Diacylglycerol kinase (DgkA) catalyzes the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria1. For half a century, this 121-residue kinase has served as a paradigm for investigating membrane protein enzymology1,3-7, folding8,9, assembly10-13, and stability1,14. Here, we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type (WT). These reveal a homo-trimeric enzyme with three transmembrane helices and an N-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site which is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are however at variance with a published solution NMR model2 in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.
Background: Despite continuous efforts of the international community to reduce the impact of malaria on developing countries, no significant progress has been made in the recent years and the discovery of new drugs is more than ever needed. Out of the many proteins involved in the metabolic activities of the Plasmodium parasite, some are promising targets to carry out rational drug discovery.
BackgroundThe kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes.ResultsA number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the γ-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH2 of the adenyl moiety, namely a "push" mechanism, and a "pull" mechanism, based on the protonation of N7. Associated with the "push" mechanism and "pull" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the γ-phosphate of the ATP and the substrate nucleophile.ConclusionsThe "push" mechanism and a "pull" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the coordination of the adenyl moiety of ATP and the C8-H of the adenyl moiety.
Bacteria adhering to newly emerged and decomposing leaves ofPotamogeton pectinatus L. were examined using scanning electron microscopy. Bacteria obtained from mature leaves were isolated, grown in pure culture, and allowed to infect bacteria-free leaves. Infected leaves and individual isolates were examined by scanning electron microscopy. The method provided detail of the forms of bacterial attachment to leaf surfaces.Supernatants from isolate cultures were assayed for cellulase activity. Six out of eight isolates produced extracellular cellulase in medium containing carboxymethyl cellulose. In one isolate cellulase was produced at discrete intervals, at the beginning and end of the growth cycle. The adhesion of the isolates toP. pectinatus leaves, as well as their possession of cellulase, are considered as evidence of an active role of bacteria in the degradation ofP. pectinatus foliage. The system is unusual in that bacteria, as opposed to fungi, are primary colonizers of the leaves.
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