Understanding Chlorocatechol 1,2-Dioxygenase Function: A Promising Player in Bioremediation Processes

Abstract

Dioxygenases are bacterial nonheme iron enzymes responsible for the aerobic catabolism of several intermediates produced by the decomposition of aromatic compounds that are industrially released in the environment and recalcitrant to biodegradation. Their paramount role in the degradation of toxic substances is the main interest in studying their mechanism of action. Pp 1,2-CCD belongs to the intradiol dioxygenase family, whose structures of its members have been only recently determined. In all those structures, an unexpected and interesting feature came up: electron density for two phospholipid molecules was found at the dimer interface, bringing up issues related to the relevance of those amphipatic molecules to enzyme catalysis. In this work, we present a series of results from our group focused on the characterization of the phospholipid binding and also on its role in enzyme function. We investigate, using a combination of experimental techniques (ESR, DSC, ITC, CD, UV-Vis absorbance), Pseudomonas putida chlorocatechol 1,2-dioxygenase (Pp 1,2-CCD) before and after delipidation procedures. Our results show that: (1) Fe(III) ion is converted to Fe(II) during catalysis and that this iron site in not influenced by the presence/absence of the phospholipid molecules; (2) Pp 1,2-CCD does not interact with several lipid model membranes; (3) delipidated Pp 1,2-CCD presents a cooperative kinetics, thus differing from lipid-containing Pp 1,2-CCD, which follows a usual Michaelis-Menten kinetics; (4) the product of the catalysis is capable of inhibiting the reaction; (5) delipidation leads to a somewhat more stable enzyme, with higher thermal-transition temperature and enthalpy. We discuss those findings in terms of Pp 1,2-CCD crystal structure that we have recently determined. These results, although regarding more fundamental aspects of the activity, shed light on features of Pp 1,2-CCD function that can be relevant towards its use in bioremediation/biotechnological processes.