Functional and Mechanistic Dissection of Protein Glutaminase\nPG3 and Its Rational Engineering for Enhanced Modification of Myofibrillar\nProteins

Protein\nglutaminases (PG; EC = 3.5.1.44) are enzymes known for\nenhancing protein functionality. In this study, we cloned and expressed\nthe gene <i>chryb</i>3 encoding protein glutaminase PG3,\nexhibiting 39.4 U/mg specific activity. Mature-PG3 featured a substrate\nchannel surrounded by aromatic and hydrophobic amino acids at positions\n38–45 and 78–84, with Val81 playing a pivotal role in\nsubstrate affinity. The dynamic opening and closing motions between\nGly65, Thr66, and Cys164 at the catalytic cleft greatly influence\nsubstrate binding and product release. Redesigning catalytic pocket\nand cocatalytic region produced combinatorial mutant MT6 showing a\n2.69-fold increase in specific activity and a 2.99-fold increase at <i>t</i><sup>65 °C</sup><sub>1/2</sub>. Furthermore,\nMT6 boosted fish myofibrillar protein (MP) solubility without NaCl.\nKey residues such as Thr3, Asn54, Val81, Tyr82, Asn107, and Ser108\nwere vital for PG3-myosin interaction, particularly Asn54 and Asn107.\nThis study sheds light on the catalytic mechanism of PG3 and guided\nits rational engineering and utilization in low-salt fish MP product\nproduction.