Choi S K, Ljungdahl L G
Department of Biochemistry and Molecular Biology, Center for Biological Resources Recovery, University of Georgia, Athens, 30602-7229, USA.
Biochemistry. 1996 Apr 16;35(15):4897-905. doi: 10.1021/bi9524629.
The cellulosome of Clostridium thermocellum JW20 consists of 14-26 different polypeptides as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intact cellulosomes hydrolyzes crystalline cellulose in the presence of Ca2+ and thiols. This activity is inhibited by ethylenediaminetetraacetic acid (EDTA). Ca is incorporated into the cellulosome and tightly bound as demonstrated using 45Ca added to the growth medium. Upon incubation in 50 mM Tris (pH 7.5), 0.1 M NaCl, and 5 mM EDTA at 37 degrees C, Ca is released from the cellulosome, which disintegrates into polypeptides. The SDS-PAGE pattern of cellulosomal polypeptides is remarkably different after the EDTA treatment when compared to this pattern of untreated cellulosomes. Polypeptide bands corresponding to molecular masses of 160, 98, 76, 91, and 54 kDa disappear, and new bands of masses 150, 132, 91, 71, 57, and 46 kDa appear. N-terminal analyses of the 98, 76, 91, and 71 kDa polypeptides show that the 91 and 71 kDa polypeptides are truncated products of the 98 and 76 kDa polypeptides, respectively. The 76 and 71 kDa polypeptides correspond to CelS [Wang, W. K., Kruus K., & Wu, J. H. D. (1993) J. Bacteriol. 175, 1293-1302]. The 71 kDa polypeptide is formed from the 76 kDa polypeptide during the EDTA treatment, by a cleavage that occurs at asparagine residue 681. It involves the removal of 60 amino acid residues from the C-terminal end. All catalytic subunits so far characterized contain an asparagine residue corresponding to residue 681 of CelS. This residue is part of the conserved duplicated region found in catalytically active subunits, and it is postulated that several of these subunits also are truncated by the EDTA treatment. The polypeptides truncated by the EDTA treatment reduced Ca binding capacities compared to their native subunits, indicating a Ca-binding site within the conserved duplicated region. This region has been implicated in the binding of the catalytic peptides to the scaffolding polypeptide CipA, which is a structural protein of the cellulosome and has a strong affinity for calcium.
嗜热栖热菌JW20的纤维小体由14 - 26种不同的多肽组成，这是通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定的。完整的纤维小体在Ca2 +和硫醇存在的情况下能水解结晶纤维素。这种活性受到乙二胺四乙酸（EDTA）的抑制。Ca被整合到纤维小体中并紧密结合，这是通过向生长培养基中添加45Ca证明的。在37℃下于50 mM Tris（pH 7.5）、0.1 M NaCl和5 mM EDTA中孵育后，Ca从纤维小体中释放出来，纤维小体分解为多肽。与未处理的纤维小体的SDS - PAGE图谱相比，EDTA处理后的纤维小体多肽的SDS - PAGE图谱有显著差异。对应于分子量160、98、76、91和54 kDa的多肽条带消失，出现了分子量为150、132、91、71、57和46 kDa的新条带。对98、76、91和71 kDa多肽的N端分析表明，91和71 kDa多肽分别是98和76 kDa多肽的截短产物。76和71 kDa多肽对应于CelS [Wang, W. K., Kruus K., & Wu, J. H. D. (1993) J. Bacteriol. 175, 1293 - 1302]。71 kDa多肽是在EDTA处理过程中由76 kDa多肽形成的，通过在天冬酰胺残基681处的切割。这涉及从C端去除60个氨基酸残基。迄今为止鉴定的所有催化亚基都含有一个对应于CelS残基681的天冬酰胺残基。该残基是在催化活性亚基中发现的保守重复区域的一部分，据推测这些亚基中的几个也会被EDTA处理截短。与它们的天然亚基相比，经EDTA处理截短的多肽降低了Ca结合能力，表明在保守重复区域内有一个Ca结合位点。该区域与催化肽与支架多肽CipA的结合有关，CipA是纤维小体的一种结构蛋白，对钙有很强的亲和力。
