scholarly journals 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

2004 ◽  
Vol 385 (1) ◽  
pp. 145-153 ◽  
Author(s):  
Oliver SCHILLING ◽  
Andreas VOGEL ◽  
Brenda KOSTELECKY ◽  
Hugo NATAL da LUZ ◽  
Daniel SPEMANN ◽  
...  

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.

2009 ◽  
Vol 96 (3) ◽  
pp. 58a
Author(s):  
Christine M. Phillips ◽  
Paul S. Nerenberg ◽  
Catherine L. Drennan ◽  
Collin M. Stultz

1992 ◽  
Vol 11 (9) ◽  
pp. 3209-3217 ◽  
Author(s):  
J. van der Oost ◽  
P. Lappalainen ◽  
A. Musacchio ◽  
A. Warne ◽  
L. Lemieux ◽  
...  

Biochemistry ◽  
2002 ◽  
Vol 41 (15) ◽  
pp. 4809-4818 ◽  
Author(s):  
Gloria C. Ferreira ◽  
Ricardo Franco ◽  
Arianna Mangravita ◽  
Graham N. George

2006 ◽  
Vol 101 (4) ◽  
pp. 354-360 ◽  
Author(s):  
Mitsutoshi Toyama ◽  
Mariko Sasaki ◽  
Noriaki Hirayama ◽  
Yoshikatsu Murooka ◽  
Mitsuo Yamashita

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