A Scavenger Hunt for a DyP-Peroxidase from a Metagenome: Curated Peroxidase Database-Assisted Primer Design and Protein Structure Elucidation

Dye-decolorizing peroxidase (DyP)-type peroxidases are heme-containing enzymes that play a role in lignin synthesis and degradation and dye decolorization. Despite numerous studies about this class of enzymes, the enzyme remains under-explored. We used 1000 DyP sequences retrieved from the NCBI database to forge a phylogenetic tree. Nodes in the tree, where sequences displayed a degree of conservation, were used to design degenerate primers to locate DyP-peroxidase sequences from the DNA extract of a tannery wastewater sample. After PCR amplification and visualization using agarose electrophoresis, a band at the expected size of a DyP peroxidase (500–700 bp) was seen. TA cloning followed by blue–white colony selection validated our finding after amplicon sequencing of the PCR product to confirm the presence of an Acinetobacter species DyP-peroxidase. Our metagenomic DyP displayed 99% similarity to the DyP-peroxidase sequence found in the Acinetobacter baumannii ATCC 19606 strain. As a result, and due to the minute differences between our found DyP and the ATCC 19606 strain DyP, we expressed the latter cloned in a pET28b(+) vector and purified it from culture medium using Escherichia coli SoluBl21 as a host strain. A crude oxidation assay using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) deemed the enzyme active as shown by the formation of a green color. The crystal structure of the enzyme was solved at 2.6 Å resolution (PDB ID 9OBR) using X-ray crystallography and presented as a hexamer in solution.