Genetic Alterations Shaping the Functional Properties of DyP Peroxidases

Dye-decolorizing peroxidases (DyPs) represent a distinct family of heme peroxidases with unique capabilities in dye decolorization, lignin synthesis, and lignin degradation. While traditional phylogenetics and structural alignments have categorized DyPs into four distinct subclasses (A-D), the secret to unlocking their industrial versatility lies in the ability to understand and re-engineer the sophisticated architecture of their active sites. This review critically analyzed findings from 35 peer-reviewed articles (2007-2025) sourced from eight electronic databases, examining 93 distinct mutations within 20 different DyPs. Moving beyond basic classification, this study identifies hotspots that influence DyP performance, examining how site-directed mutagenesis affects structural integrity, H₂O₂ tolerance, and catalytic potential. Central among these is the histidine-arginine-aspartate (H-R-D) triad, which mediates the formation of high-valent intermediates in a class-specific manner. Analysis reveals that 29% of the studied variants surpassed their wild-type counterparts in catalytic efficiency, with a few variants achieving rare and desirable improvements in stability and an alkaline shift in optimal working pH. Mapping these mutational effects identifies the critical surface-exposed motifs and gatekeeping amino acids that govern long-range electron transfer and substrate accessibility. Since rate-limiting steps are substrate-dependent, a single mutation might produce diverse catalytic outcomes. Beyond clarifying structure-function relationships, these findings aim to provide a strategic blueprint for directed evolution. Identifying these catalytic hotspots guides the precision engineering of DyPs with tailored specificity and enhanced characteristics. Overall, this work acts as a catalyst for future protein engineering, unlocking a new era of robust biocatalysts capable of withstanding the harsh environments of modern industrial biotechnology.