Functional and Structural characterization of Four Novel Protein Glutaminases

Protein glutaminase (PG) is a deamidating enzyme that can specifically convert glutamine to glutamic acid residues in proteins or peptides without any side products. The deamidation reactions can remarkably improve the functionalities (solubility, emulsification and foaming properties, etc.) of food proteins, resulting in the great application prospects of PG in the food industry. However, PG has thus far been primarily produced from Chryseobacterium proteolyticum in the food industry; only PG from Chryseobacterium proteolyticum (PG(CP)) has been well characterized, which greatly restricts its application. Here, a simple one-step purification method for the mass production of PG was developed, wherein GST-3C protease and pro-PG with the HRV 3C protease cleavage site were coexpressed in E. coli. Moreover, four novel PG proteins (namely, PG(JM1), PG(G311), PG(CL) and PG(ATCC)) were discovered from other Chryseobacterium species. Enzymatic assays showed that PG(ATCC) had a rather high T50 value of 65.7℃ and a Tm value of 70℃, making it the most thermostable of all the identified PG proteins. Moreover, these PG proteins, especially PG(JM1), showed high deamidation degrees (>66%) on gliadin, resulting in a dramatic increase in solubility of gliadin compared with the control group. Furthermore, we determined the sequence specificity of these four PG proteins by performing a proteomic analysis, which revealed the specificity pattern: a strong preference for nonpolar amino acid residues (Leu or Ala, etc.) flanking the deamidation site. The crystal structures of mature PG(ATCC) and the pro-forms of PG(JM1), PG(G311), PG(CL) and PG(ATCC) were refined at 2.00 Å, 1.92 Å, 1.85 Å, 1.75 Å and 2.40 Å, respectively. The higher enzymatic and structural stabilities of PG(ATCC) might have contributed to the higher helix content and lower number of Asn residues on the protein surface. Two docking models for PG binding to the peptide substrate, Cbz-Gln-Gly or LLAQA, were simulated. The data revealed that Cbz-Gln-Gly or LLAQA was stretched along the PG active cleft and the hydrophobic residues directly interacting with the substrate. This study provides a practical route for the mass production of PG and lays the foundation for both enzymatic activity and structural stability optimizations.