Three catalytic frameworks, one engineering logic: Bottlenecks and design levers in pesticide-degrading hydrolases

The widespread use of pesticides has improved agricultural productivity; however, the long-term persistence of their residues and transformation products in soils and aquatic environments can pose ecological and human health risks. Microbial degradation represents an important environmental fate process, and hydrolysis often serves as one of the key initiating steps in the biotransformation of multiple pesticide classes. Along the central thread of mechanism-bottlenecks-engineering levers, this review focuses on microbial pesticide-degrading hydrolases and organizes the evidence and representative case studies according to three common catalytic frameworks: (i) Ser-His-Asp/Glu serine hydrolases with the α/β-hydrolase (ABH) fold; (ii) Ser-cisSer-Lys serine amidases from the amidase signature (AS) family; and (iii) metal-dependent hydrolases, represented by the amidohydrolase superfamily (TIM-barrel) and the metallo-β-lactamase (MBL) superfamily. Although these three frameworks differ substantially in fold and catalytic configuration, a recurring principle in engineering practice is that the key components governing the core chemical step are typically more conserved and more tightly constrained by geometric and electrostatic requirements. By contrast, peripheral determinants-such as access at the active-site entrance, substrate positioning, conformational gating, and remote coupling networks in the second/third shells-tend to be more malleable and often constitute the primary engineering levers for improving efficiency and expanding substrate scope. On this basis, this review maps the major engineering levers of pesticide-degrading hydrolases-gating elements, substrate-binding pockets, distal sites or outer-shell networks, and substrate access channels, while also clarifying the constraints imposed by the catalytic core. It further discusses how evolutionary analysis, rational or semi-rational design, directed evolution, and computation and data-driven tools can be selected and combined according to available mechanistic information and screening capacity. By linking conserved catalytic frameworks, tunable structural determinants, and task-relevant validation, this review aims to provide mechanism-guided principles for the discovery and engineering of pesticide-degrading hydrolases with improved interpretability and translational potential.