Metabolic Mechanism of Mannan in a Ruminal Bacterium, Ruminococcus albus, Involving Two Mannoside Phosphorylases and Cellobiose 2-Epimerase DISCOVERY OF A NEW CARBOHYDRATE PHOSPHORYLASE, β-1,4-MANNOOLIGOSACCHARIDE PHOSPHORYLASE
Metabolic Mechanism of Mannan in a Ruminal Bacterium, Ruminococcus albus, Involving Two Mannoside Phosphorylases and Cellobiose 2-Epimerase DISCOVERY OF A NEW CARBOHYDRATE PHOSPHORYLASE, β-1,4-MANNOOLIGOSACCHARIDE PHOSPHORYLASE
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DOI：
10.1074/jbc.m112.390336
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发表时间：
2012-12-07
影响因子：
4.8
通讯作者：
Matsui, Hirokazu
中科院分区：
文献类型：
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作者：
Kawahara, Ryosuke;Saburi, Wataru;Matsui, Hirokazu
白色瘤胃球菌是典型的消化纤维素和半纤维素的瘤胃细菌。纤维二糖2-差向异构酶（CE；EC 5.1.3.11）可将纤维二糖转化为4-O-β-D-葡萄糖基-D-甘露糖，是R. albus中一种特别独特的酶，但其生理功能尚不清楚。最近，针对另一种产生 CE 的细菌脆弱拟杆菌，提出了一种涉及 CE 的新甘露聚糖代谢途径。在此途径中，β-1,4-甘露二糖通过 CE 差向异构化为 4-O-β-D-甘露糖基-D-葡萄糖 (Man-Glc)，并且 Man-Glc 通过 Man-Glc 磷酸化酶 (MP; EC 2.4.1.281) 磷酸化为 α-D-甘露糖基 1-磷酸 (Man1P) 和 D-葡萄糖。白色瘤胃球菌NE1表现出胞内MP活性，从无细胞提取物中获得了两种MP同工酶RaMP1和RaMP2。这些酶对底物非还原端的甘露糖残基具有高度特异性，并通过连续的 Bi Bi 机制催化 Man-Glc 的磷酸解和合成。在合成反应中，在 Man1P 存在的情况下，RaMP1 仅对 D-葡萄糖和 6-脱氧-D-葡萄糖表现出高活性，而 RaMP2 表现出与 RaMP1 显着不同的受体特异性。 RaMP2 作用于 C2 和 C3 位的 D-葡萄糖衍生物，包括脱氧和脱氧氟类似物以及差向异构体，但不作用于 C6 位取代的那些。此外，RaMP2 对以下寡糖具有高合成活性：β-连接葡萄糖二糖、麦芽糖、N,N'-二乙酰壳二糖和 β-1,4-甘露寡糖。特别是，β-1,4-甘露寡糖作为 RaMP2 的受体底物明显优于 D-葡萄糖。在磷酸解反应中，RaMP2 对 β-1,4-甘露二糖的活性较弱，但有效降解 β-1,4-甘露寡糖的时间长于 β-1,4-甘露二糖。因此，RaMP2被认为比β-1,4-甘露二糖更能催化β-1,4-甘露寡糖的磷酸解，产生Man1P和β-1,4-甘露二糖。
Ruminococcus albus is a typical ruminal bacterium digesting cellulose and hemicellulose. Cellobiose 2-epimerase (CE; EC 5.1.3.11), which converts cellobiose to 4-O-beta-D-glucosyl-D-mannose, is a particularly unique enzyme in R. albus, but its physiological function is unclear. Recently, a new metabolic pathway of mannan involving CE was postulated for another CE-producing bacterium, Bacteroides fragilis. In this pathway, beta-1,4-mannobiose is epimerized to 4-O-beta-D-mannosyl-D-glucose (Man-Glc) by CE, and Man-Glc is phosphorolyzed to alpha-D-mannosyl 1-phosphate (Man1P) and D-glucose by Man-Glc phosphorylase (MP; EC 2.4.1.281). Ruminococcus albus NE1 showed intracellular MP activity, and two MP isozymes, RaMP1 and RaMP2, were obtained from the cell-free extract. These enzymes were highly specific for the mannosyl residue at the non-reducing end of the substrate and catalyzed the phosphorolysis and synthesis of Man-Glc through a sequential Bi Bi mechanism. In a synthetic reaction, RaMP1 showed high activity only toward D-glucose and 6-deoxy-D-glucose in the presence of Man1P, whereas RaMP2 showed acceptor specificity significantly different from RaMP1. RaMP2 acted on D-glucose derivatives at the C2- and C3-positions, including deoxy- and deoxyfluoro-analogues and epimers, but not on those substituted at the C6-position. Furthermore, RaMP2 had high synthetic activity toward the following oligosaccharides: beta-linked glucobioses, maltose, N, N'-diacetylchitobiose, and beta-1,4-mannooligosaccharides. Particularly, beta-1,4-mannooligosaccharides served as significantly better acceptor substrates for RaMP2 than D-glucose. In the phosphorolytic reactions, RaMP2 had weak activity toward beta-1,4-mannobiose but efficiently degraded beta-1,4-mannooligosaccharides longer than beta-1,4-mannobiose. Consequently, RaMP2 is thought to catalyze the phosphorolysis of beta-1,4-mannooligosaccharides longer than beta-1,4-mannobiose to produce Man1P and beta-1,4-mannobiose.