Pseudodata-Guided Invariant Representation Learning Boosts the Out-of-Distribution Generalization in Enzymatic...

Accurate prediction of enzyme kinetic parameters is essential for understanding catalytic mechanisms and guiding enzyme engineering. However, existing deep learning-based enzyme-substrate interaction (ESI) predictors often exhibit performance degradation on sequence-divergent, out-of-distribution (OOD) cases, limiting robustness under biologically relevant perturbations. We propose O²DENet, a lightweight, plug-and-play module that enhances OOD generalization via biologically and chemically informed perturbation augmentation and invariant representation learning. O²DENet introduces enzyme-substrate perturbations and enforces consistency between original and augmented enzyme-substrate-pair representations to encourage invariance to distributional shifts. When integrated with representative ESI models, O²DENet consistently improves predictive performance for both k_cat and K_m across stringent sequence-identity-based OOD benchmarks, achieving state-of-the-art results among the evaluated methods in terms of accuracy and robustness metrics. Overall, O²DENet provides a general and effective strategy to enhance the stability and deployability of data-driven enzyme kinetics predictors for real-world enzyme engineering applications.