Structure of the Cellulose Synthase Complex of Gluconacetobacter hansenii at 23.4 Å Resolution.
Structure of the Cellulose Synthase Complex of Gluconacetobacter hansenii at 23.4 Å Resolution.
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DOI：
10.1371/journal.pone.0155886
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Nixon BT
中科院分区：
文献类型：
--
作者：
Du J;Vepachedu V;Cho SH;Kumar M;Nixon BT
细菌结晶纤维素广泛应用于生物医学和工业领域，但其合成的分子机制尚不清楚。与大多数制造非晶态纤维素的细菌不同，汉森葡萄糖乙酸杆菌能分泌大量的晶态纤维素。它的纤维素合成酶(ACSA)与辅助蛋白AcsB形成一个‘末端复合体’(TC)，并在结晶纤维素带的底部用冷冻断裂的电子显微镜观察到。催化的AcsAB复合体被包埋在细胞膜中。推测ACSC的C末端部分在外膜上形成一个易位通道，其余的ACSC可能在周质中与ACSD相互作用。因此，人们认为，在ACSC和ACSD挤压的配合下，从一系列有组织的TC合成能够使这种细菌产生结晶纤维素。该体系唯一的结构数据是上述冷冻断口的电子显微镜图像，荧光显微镜图像显示TCS排列成一排，ACSD的晶体结构与纤维五糖结合，以及ACSA的Pilz结构域的晶体结构。在这里，我们通过确定高纯度的AcsAB络合物的负染色结构来推进我们对细菌纤维素合成酶生产结晶纤维素的结构基础的理解，该络合物催化UDP-葡萄糖进入β-1，4-葡聚糖链，并对变构激活剂环二鸟苷的存在做出反应。虽然AcsAB复合体在体外具有功能，但在透射电子显微镜下看不到合成的纤维素。阴性染色结构表明，AcsAB与非晶态纤维素产生菌球形红杆菌的BcsAB合成酶非常相似。结果表明，结晶纤维素产生菌和非结晶纤维素产生菌具有保守的催化和膜转位成分，支持线性排列的TCS中的挤出机制和顺序使结晶纤维素产生的假说。
Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into β-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose.