Structural and biochemical characterization of β-agarase YM01-3 from Catenovulum agarivorans: Functional insights...

Truncated variants of Catenovulum agarivorans β-agarase YM01-3 (GH, R1, R2 domains) were constructed: Y3GH (GH only) and Y3R1 (GH + R1). Y3R1 exhibited 105.2 % relative activity versus YM01-3 (100 %) and Y3GH (49.3 %). Its optimal temperature rose 5 °C to 65 °C, while its optimal pH was unchanged. Thermal stability improved significantly, with Y3R1 retaining 71.6 % activity after 1 h at 60 °C versus 11.7 % (YM01-3) and 21.9 % (Y3GH). Kinetic analysis showed K_m values of 7.52 (YM01-3), 18.04 (Y3R1), and 1.30 mg/mL (Y3GH), and V_max values of 5000.7 (YM01-3), 10000.7 (Y3R1), and 1000.0 U/mg (Y3GH). These results demonstrated that the R1 domain enhanced enzyme activity, thermostability, and V_max value, while the R2 domain reduced thermostability and V_max value but improved the substrate affinity. Molecular dynamics simulations revealed that Y3R1's structural integrity at high temperatures due to the increased enzyme compactness and rigid β-sheets near catalytic sites. This heightened conformational stability likely underlay the improved thermotolerance of the truncated enzyme Y3R1. LC-MS confirmed Y3R1 produced the same neoagaro-oligosaccharides (NA2, NA4, NA6) as wild-type. This study elucidated the structure-function relationship between the non-catalytic R domain and the enzymatic properties of C. agarivorans β-agarase, aiding future enzyme structure-activity relationships research and applications.