Structural Analysis of Marine <i>Flavobacterium</i> Protein Glutaminase Reveals a “Gatekeeper” Residue Affecting Its Catalytic Activity

A novel protein glutaminase (PG) named FBPG, derived from <i>Flavobacterium</i> sp. 316, was the focus of analysis for the relationship between its structure and function. Through successful heterologous expression in <i>E. coli</i> BL21 (DE3), a mature peptide (mFBPG) was purified following trypsin digestion, demonstrating a specific activity of 1.43 U/mg and a mass of 20.13 kDa. The elucidation of its crystal structure via X-ray diffraction unveiled a composition comprising seven α-helices and 14 β-sheets. Molecular docking studies identified residue R225 as a pivotal "gatekeeper" regulating substrate entry into the catalytic site. Furthermore, the specific activity of the modified variant was 10.38 U/mg after substituting R225 with lysine (R225K), a remarkable 6.37-fold enhancement over that of wild-type mFBPG. These observations enhance our comprehension of the PG enzyme family and lay the foundation for improving their performance.