Engineering A-type Dye-Decolorizing Peroxidases by Modification of a Conserved Glutamate Residue.

Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many open questions. Their heme-containing active site is buried by three conserved flexible loops with a putative role in modulating substrate access and enzyme catalysis. Here, we investigated the role of a conserved glutamate residue in stabilizing interactions in loop 2 of A-type DyPs. First, we did site saturation mutagenesis of this residue, replacing it with all possible amino acids in bacterial DyPs from Bacillus subtilis (BsDyP) and from Kitasatospora aureofaciens (KaDyP1), the latter being characterized here for the first time. We screened the resulting libraries of variants for activity towards ABTS and identified variants with increased catalytic efficiency. The selected variants were purified and characterized for activity and stability. We furthermore used Molecular Dynamics simulations to rationalize the increased catalytic efficiency and found that the main reason is the electron channeling becoming easier from surface-exposed tryptophans. Based on our findings, we also propose that this glutamate could work as a pH switch in the wild-type enzyme, preventing intracellular damage.

Purification of proteins with native terminal sequences using a Ni(II)-cleavable C-terminal hexahistidine affinity tag.

The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a metallonuclease with its precisely determined native termini. First, the gene encoding the target protein is inserted into a newly designed cloning site, which contains two self-eliminating BsmBI restriction enzyme sites. As a consequence, the engineered DNA code of Ni(II)-sensitive Ser-X-His-X motif is fused to the 3'-end of the inserted gene followed by the gene of an affinity tag for protein purification purpose. The C-terminal segment starting from Ser mentioned above is cleaved off from purified protein by a Ni(II)-induced protease-like action. The success of the purification and cleavage was confirmed by gel electrophoresis and mass spectrometry, while structural integrity of the purified protein was checked by circular dichroism spectroscopy. Our new protein expression DNA construct is an advantageous tool for protein purification, when the complete removal of affinity or other tags, without any remaining amino acid residue is essential. The described procedure can easily be generalized and combined with various affinity tags at the C-terminus for chromatographic applications.