Cloning, overexpression, purification, characterization and structural modelling of a metabolically active Fe2+ dependent 2,6-dichloro-p-hydroquinone 1,2-dioxygenase (CpsA) from Bacillus cereus strain AOA-CPS_1

2,6-Dichloro-p-hydroquinone (DiCHQ) aromatic-ring cleavage by DiCHQ 1,2-dioxygenase (CpsA) is very crucial for complete transformation of pentachlorophenol (PCP) to 2-chloromaleylacetate in Bacillus cereus AOA-CPS_1 (BcAOA). The 978 bp gene (cpsA) was detected and amplified in the genome of BcAOA; cloned, overexpressed and purified to homogeneity. CpsA showed a single approximately equal to congruent to 36.9 kDa protein band on SDS-PAGE and exhibited optimum activity at 30 degrees C and pH 9.0. CpsA was stable between 20 degrees C and 40 degrees C, and also retained about 90% of its activity at 60 degrees C for 120 min. The enzyme retained about 90% activity between pH 9.0 and 11.5 and 60% activity at pH 13.0. CpsA was found to be Fe2+ dependent as about 90% increased activity was observed in the presence of FeSO4. CpsA showed apparent v(max), K-m, k(cat) and k(cat)/K-m of 27.77 +/- 0.9 mu Ms-1, 0.990 +/- 0.03 mM, 4.20 +/- 0.04 s(-1) and 4.24 +/- 0.03 s(-1) mM(-1), respectively at pH 9.0. Analysis of the reaction products via GC-MS confirmed 2-chloromaleylacetate as the ring-cleavage product. CpsA 3D structure revealed a conserved 2-His-1-carboxylate facial triad motif (His 9, His 244 and Thr 11), with Fe3+ at the centre. Findings from this study provide new insights into the involvement of this enzyme in PCP degradation and suggests alternate possible mechanism of ring-cleavage by dioxygenases. (C) 2020 Elsevier B.V. All rights reserved.