Targeted next-generation sequencing identifies novel variants in candidate genes for Parkinson's disease in Black South African and Nigerian patients.

BACKGROUND: The prevalence of Parkinson's disease (PD) is increasing in sub-Saharan Africa, but little is known about the genetics of PD in these populations. Due to their unique ancestry and diversity, sub-Saharan African populations have the potential to reveal novel insights into the pathobiology of PD. In this study, we aimed to characterise the genetic variation in known and novel PD genes in a group of Black South African and Nigerian patients. METHODS: We recruited 33 Black South African and 14 Nigerian PD patients, and screened them for sequence variants in 751 genes using an Ion AmpliSeq™ Neurological Research panel. We used bcftools to filter variants and annovar software for the annotation. Rare variants were prioritised using MetaLR and MetaSVM prediction scores. The effect of a variant on ATP13A2's protein structure was investigated by molecular modelling. RESULTS: We identified 14,655 rare variants with a minor allele frequency ≤ 0.01, which included 2448 missense variants. Notably, no common pathogenic mutations were identified in these patients. Also, none of the known PD-associated mutations were found highlighting the need for more studies in African populations. Altogether, 54 rare variants in 42 genes were considered deleterious and were prioritized, based on MetaLR and MetaSVM scores, for follow-up studies. Protein modelling showed that the S1004R variant in ATP13A2 possibly alters the conformation of the protein. CONCLUSIONS: We identified several rare variants predicted to be deleterious in sub-Saharan Africa PD patients; however, further studies are required to determine the biological effects of these variants and their possible role in PD. Studies such as these are important to elucidate the genetic aetiology of this disorder in patients of African ancestry.

Crystal structure of the integral membrane diacylglycerol kinase.

Diacylglycerol kinase catalyses 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 bacteria. For half a century, this 121-residue kinase has served as a model for investigating membrane protein enzymology, folding, assembly and stability. Here we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type. These reveal a homo-trimeric enzyme with three transmembrane helices and an amino-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site that 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 model in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.

The role of the C8 proton of ATP in the catalysis of shikimate kinase and adenylate kinase.

BACKGROUND: It has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 µM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP) as a molecular probe with site directed mutagenesis (SDM) of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings. RESULTS: We have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK) and adenylate kinase 1 (AK1), are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP. CONCLUSIONS: The data outlined serves to demonstrate the "push" mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It is therefore conceivable that kinase enzymes achieve the observed 2,500-fold variation in KM through a combination of the various conserved "push" and "pull" mechanisms associated with the release of C8-H, the proton transfer cascades unique to the class of kinase in question and the resultant/concomitant creation of a pentavalent species from the γ-phosphate group of ATP. Also demonstrated is the interplay between the role of the C8-H of ATP and the ATP concentration in the observed enzyme activity. The lability of the C8-H mediated by active site residues co-ordinated to the purine ring of ATP therefore plays a significant role in explaining the broad KM range associated with kinase steady state enzyme activities.

The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases.

BACKGROUND: The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent Km of the kinases ranges from less than 0.4 µM to in excess of 1000 µM for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude. RESULTS: We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP) as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE) but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold. CONCLUSIONS: Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the broad range of Km associated with kinase enzymes.

Conserved phosphoryl transfer mechanisms within kinase families and the role of the C8 proton of ATP in the activation of phosphoryl transfer.

BACKGROUND: The 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. RESULTS: A 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. CONCLUSIONS: The "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.