2WO0

EDTA treated E. coli copper amine oxidase

Summary for 2WO0

• Entry DOI: 10.2210/pdb2wo0/pdb
• Related: 1D6U 1D6Y 1D6Z 1DYU 1JRQ 1LVN 1OAC 1QAF 1QAK 1QAL 1SPU 2W0Q 2WGQ
• Descriptor: PRIMARY AMINE OXIDASE, COPPER (II) ION, SODIUM ION, ... (4 entities in total)
• Functional Keywords: oxidoreductase, amine oxidation, copper amine oxidase, tpq, metal-binding
• Biological source: ESCHERICHIA COLI
• Cellular location: Periplasm: P46883
• Total number of polymer chains: 2
• Total formula weight: 162952.51

Authors

Smith, M.A.,Pirrat, P.,Pearson, A.R.,Knowles, P.F.,Phillips, S.E.V.,McPherson, M.J. (deposition date: 2009-07-21, release date: 2010-05-05, Last modification date: 2011-07-13)

Primary citation

Smith, M.A.,Pirrat, P.,Pearson, A.R.,Kurtis, C.R.,Gaule, T.G.,Knowles, P.F.,Phillips, S.E.,McPherson, M.J.
Exploring the Roles of the Metal Ions in Escherichia Coli Copper Amine Oxidase.
Biochemistry, 49:1268-, 2010

PubMed Abstract: To investigate the role of the active site copper in Escherichia coli copper amine oxidase (ECAO), we initiated a metal-substitution study. Copper reconstitution of ECAO (Cu-ECAO) restored only approximately 12% wild-type activity as measured by k(cat(amine)). Treatment with EDTA, to remove exogenous divalent metals, increased Cu-ECAO activity but reduced the activity of wild-type ECAO. Subsequent addition of calcium restored wild-type ECAO and further enhanced Cu-ECAO activities. Cobalt-reconstituted ECAO (Co-ECAO) showed lower but significant activity. These initial results are consistent with a direct electron transfer from TPQ to oxygen stabilized by the metal. If a Cu(I)-TPQ semiquinone mechanism operates, then an alternative outer-sphere electron transfer must also exist to account for the catalytic activity of Co-ECAO. The positive effect of calcium on ECAO activity led us to investigate the peripheral calcium binding sites of ECAO. Crystallographic analysis of wild-type ECAO structures, determined in the presence and absence of EDTA, confirmed that calcium is the normal ligand of these peripheral sites. The more solvent exposed calcium can be easily displaced by mono- and divalent cations with no effect on activity, whereas removal of the more buried calcium ion with EDTA resulted in a 60-90% reduction in ECAO activity and the presence of a lag phase, which could be overcome under oxygen saturation or by reoccupying the buried site with various divalent cations. Our studies indicate that binding of metal ions in the peripheral sites, while not essential, is important for maximal enzymatic activity in the mature enzyme.

PubMed: 20052994
DOI: 10.1021/BI901738K

Experimental method

X-RAY DIFFRACTION (2.6 Å)