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.
Micalella
Chiara
C
Department of Biochemistry and Molecular Biology, University of Parma, Viale GP. Usberti 23/A, 43100 Parma, Italy.
Martignon
Sara
S
Bruno
Stefano
S
Pioselli
Barbara
B
Caglio
Raffaella
R
Valetti
Francesca
F
Pessione
Enrica
E
Giunta
Carlo
C
Rizzi
Menico
M
D000150:Acinetobacter / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D050548:Catechol 1,2-Dioxygenase / Q000737:chemistry* / Q000378:metabolism; D002396:Catechols / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D015591:Microspectrophotometry / Q000379:methods; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding
D001426:Bacterial Proteins; D002396:Catechols; D017261:Glycosylphosphatidylinositols; D050548:Catechol 1,2-Dioxygenase; C034221:catechol