Structure of the extended-spectrum class C beta-lactamase of Enterobacter cloacae GC1, a natural mutant with a tandem tripeptide insertion.
Structure of the extended-spectrum class C beta-lactamase of Enterobacter cloacae GC1, a natural mutant with a tandem tripeptide insertion.
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阴沟肠杆菌 GC1 的超广谱 C 类 β-内酰胺酶的结构，具有串联三肽插入的天然突变体。
作者：
G. Crichlow;A. Kuzin;M. Nukaga;K. Mayama;T. Sawai;J. Knox
通过X射线晶体学分析了来自临床分离的阴沟肠杆菌菌株GC 1的C类β-内酰胺酶，该酶对氧亚氨基β-内酰胺抗生素具有改进的水解活性，分辨率为1.8 A。相对于野生型P99 β-内酰胺酶，该天然突变体含有高度独特的串联重复Ala 211-Val 212-Arg 213 [Nugaka等（1995）J.Biol.Chem.270，5729-5735]。39.4 kDa的染色体β-内酰胺酶在磷酸钾中从聚（乙二醇）8000中结晶，空间群为P2（1）2（1）2，细胞尺寸a = 78.0 A，B = 69.5 A和c = 63.1 A。用分子置换法求解晶体结构，并对8 ~ 1.8 A范围内的所有非零数据将模型修正到R因子为0.20。模型键和角度与理想值的偏差分别为0.008 A和1.4度。GC 1和P99 β-内酰胺酶中α-碳原子的重叠导致均方根偏差为0.6 A。最大的偏差发生在含有Gln 120的环和Omega环区域（200-218）中，其中三个残基213-215是无序的。可能由于这种紊乱，从318-324 β链到另一侧含有Gln 120和Tyr 150的两个环测量的底物结合腔的开口宽度比P99宽0.6-1.4 A。有人建议，构象的灵活性，在扩大欧米茄环，其对邻近蛋白质结构的影响，可能会促进水解的羟亚氨基β-内酰胺，使酰基中间体更开放的攻击水。然而，核心催化位点残基Ser 64、Lys 67、Tyr 150、Asn 152、Lys 318和Ser 321中的骨架原子与P99中的原子仅偏离0.4 A（rmsd）。在pH 8下，潜在催化碱Tyr 150相对于P99的旋转与该残基低于正常pK（a）的要求一致。
A class C beta-lactamase from a clinical isolate of Enterobacter cloacae strain GC1 with improved hydrolytic activity for oxyimino beta-lactam antibiotics has been analyzed by X-ray crystallography to 1.8 A resolution. Relative to the wild-type P99 beta-lactamase, this natural mutant contains a highly unique tandem repeat Ala211-Val212-Arg213 [Nugaka et al. (1995) J. Biol. Chem. 270, 5729-5735]. The 39.4 kDa chromosomal beta-lactamase crystallizes from poly(ethylene glycol) 8000 in potassium phosphate in space group P2(1)2(1)2 with cell dimensions a = 78.0 A, b = 69.5 A, and c = 63.1 A. The crystal structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.20 for all nonzero data from 8 to 1.8 A. Deviations of model bonds and angles from ideal values are 0.008 A and 1.4 degrees, respectively. Overlay of alpha-carbon atoms in the GC1 and P99 beta-lactamases results in an rms deviation of 0.6 A. Largest deviations occur in a loop containing Gln120 and in the Omega loop region (200-218) where the three residues 213-215 are disordered. Possibly as a result of this disorder, the width of the opening to the substrate binding cavity, as measured from the 318-324 beta-strand to two loops containing Gln120 and Tyr150 on the other side, is 0.6-1.4 A wider than in P99. It is suggested that conformational flexibility in the expanded Omega loop, and its influence on adjacent protein structure, may facilitate hydrolysis of oxyimino beta-lactams by making the acyl intermediate more open to attack by water. Nevertheless, backbone atoms in core catalytic site residues Ser64, Lys67, Tyr150, Asn152, Lys318, and Ser321 deviate only 0.4 A (rmsd) from atoms in P99. A rotation of a potential catalytic base, Tyr150, relative to P99 at pH 8, is consistent with the requirement for a lower than normal pK(a) for this residue.