Exploring the Roles of the Metal Ions in Escherichia coli Copper Amine Oxidase^,

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 ∼12% wild-type activity as measured by kcat(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.

Effects of ABA and CaCl2 on GABA accumulation in fava bean germinating under hypoxia-NaCl stress

“…14,17) Ca 2+ is necessary for activating GAD activity in plants; meanwhile, there is a Ca 2+ binding site in DAO. 18) Hence, EDTA decreased the GABA accumulation by chelating endogenous Ca 2+ and then inhibiting GAD and DAO activities.…”

“…Ca 2+ is necessary for GAD and DAO activation since GAD and DAO are Ca 2+ binding proteins. 18) The unimpeded metabolism of GABA shunt and polyamine degradation pathway contributed to enhance the resistance ability of plants. In the present study, exogenous ABA and Ca 2+ treatments led to a sharp increase in GABA content in germinated fava bean (Fig.…”

High-resolution crystal structure of copper amine oxidase fromArthrobacter globiformis: assignment of bound diatomic molecules as O2

“…Furthermore, the metal-coordination distances to main-chain carbonyl groups (2.5 Å ), carboxyl groups (2.4 Å ) or the water molecule (2.7 Å ) were more preferable for Na + than for K + (Harding, 2006). In the structures of AGAO GL2 (Langley et al, 2006) and ECAO (Smith et al, 2010), an Na + and a Ca 2+ ion is bound in this site, respectively, whereas this site is vacant in AGAO RT (Wilce et al, 1997) and AGAO GL (Kishishita et al, 2003). The second Cu 2+ ion identified in the vicinity of Asp161, Asp165, His170 and His201 located on the protein surface of AGAO GL2 (Langley et al, 2006) is replaced by a water molecule in AGAO PG .…”

“…6). Although it is controversial whether the bound copper(II) atom plays a direct innersphere role or an indirect outer-sphere role in the re-oxidation of reduced TPQ by O 2 depending on the source of the CuAO (Smith et al, 2010;Mukherjee et al, 2008), a recent kinetic and spectroscopic study has demonstrated that the bound copper(II) atom in AGAO mediates electron transfer to O 2 through the copper(I)-semiquinone intermediate (direct inner-sphere mechanism; Shepard et al, 2008). We have also determined the crystal structure of AGAO anaerobically reduced with substrate and found that the TPQ in the copper(I)-semiquinone state is in the on-copper conformation (T. Okajima, unpublished results).…”

Recent advances in γ‐aminobutyric acid (GABA) properties in pulses: an overview

“…Other effective factors for modifying GABA production in pulses were reported to be Ca 2+ and a chelating agent such as ethylene‐diamine‐tetra‐acetic acid (EDTA). Calcium ions are required for GAD and DAO activation since these enzymes are Ca 2+ binding proteins, therefore Ca 2+ addition (in CaCl 2 form) can lead to their activation, thus resulting in GABA content enhancement. In terms of GAD activity in pulses, environmental conditions (e.g.…”