Int J Biol Macromol. 2026 Oct 26:155049. doi: 10.1016/j.ijbiomac.2026.155049. Online ahead of print.ABSTRACTImine reductases (IREDs) are promising biocatalysts for chiral amine synthesis, yet their application is often limited by low catalytic turnover. Here, we developed a tunnel-directed engineering strategy for the IRED from Paenibacillus mucilaginosus (PmIR) by systematically mutating 24 residues lining its intersubunit tunnel. The resulting variants M6 (Q138M/P140A/Q190S/W195M /S228N/R251N) and M3-3 (Q138L/P140A/A184L/Q190S/S228A/R251N/G255S) exhibited approximately 20-fold and 15-fold increases in kcat toward two bulky imines, respectively, with little change in KM. To investigate the mechanistic basis of the increased turnover of M6, computational analyses focused on the WT and M6 using 3-(3,4-dihydro-2H-pyrrol-5-yl)-5-fluoro-2-methoxypyridine as the model substrate. Molecular docking, conventional molecular dynamics, binding free energy estimation, random-acceleration molecular dynamics, contact-fingerprint analysis, and steered molecular dynamics supported a model in which the activity enhancement was not supported by stronger ground-state substrate binding or improved hydride-transfer geometry. Instead, tunnel remodeling disrupted persistent product-retaining contacts in WT, introduced transient guiding contacts, and produced a more direct egress trajectory, with path tortuosity decreasing from 5.22 in WT to 2.58 in M6. In the M6-DPFM system, R251N, a distal and solvent-facing substitution did not contact the substrate in catalytically competent docking poses but became involved in transient contacts during product dissociation, supporting a mechanistic separation between catalytic positioning and post-catalytic transport. At increased substrate concentrations, the corresponding variants achieved >99% conversion within 3 h, with >99% enantiomeric excess. These results suggest dynamic remodeling of the intersubunit product-egress pathway as a complementary strategy for improving IRED turnover without compromising stereoselectivity.PMID:42933612 | DOI:10.1016/j.ijbiomac.2026.155049