were the minimum substrate chain lengths needed to accommodate enzyme activities. The fourth M residue from the non-reducing end is epimerized first by both enzymes. When acting on MG-oligomers, AlgE1 needed a decamer while AlgE6 an octamer to accommodate activity. By performing FIA (flow injection analysis)-MS on the lyase digests of epimerized and standard MG-oligomers, the M residue in position 5 from the non-reducing end was preferentially attacked by both enzymes, creating an MGMG (G) under barG-sequence (underlined and boldface indicate the epimerized residue).