Purification, folding, activity analysis and substrate specificity of Pseudomonas diacylglycerol kinase

The structural and functional investigation of bacterial membrane proteins is crucial to the development of antibiotics. Diacylglycerol kinase (DAGK) from Escherichia coli (E. coli) has been extensively studied as a model membrane protein. However, the DAGK from Pseudomonas aeruginosa (PAO1-DAGK) with a 44 % sequence identity to E. coli-DAGK is not well characterized. To explore the properties of PAO1-DAGK, it was successfully expressed in E. coli and was purified in Decyl-β-D-maltoside (DM) micelles followed with characterizations. Chemical cross-linking studies revealed that PAO1-DAGK in DM micelles could form dimers and trimers. The kinase activity of PAO1-DAGK was determined to be 24.2 ± 2.2 U/mg protein in a mixed-micelle system. The effects of pH and temperature on the activity of PAO1-DAGK were also investigated, respectively. PAO1-DAGK in DM micelles exhibited good stability at pH 6.0-10.0 and below 45 °C. Substrate specificity measurements indicated that PAO1-DAGK demonstrated a clear preference for medium-chain diacylglycerols (DAGs) in the mixed-micelle system, with sn-1,2-Dihexanoylglycerol (DiC6) being the most favored substrate. Molecular docking results demonstrated the interactions between DAGs and PAO1-DAGK.