Understanding the importance of glycosylated threonine and stereospecific action of Drosocin, a Proline rich antimicrobial peptide.

Glycosylation is an essential post-translational modification for few antimicrobial peptides of Proline rich class. In the present study we have shown the importance of Thr glycosylation over Ser glycosylation in Drosocin. Difference of a methyl group makes glycosylated-Thr preferred over glycosylated-Ser and renders higher activity to the peptide, probably due to the rigid conformation provided by the glycosylated-Thr. The structural rigidity provided by glycosylated-Thr to Drosocin backbone was mimicked by substituting glycosylated-Thr11, Ser7 and Ser12 with Pro residues. The designed non-glycosylated analogue, P(7)P(11)P(12)-Drosocin, exhibited functional and structural properties similar to that of the native monoglycosylated peptide. The functional importance of stereospecificity of amino acids and sugar was further explored. Interestingly, (all D) p(7)p(11)p(12)-Drosocin failed to exhibit antimicrobial activity but had comparable binding affinity to DnaK, one of the proposed targets for Proline rich class of antibacterial peptides, as that of its L counterpart. However, Drosocin containing either L or D enantiomeric sugar, displayed antimicrobial activity and binding affinity to bacterial heat shock protein, DnaK. The flow cytometry (FACS) experiments revealed the internalization of Drosocins bearing enantiomeric sugars and P(7)P(11)P(12)-Drosocin but not of its d-enantiomer into bacteria suggesting the importance of stereospecificity of amino acids for membrane entry. Once internalized both enantiomeric peptides may behave similarly. This assumption was corroborated by in vitro activity of (all D) p(7)p(11)p(12)-Drosocin in cell free assay where it abrogated transcription/translation pathway similar to l-enantiomer but could not inhibit the same in whole cell assay. These research findings provide insights into the mode of action of Proline rich class of antibacterial peptides and guidelines for designing functionally equivalent non-glycosylated analogues of glycosylated antibacterial peptides.

Glycosylated analogs of formaecin I and drosocin exhibit differential pattern of antibacterial activity.

The synthetic glycopeptides are interesting model systems to study the effect of O-glycosylation in modulating their function and structure. A series of glycosylated analogs of two antibacterial peptides, formaecin I and drosocin, were synthesized by varying the nature of sugar and its linkage with bioactive peptides to understand the influence of structure variation of glycosylation on their antibacterial activities. Higher antibacterial activities of all glycopeptides compared to their respective non-glycosylated counterparts emphasize in part the importance of sugar moieties in functional implications of these peptides. The consequences of the unique differences among the analogs were apparent on their antibacterial activities but not evident structurally by circular dichroism studies. We have shown that differently glycosylated peptides exhibit differential effect among each other when tested against several Gram-negative bacterial strains. The change of monosaccharide moiety and/or its anomeric configuration in formaecin I and drosocin resulted into decrease in the antibacterial activity in comparison to that of the native glycopeptide, but the extent of decrease in antibacterial activity of glycosylated drosocin analogs was less. Probably, the variation in peptide conformation arising due to topological dissimilarities among different sugars in the same peptide resulting in possible modulation in binding properties appears to be responsible for differences in their antibacterial activities. Indeed, these effects of glycosylation are found to be sequence-specific and depend in the milieu of amino acid residues. Interestingly, none of the carbohydrate variants affected the basic property of these peptides, which is non-hemolytic and non-toxicity to eukaryotic cells.

Phospholipase D from Allium sativum bulbs: A highly active and thermal stable enzyme.

This is the first report on the identification and partial characterization of phospholipase D (EC 3.1.4.4) from Allium sativum (garlic) bulbs (PLD(GB)). The enzyme shares the phenomenon of interfacial activation with other lipolytic enzymes, i.e. the hydrolytic rate increases when the substrate changes to a more aggregated state. The enzyme activity is highly temperature tolerant and the temperature optimum was measured to be 70 degrees C. PLD(GB) unlike many plant PLDs exhibited high thermal stability. It was activated further after exposure to high temperatures, i.e. 80 degrees C, indicating that the enzyme refolds better upon cooling back to room temperature after short exposure to thermal stress. The activity of PLD(GB) is optimum in 70mM calcium ion concentration and the enzyme is activated further in the presence of phosphatidyl-4,5-bisphosphate (PIP(2)). PLD(GB) exhibited both hydrolytic and transphosphatidylation activities, both of which appear to be higher than those of PLD from cabbage leaves (PLD(CL)).

Identification and partial characterization of a highly active and stable phospholipase D from Brassica juncea seeds.

Phospholipase D (PLD) activity has been identified in some new plant sources i.e. Brassica juncea (mustard) seeds, Zingibar officinale (ginger) rhizomes and Azadirachta indica (neem) leaves with the aim of identifying PLDs that possess high catalytic activity and stability. PLD from mustard seeds (PLD(ms)) exhibited the highest PLD specific activity, which was highly pH and temperature tolerant. PLD(ms) unlike many plant PLDs exhibited high thermal stability. The activity of PLD(ms) is optimum in the millimolar concentration of calcium ions and is independent of phosphatidylinositol-4,5-bisphosphate (PIP2). An active and stable enzyme like PLD(ms) may be utilized in the lipid industry.