Classic Pentachlorophenol Hydroxylating Phenylalanine 4-Monooxygenase from Indigenous Bacillus tropicus Strain AOA-CPS1: Cloning, Overexpression, Purification, Characterization and Structural Homology Modelling

The metabolically promiscuous pentachlorophenol (PCP) hydroxylating Phe4MO (represented as CpsB) was detected, amplified (from the genome of Bacillus tropicus strain AOA-CPS1), cloned, overexpressed, purified and characterized here. The 1.755-kb gene cloned in the pET15b vector expressed a approximately equal to 64 kDa monomeric protein which was purified to homogeneity by single-step affinity chromatography, with a total yield of 82.1%. The optimum temperature and pH of the enzyme were found to be 30 degrees C and 7.0, respectively. CpsB showed functional stability between pH 6.0-7.5 and temperature 25-30 degrees C. The enzyme-substrate reaction kinetic studies showed the allosteric nature of the enzyme and followed pre-steady state using NADH as a co-substrate with apparent v(max), K-m, k(cat) and k(cat)/K-m values of 0.465 mu M(.)s(-1), 140 mu M, 0.099 s(-1) and 7.07 x 10(-4) mu M(-1.)s(-1), respectively, for the substrate PCP. The in-gel trypsin digestion experiments and bioinformatic tools confirmed that the reported enzyme is a Phe4MO with multiple putative conserved domains and metal ion-binding site. Though Phe4MO has been reported to have a diverse catalytic function, here we report, for the first time, that it functions as a PCP dehalogenase or PCP-4-monooxygenase by hydroxylating PCP. Hence, the use of this enzyme may be further explored in the bioremediation of PCP and other related xenobiotics.

Automated summary

The metabolically promiscuous pentachlorophenol (PCP) hydroxylating Phe4MO, represented as CpsB, was detected and cloned from the genome of Bacillus tropicus strain AOA-CPS1. The enzyme functions as a PCP dehalogenase or PCP-4-monooxygenase, showing optimal activity at 30 degrees C and pH 7.0.