Compartmentalization of the Carbaryl Degradation Pathway: Molecular Characterization of Inducible Periplasmic Carbaryl Hydrolase from Pseudomonas spp

Abstract

Pseudomonas sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of mcbA encoding CH in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4 to 6-fold higher in the periplasm as compared to the cytoplasm. The recombinant CH (rCH) showed 4-fold higher activity in the periplasm of Escherichia coli. The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of Tmd+Sp resulted in expression of the inactive protein. Confocal microscopic analysis of E. coli expressing (Tmd+Sp)-GFP fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. N-terminal sequence of the mature periplasmic CH confirms the absence of Tmd+Sp region and the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7 and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68-76 kDa, catalyze hydrolysis of ester bond with apparent K_m and V_max in the range of 98-111 μM and 69-73 μmol.min^-1.mg^-1, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol metabolizing enzymes in the cytoplasm of Pseudomonas spp. suggests the compartmentalization of metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentration without cellular toxicity of 1-naphthol.