RIVER: Evidence-guided multi-agent retrieval for enzyme--reaction discovery

Abstract

Enzyme function annotation and the identification of candidate enzymes for a given reaction are essential steps in connecting genomic information to biochemical reaction networks. However, existing methods rely on sequence or structural similarity or on training with known enzyme--reaction pairs, making it difficult to integrate biochemical evidence across large candidate spaces; they also struggle with retrieval tasks involving enzyme families whose members are highly similar. Here, we introduce RIVER (Reaction--enzyme Inference via Evidence-guided Retrieval), a multi-agent framework for bidirectional enzyme--reaction retrieval that identifies potentially catalyzed reactions for a given enzyme and ranks candidate enzymes for a given reaction. RIVER coordinates large language models, specialized biochemical computational tools, and external knowledge resources to assemble evidence at multiple levels, including reaction chemistry, protein sequences and domains, catalytic mechanisms, and database annotations. Dedicated validation modules further examine evidence gaps and potential conflicts and rerank candidates using traceable evidence records. On public benchmarks, RIVER achieves the highest success rates in both enzyme retrieval and enzyme annotation. In family-specific enzyme retrieval, RIVER outperforms the evaluated baselines on the selected datasets and metrics. In Escherichia coli enzyme retrieval tasks constructed from recent literature, RIVER achieves the best retrieval results with closed-source and open-source model configurations, substantially outperforming closed-source general-purpose and biology-specialized multi-agent systems, including Claude Code, Codex, and Phylo Max. These results suggest that evidence-guided multi-agent retrieval can prioritize biochemically supported enzyme--reaction relationships, providing a basis for subsequent experimental validation.