Bioinformatics Analysis of Diadenylate Cyclase Regulation on Cyclic Diadenosine Monophosphate Biosynthesis in Exopolysaccharide Production by Leuconostoc mesenteroides DRP105

Lactic acid bacteria exopolysaccharides (EPS) have a variety of excellent biological functions and are widely used in the food and pharmaceutical industries. The complex metabolic system of lactic acid bacteria and the mechanism of EPS biosynthesis have not been fully analyzed, which limits the wider application of EPS. EPS synthesis is regulated by cyclic diadenosine monophosphate (c-di-AMP), but the exact mechanism remains unclear. Dac and pde are c-di-AMP anabolic genes, gtfA, gtfB and gtfC are EPS synthesis gene clusters, among which gtfC was the key gene for EPS synthesis in Leuconostoc mesenteroides DRP105. In order to explore whether diadenylate cyclase (DAC) can catalyze the synthesis of c-di-AMP from ATP, the sequence of DAC was analyzed by bioinformatics based on the whole genome sequence. DAC was a CdaA type diadenylate cyclase containing the classical domain DisA_N and DGA and RHR motifs. The secondary structure was mainly composed of α-helices, and AlphaFold2 was used to model the 3D structure of the protein and evaluate the rationality of the DAC protein structure model. A total of 8 salt bridges, 21 hydrogen bonds and 221 non-bonded interactions were found between DAC and GtfC. Molecular docking simulations revealed ATP1 and ATP2 fully occupied the binding pocket of DAC and interacted directly with the binding site residues of DAC. The molecular dynamics simulations showed that the binding of DAC to ATP molecules was relatively stable. Gene and enzyme correlation analysis found that dac and gtfC gene expression were significantly positively correlated with DAC enzyme activity, c-di-AMP content and EPS production, and had no significant correlation with PDE enzyme activity responsible for c-di-AMP degradation. Bioinformatics analysis of the regulatory role of DAC in the synthesis of EPS by lactic acid bacteria was helpful to fully reveal the biosynthetic mechanism of EPS and provide theoretical basis for large-scale industrial production of EPS.