Molecular mechanism of γ-glutamyl transferase derived from Rhodotorula mucilaginosa on γ-glutamyl peptide biosynthesis : γ-glutamyl transferase-substrate recognition, substrate affinity and γ-glutamyl peptide profiling

Abstract

AbstractTo elucidate the molecular mechanism of γ-glutamyl transferase (GGT) from Rhodotorula mucilaginosa in catalyzing γ-glutamyl peptide synthesis, the enzymatic characteristics, sequence identification, substrate recognition and selectivity of GGT, and peptide profiling were systematically investigated. GGT from R. mucilaginosa EIODSF019 (RE) retained above 40% and 90% of its maximum activities at pH 5.0 and 40 °C, respectively. The Ea and Km values of GGT in RE were significantly lower than those of Pichia kudriavzevii XS-5 (PK) and R. mucilaginosa XZY63–3 (RX), indicating higher substrate affinity and catalytic efficiency, and Fe2+ (10 mmol/L) enhanced the GGT activities of RE by approximately 16%. The molecular weight of GGT of RE was 69.33 kDa, matching the UniProt protein A0A2T0A164. Molecular docking and molecular dynamics simulation demonstrated that the site of Glu-428 in GGT could be the key residue for Cys and Gln binding, and the complexes formed by the Glu-GGT binding with Cys and Gln (Glu-GGT-Cys and Glu-GGT-Gln) exhibited lower binding energies and higher stability. RE-GGT efficiently catalyzed the synthesis of γ-Glu-Cys, γ-Glu-Gln, γ-Glu-Glu, γ-Glu-Lys, γ-Glu-Ala and GSH, while Cys and Gln could maintain high conversion efficiency at 50 °C and 1.2 M NaCl, respectively, contributing to more accumulation of γ-Glu-Cys and γ-Glu-Gln. This study provided valuable insights for the efficient biocatalytic synthesis of γ-glutamyl peptides in fermented meat processing.