ABSTRACT
Amylomaltases (AMs) play important roles in glycogen and maltose metabolism. However, the molecular mechanism is elusive. Here, we investigated the conformational dynamics of the 250s loop and catalytic mechanism of Thermus aquaticus TaAM using path-metadynamics and QM/MM MD simulations. The results demonstrate that the transition of the 250s loop from an open to closed conformation promotes polysaccharide sliding, leading to the ideal positioning of the acid/base. Furthermore, the conformational dynamics can also modulate the selectivity of hydrolysis and transglycosylation. The closed conformation of the 250s loop enables the tight packing of the active site for transglycosylation, reducing the energy penalty and efficiently preventing the penetration of water into the active site. Conversely, the partially closed conformation for hydrolysis results in a loosely packed active site, destabilizing the transition state. These computational findings guide mutation experiments and enable the identification of mutants with an improved disproportionation/hydrolysis ratio. The present mechanism is in line with experimental data, highlighting the critical role of conformational dynamics in regulating the catalytic reactivity of GHs.
ABSTRACT
Gastric cancer is one of the leading prevalent and malignant cancers worldwide, especially in east Asia. However, the in-depth molecular mechanism underlying gastric cancer progression remains uncertain. Recently, studies have identified that long non-coding RNA (lncRNA) could play critical roles in the tumorigenesis of multiple types of cancer. Studies on long non-coding RNA BLACAT2 have proven that it participates in bladder cancer and colorectal cancer regulation and was identified as highly expressed using the cBioPortal for Cancer Genomics in gastric cancer. However, the precise function of lncRNA-BLACAT2 in the carcinogenesis and progression of gastric cancer remains largely unexplored. Our study discovered that lncRNA-BLACAT2 was significantly upregulated in gastric cancer. Different studies have illustrated that BLACAT2 promoted gastric cancer progression through regulating proliferation, migration, invasion, and apoptosis in terms of biological function. Furthermore, BLACAT2 was verified to perform its function through interaction with miR-193b-5p using a luciferase reporter assay. On the other hand, MiR-193b-5p specific inhibitor treatment reversed the inhibitory effect of BLACAT2 on cell biological functions. Additional studies also discovered that Methyltransferase Like 3 (METTL3) was the downstream target of miR-193b-5p. Subsequently, restoration of METTL3 eliminated the suppressive effect of proliferation or the promotive effect of apoptosis caused by BLACAT2 knockdown. To sum up, these experimental results demonstrated that BLACAT2 acted as an oncogene in gastric cancer progression through the regulation of the miR-193b-5p/METTL3 pathway, hence providing new insights regarding the pathogenesis of gastric cancer.
