Structure of the Extended-Spectrum Class C -Lactamase of Enterobacter cloacae GC1, a Natural Mutant with a Tan
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- 作者：Gregg V. Crichlow ; Alexandre P. Kuzin ; Michiyoshi Nukaga ; Kayoko Mayama ; Tetsuo Sawai ; and James R. Knox
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：August 10, 1999
- 年：1999
- 卷：38
- 期：32
- 页码：10256 - 10261
- 全文大小：297K
- 年卷期：v.38,no.32(August 10, 1999)
- ISSN：1520-4995
文摘
A class C 
-lactamase from a clinical isolate of Enterobacter cloacae strain GC1 with improvedhydrolytic activity for oxyimino -lactam antibiotics has been analyzed by X-ray crystallography to 1.8Å resolution. Relative to the wild-type P99 -lactamase, this natural mutant contains a highly uniquetandem repeat Ala211-Val212-Arg213 [Nugaka et al. (1995) J. Biol. Chem. 270, 5729-5735]. The 39.4kDa chromosomal -lactamase crystallizes from poly(ethylene glycol) 8000 in potassium phosphate inspace group P21212 with cell dimensions a = 78.0 Å, b = 69.5 Å, and c = 63.1 Å. The crystal structurewas solved by the molecular replacement method, and the model has been refined to an R-factor of 0.20for all nonzero data from 8 to 1.8 Å. Deviations of model bonds and angles from ideal values are 0.008Å and 1.4
, respectively. Overlay of 
pha.gif" BORDER=0>-carbon atoms in the GC1 and P99 -lactamases results in an rmsdeviation of 0.6 Å. Largest deviations occur in a loop containing Gln120 and in the 
loop region (200-218) where the three residues 213-215 are disordered. Possibly as a result of this disorder, the width ofthe opening to the substrate binding cavity, as measured from the 318-324 -strand to two loops containingGln120 and Tyr150 on the other side, is 0.6-1.4 Å wider than in P99. It is suggested that conformationalflexibility in the expanded loop, and its influence on adjacent protein structure, may facilitate hydrolysisof oxyimino -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 deviateonly 0.4 Å (rmsd) from atoms in P99. A rotation of a potential catalytic base, Tyr150, relative to P99 atpH 8, is consistent with the requirement for a lower than normal pKa for this residue.
-lactamase from a clinical isolate of Enterobacter cloacae strain GC1 with improvedhydrolytic activity for oxyimino -lactam antibiotics has been analyzed by X-ray crystallography to 1.8Å resolution. Relative to the wild-type P99 -lactamase, this natural mutant contains a highly uniquetandem repeat Ala211-Val212-Arg213 [Nugaka et al. (1995) J. Biol. Chem. 270, 5729-5735]. The 39.4kDa chromosomal -lactamase crystallizes from poly(ethylene glycol) 8000 in potassium phosphate inspace group P21212 with cell dimensions a = 78.0 Å, b = 69.5 Å, and c = 63.1 Å. The crystal structurewas solved by the molecular replacement method, and the model has been refined to an R-factor of 0.20for all nonzero data from 8 to 1.8 Å. Deviations of model bonds and angles from ideal values are 0.008Å and 1.4, respectively. Overlay of pha.gif" BORDER=0>-carbon atoms in the GC1 and P99 -lactamases results in an rmsdeviation of 0.6 Å. Largest deviations occur in a loop containing Gln120 and in the loop region (200-218) where the three residues 213-215 are disordered. Possibly as a result of this disorder, the width ofthe opening to the substrate binding cavity, as measured from the 318-324 -strand to two loops containingGln120 and Tyr150 on the other side, is 0.6-1.4 Å wider than in P99. It is suggested that conformationalflexibility in the expanded loop, and its influence on adjacent protein structure, may facilitate hydrolysisof oxyimino -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 deviateonly 0.4 Å (rmsd) from atoms in P99. A rotation of a potential catalytic base, Tyr150, relative to P99 atpH 8, is consistent with the requirement for a lower than normal pKa for this residue.