X-ray crystallography, mass spectrometry and single crystal microspectrophotometry: A multidisciplinary characterization of catechol 1,2 dioxygenase

Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

Research Highlights
► Microspectrophotometric analysis reveals that Acinetobacter radioresistens catechol 1,2-dioxygenase behaves in a similar way in solution and in the crystalline state with respect to both Fe(III) coordination and ligand binding properties.
► By combining X-ray crystallography and mass spectrometry, the phospholipid tightly bound at the dimer interface was identified to be either a glycerophosphoinositol or a glycerophosphoinositol monophosphate with hydrophobic tail C18:0/C16:1(9Z).
► Crystallographic investigation provides an explanation for the inversion of specificity shown by the L69A and A72G variants in catechol 1,2-dioxygenase from Acinetobacter radioresistens.