[P001] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P01-035] Functional Characterization and Structure-Based Analysis of Diverse Lid-Loop-Negative γ-Glutamyltranspeptidases

○Yu Jin Park¹, Min Ho Jeon¹, Hye Bin Kim¹, Eunseo Bae¹, In Hwan Jang¹, Ju Hee Hwang¹, Tae-Jip Kim¹ (1. Chungbuk National University, Division of Animal, Horticultural and Food Sciences (Korea))

Keywords:

Lid-loop-negative γ-glutamyltranspeptidases,Transpeptidation,Hydrolysis

γ-Glutamyltranspeptidases (GGTs; EC 2.3.2.2) are heterodimeric enzymes that undergo autocatalytic cleavage to generate large and small subunits. GGT catalyzes the hydrolysis of the γ-glutamyl bond of γ-glutamyl compounds or transfers the γ-glutamyl moiety to acceptor molecules such as amino acids and peptides, thereby producing various γ-glutamyl derivatives. The resulting L-glutamate and γ-glutamyl compounds play important industrial roles by enhancing umami and kokumi tastes in food, respectively. Previous structural studies of GGTs from Escherichia coli and Helicobacter pylori revealed that the small subunit contains a lid-loop conformation, which restricts their ability to degrade high-molecular-weight substrates such as poly-γ-glutamate (γ-PGA). In contrast, GGTs from Bacillus species are known to lack the lid-loop conformation and instead possess an additional amino acid sequence in the C-terminal region of the large subunit, which is thought to enable more efficient γ-PGA degradation compared with lid-loop-positive GGTs. To date, lid-loop-negative GGTs have been studied primarily from Bacillus subtilis (BsGGT) and Bacillus licheniformis (BlGGT). In this study, the enzymatic properties of lid-loop-negative GGTs from Bacillus atrophaeus (BatGGT), B. zhangzhouensis (BzhGGT), B. sonorensis (BsoGGT), B. mojavensis (BmoGGT), and Cytobacillus depressus (CdeGGT) were investigated and compared. These enzymes consist of 558–589 amino acid residues, forming large (~41 kDa) and small (~20 kDa) subunits, which share 70–93% amino acid sequence identity with BsGGT and 69–88% identity with BlGGT. Using the artificial substrate γ-glutamyl-p-nitroanilide (γ-GpNA) with diglycine as an acceptor, most GGTs exhibited high transpeptidation activity at pH 9–10 and high hydrolytic activity at pH 8–9. While lid-loop-negative GGTs showed similar γ-GpNA hydrolytic activities of approximately 7 U/mg, their transpeptidation activities varied substantially, ranging from 15 to 41 U/mg. These results indicate that the transpeptidation activity of GGTs toward γ-GpNA is generally higher than the hydrolytic activity. These GGTs also exhibited significant differences in hydrolytic activity when γ-PGA polymers were used as substrates. Notably, BatGGT (4.4 U/mg) exhibited more than eightfold higher γ-PGA hydrolytic activity than CdeGGT (0.5 U/mg). To elucidate why structurally similar lid-loop-negative GGTs show such variation in hydrolytic activity, the three-dimensional structures of the enzymes were predicted using AlphaFold and compared with their amino acid sequences and enzymatic functions.