Direct Tunnel Channeling of Unstable Amino Aldehyde Intermediates Enables Stepwise Diamination in Transaminases

Amino aldehyde intermediates exhibit extreme chemical lability, undergoing nonproductive cyclization and polymerization in bulk solvent at rates orders of magnitude faster than enzymatic recapture. This kinetic mismatch has posed a fundamental bottleneck for transaminase-mediated stepwise diamination. Here, we resolve this long-standing challenge by discovering a previously unrecognized trans-subunit tunnel within a homodimeric transaminase that directly channels the unstable amino aldehyde intermediate between the two pyridoxal-5′-phosphate (PLP) active sites, bypassing release and rebinding. Mechanism-guided semi-rational engineering of the trans-subunit tunnel in Shimia marina transaminase (SMTA) yielded the S122G/Y324N with 1.98-fold enhanced catalytic efficiency (kcat/KM) for the stepwise diamination of 2,5-diformylfuran (DFF) to bis(aminomethyl)furan (BAMF). Stopped-flow kinetics and molecular dynamics simulations revealed that the mutations shift the tunnel from a tight-binding locked state to a dynamic-transport regime, reducing steric hindrance and optimizing electrostatic guidance to accelerate inter-subunit intermediate transit. A whole-cell cascade converting renewable 5-hydroxymethylfurfural (5-HMF) to BAMF—a promising bio-based diamine for advanced polyamides—was subjected to techno-economic (TEA) and life cycle (LCA) analyses. Together, these findings establish tunnel engineering as a generalizable paradigm for sequential biocatalytic amination. This work provides a mechanism-driven blueprint for efficient transaminase-mediated stepwise diamination, enabling biocatalytic production of bio-based diamines from renewable feedstocks.