ZiPD (zinc phosphodiesterase; synonyms are ElaC, ecoZ, RNaseZ and 3′ tRNase) and the iron-dependent redox enzyme FlRd (flavorubredoxin) from Escherichia coli represent prototypical cases of proteins sharing the metallo-β-lactamase fold that require strict metal selectivity for catalytic activity, yet their metal selectivity has only been partially understood. In contrast with hydrolytic metallo-β-lactamase proteins, iron-dependent FlRd-like enzymes have an atypical glutamate ligand, which replaces one otherwise conserved histidine ligand. X-ray absorption spectroscopy revealed that the FlRd metallo-β-lactamase domain is capable of incorporating two zinc ions into the binuclear metal-binding site. Zinc dissociation constants, determined by isothermal titration calorimetry are similar for zinc binding to E. coli ZiPD (Kd1=2.2±0.2 μM and Kd2=23.0±0.6 μM) and to the E. coli FlRd metallo-β-lactamase domain (Kd1=0.7±0.1 μM and Kd2=26.0±0.1 μM). In good correspondence, apo-ZiPD requires incubation with 10 μM zinc for full reconstitution of the phosphodiesterase activity. Accordingly, metal selectivity of ZiPD and FlRd only partially relies on first shell metal ligands. Back mutation of the atypical glutamate in FlRd to a histidine unexpectedly resulted in an increased first zinc dissociation constant (Kd1=30±4 μM and Kd2=23±2 μM). In combination with a recent mutational study on ZiPD [Vogel, Schilling and Meyer-Klaucke (2004) Biochemistry 43, 10379–10386], we conclude that the atypical glutamate does not guide metal selectivity of the FlRd metallo-β-lactamase domain but suppresses possible hydrolytic cross-activity.
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Research Article|
December 14 2004
Zinc- and iron-dependent cytosolic metallo-β-lactamase domain proteins exhibit similar zinc-binding affinities, independent of an atypical glutamate at the metal-binding site
Oliver SCHILLING;
Oliver SCHILLING
*EMBL Outstation Hamburg, Notkestrasse 85, D-22603 Hamburg, Germany
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Andreas VOGEL;
Andreas VOGEL
1
*EMBL Outstation Hamburg, Notkestrasse 85, D-22603 Hamburg, Germany
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Brenda KOSTELECKY;
Brenda KOSTELECKY
*EMBL Outstation Hamburg, Notkestrasse 85, D-22603 Hamburg, Germany
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Hugo NATAL da LUZ;
Hugo NATAL da LUZ
†Institute for Experimental Physics II, University of Leipzig, Linnéstr. 5, D-04103 Leipzig, Germany
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Daniel SPEMANN;
Daniel SPEMANN
†Institute for Experimental Physics II, University of Leipzig, Linnéstr. 5, D-04103 Leipzig, Germany
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Bettina SPÄTH;
Bettina SPÄTH
‡Molekulare Botanik, Universität Ulm, D-89069 Ulm, Germany
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Anita MARCHFELDER;
Anita MARCHFELDER
‡Molekulare Botanik, Universität Ulm, D-89069 Ulm, Germany
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Wolfgang TRÖGER;
Wolfgang TRÖGER
†Institute for Experimental Physics II, University of Leipzig, Linnéstr. 5, D-04103 Leipzig, Germany
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Wolfram MEYER-KLAUCKE
Wolfram MEYER-KLAUCKE
2
*EMBL Outstation Hamburg, Notkestrasse 85, D-22603 Hamburg, Germany
2To whom correspondence should be addressed (email wolfram@embl-hamburg.de).
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Publisher: Portland Press Ltd
Received:
May 11 2004
Revision Received:
August 10 2004
Accepted:
August 23 2004
Accepted Manuscript online:
August 23 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 385 (1): 145–153.
Article history
Received:
May 11 2004
Revision Received:
August 10 2004
Accepted:
August 23 2004
Accepted Manuscript online:
August 23 2004
Citation
Oliver SCHILLING, Andreas VOGEL, Brenda KOSTELECKY, Hugo NATAL da LUZ, Daniel SPEMANN, Bettina SPÄTH, Anita MARCHFELDER, Wolfgang TRÖGER, Wolfram MEYER-KLAUCKE; Zinc- and iron-dependent cytosolic metallo-β-lactamase domain proteins exhibit similar zinc-binding affinities, independent of an atypical glutamate at the metal-binding site. Biochem J 1 January 2005; 385 (1): 145–153. doi: https://doi.org/10.1042/BJ20040773
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