Spectroscopic Characterization of a Binuclear Metal Site in Bacillus cereus -Lactamase II
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- 作者：Elena G. Orellano ; Javier E. Girardini ; Julia A. Cricco ; Eduardo A. Ceccarelli ; and Alejandro J. Vila
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：July 14, 1998
- 年：1998
- 卷：37
- 期：28
- 页码：10173 - 10180
- 全文大小：98K
- 年卷期：v.37,no.28(July 14, 1998)
- ISSN：1520-4995
文摘
The zinc metalloenzyme 
-lactamase II (LII) from Bacillus cereus has been overexpressedin Escherichia coli as a fusion protein with glutathione-S-transferase, and the metal binding properties ofrecombinant LII toward Zn(II) and Co(II) have been studied by fluorescence and activity measurements.The apoenzyme is able to bind two metal ion equivalents, which confer on LII its maximum enzymaticefficiency. The enzyme is partially active with one metal ion equivalent. The diCo(II) and a mixedZn(II)Co(II) derivative of LII were obtained and probed by electronic and paramagnetic NMRspectroscopy. In the high-affinity site, the metal is bound to three His residues and a solvent molecule,adopting a tetrahedral geometry. A Cys, a His, and an Asp residue are coordinated to the low-affinitymetal site, together with two or three solvent molecules. This coordination polyhedron resembles thebinuclear metal site of the Bacteroides fragilis -lactamase [Concha, N., Rasmussen, B. A., Bush, K.,and Herzberg, O. (1996) Structure 4, 823-836; Carfi, A., Duée, E., Paul-Soto, R., Galleni, M., Frère, J.M., and Dideberg, O. (1998) Acta Crystallogr. D54, 47-57] but differs from that resulting from theX-ray study of LII [Carfi, A., Pares, S., Duée, E., Galleni, M., Duez, C., Frère, J. M., and Dideberg, O.(1995) EMBO J. 14, 4914-4921]. These results suggest that this binuclear metal site may be a generalfeature of metallo--lactamases.
-lactamase II (LII) from Bacillus cereus has been overexpressedin Escherichia coli as a fusion protein with glutathione-S-transferase, and the metal binding properties ofrecombinant LII toward Zn(II) and Co(II) have been studied by fluorescence and activity measurements.The apoenzyme is able to bind two metal ion equivalents, which confer on LII its maximum enzymaticefficiency. The enzyme is partially active with one metal ion equivalent. The diCo(II) and a mixedZn(II)Co(II) derivative of LII were obtained and probed by electronic and paramagnetic NMRspectroscopy. In the high-affinity site, the metal is bound to three His residues and a solvent molecule,adopting a tetrahedral geometry. A Cys, a His, and an Asp residue are coordinated to the low-affinitymetal site, together with two or three solvent molecules. This coordination polyhedron resembles thebinuclear metal site of the Bacteroides fragilis -lactamase [Concha, N., Rasmussen, B. A., Bush, K.,and Herzberg, O. (1996) Structure 4, 823-836; Carfi, A., Duée, E., Paul-Soto, R., Galleni, M., Frère, J.M., and Dideberg, O. (1998) Acta Crystallogr. D54, 47-57] but differs from that resulting from theX-ray study of LII [Carfi, A., Pares, S., Duée, E., Galleni, M., Duez, C., Frère, J. M., and Dideberg, O.(1995) EMBO J. 14, 4914-4921]. These results suggest that this binuclear metal site may be a generalfeature of metallo--lactamases.