Sequence and structure-based method to predict diacylglycerol lipases in protein sequence.

Lipase enzymes play a central role in biotechnology and the food industry. Diacylglyceride lipases (DAG) have received considerable attention due to their physiological significance and potential industrial usage. However, compared to the wide application of triacylglycerol (TAG) lipases, DAG lipases have a limited application due to their low thermostability and specific activity. The molecular basis of substrate specificity of DAG lipases remains elusive, making structure-guided engineering of TAG to DAG lipase difficult. Besides, the number of available DAG lipases is limited compared to TAG lipases. In the current study, we identified structural consensus motifs of DAG lipases that contribute to their DAG specificity on a structural comparison of DAG and TAG lipases. To find potential DAG lipases, sequence motifs and predicted secondary structures were used to screen millions of protein sequences and predict new DAG lipases. In total, 83 new putative DAG lipases were identified. The predicted DAG lipases were validated by expression of randomly chosen putative DAG lipases followed by functional assay for their DAG and TAG specific activity. The reported method is efficient and cost-effective for discovering new DAG lipases used in the food industry after the required characterization to meet potential application needs.

Molecular modeling and simulation of the human eNOS reductase domain, an enzyme involved in the release of vascular nitric oxide.

Homology modeling of the reductase domain of endothelial nitric oxide synthase (eNOS), which regulates the catalytic activity of eNOS, and molecular dynamics studies focusing especially on the serine residues S615, S633, and S1177 were performed. MD analysis of this structure revealed that S633 is highly flexible and accessible to solvent molecules, while S1177 becomes highly flexible when S633 is phosphorylated. The presence of intramolecular interactions between S1177 among the major serine residues underscores its structural importance to the efficient synthesis of nitric oxide in endothelium. In order to evaluate the appropriateness of phosphomimetic (for phosphorylation) and phosphomutant (for dephosphorylation) eNOSs for use as experimental model systems, the structural dynamics and conformational changes in phosphomimetic (S615D, S633D, S1177D) and phosphomutant (S615A, S633A, S1177A) eNOSs were investigated. Phosphomimetic and phosphomutant eNOSs portrayed S633 as a modulator of S1177, whereas such correlations could not be observed in native and phosphorylated eNOSs. Computational analysis of the docked complex revealed that phosphorylated pS1177 and pS615 have high affinity for Akt (one of the key kinases in the eNOS activation pathway), with a significant number of hydrogen bonds and salt bridges observed between these residues and Akt . This work therefore provides evidence of the subtle structural changes that occur within the reductase domain which contribute to the stability-flexibility-activity relationship of eNOS. Such subtle changes are of great importance in the context of regulated nitric oxide release by different phosphorylated forms of eNOS and the need to account for the existence of subtle differences between real proteins and experimental model systems.