4AKP

Mutations in the neighbourhood of CotA-laccase trinuclear site: E498T mutant

Summary for 4AKP

• Entry DOI: 10.2210/pdb4akp/pdb
• Related: 1GSK 1OF0 1UVW 1W6L 1W6W 1W8E 2BHF 2X87 2X88 4A66 4A67 4A68 4AKO 4AKQ
• Descriptor: SPORE COAT PROTEIN A, COPPER (II) ION, OXYGEN MOLECULE, ... (5 entities in total)
• Functional Keywords: oxidoreductase, multi-copper oxidase, trinuclear cluster oxygen reduction
• Biological source: BACILLUS SUBTILIS
• Total number of polymer chains: 1
• Total formula weight: 59593.78

Authors

Silva, C.S.,Chen, Z.,Durao, P.,Pereira, M.M.,Todorovic, S.,Hildebrandt, P.,Martins, L.O.,Lindley, P.F.,Bento, I. (deposition date: 2012-02-28, release date: 2012-03-14, Last modification date: 2023-12-20)

Primary citation

Chen, Z.,Durao, P.,Silva, C.S.,Pereira, M.M.,Todorovic, S.,Hildebrandt, P.,Bento, I.,Lindley, P.F.,Martins, L.O.
The Role of Glu498 in the Dioxygen Reactivity of Cota-Laccase from Bacillus Subtilis.
Dalton Trans, 39:2875-, 2010

PubMed Abstract: The multicopper oxidases couple the one-electron oxidation of four substrate molecules to the four electron reductive cleavage of the O-O bond of dioxygen. This reduction takes place at the trinuclear copper centre of the enzyme and the dioxygen approaches this centre through an entrance channel. In this channel, an acidic residue plays a key role in steering the dioxygen to the trinuclear copper site, providing protons for the catalytic reaction and giving overall stability to this site. In this study, the role of the Glu(498) residue, located within the entrance channel to the trinuclear copper centre, has been investigated in the binding and reduction of dioxygen by the CotA-laccase from Bacillus subtilis. The absence of an acidic group at the 498 residue, as in the E498T and E498L mutants, results in a severe catalytic impairment, higher than 99%, for the phenolic and non-phenolic substrates tested. The replacement of this glutamate by aspartate leads to an activity that is around 10% relative to that of the wild-type. Furthermore, while this latter mutant shows a similar K(m) value for dioxygen, the E498T and E498L mutants show a decreased affinity, when compared to the wild-type. X-ray structural and spectroscopic analysis (UV-visible, electron paramagnetic resonance and resonance Raman) reveal perturbations of the structural properties of the catalytic centres in the Glu(498) mutants when compared to the wild-type protein. Overall, the results strongly suggest that Glu(498) plays a key role in the protonation events that occur at the trinuclear centre and in its stabilization, controlling therefore the binding of dioxygen and its further reduction.

PubMed: 20200715
DOI: 10.1039/B922734B

Experimental method

X-RAY DIFFRACTION (2 Å)