2018Al-EthariAYHPhD.pdf (5.82 MB)
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Phosphoglycerate kinase and phosphoenolpyruvate synthase of the enteric pathogen Helicobacter pylori

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posted on 17.09.2018, 10:06 authored by Aziz Yasir Hasan Al-Ethari
Helicobacter pylori is a globally distributed enteric pathogen implicated in several serious diseases. Understanding the genetics and metabolism of the pathogen is of significant importance to developing new therapies for eradication. However, its metabolism is poorly characterised. The genome lacks coding sequences of some key glycolytic enzymes, however the gluconeogenic enzymes fructose-1,6-bisphosphatase and phosphoenolpyruvate synthase (hpPPSA) are present. This suggests H. pylori uses the glycolytic/gluconeogenic pathway for anabolic biosynthesis rather than for catabolic energy production. This study examines the structure and function of hpPPSA and phosphoglycerate kinase (hpPGK) and investigates the conditional essentiality of these genes, which were identified by in silico double deletion mutational studies of H. pylori. The ppsA and pgk mutants (with controls) were constructed using experimental knock out strategies, and their role in synthetic lethality was investigated. The ppsA-mutated allele alone showed evidence of essentiality. The Krebs cycle in H. pylori deviates from the text book examples such as in humans and E. coli, thus pgk may be essential alone or in combination with other enzymes. The X-ray crystal structure of apo hpPGK was determined and compared to human PGK. Structural superposition showed that both the substrate and the nucleotide binding residues are well conserved. Four sulphate ions were identified bound in the hpPGK dimer. The positions of these molecules allowed the path of phosphoryl transfer during catalysis to be modelled. The enzyme was further characterised using kinetic techniques and compared to homologous enzymes. Prediction of the hpPPSA structure by homology modelling analysis located the essential His and Cys catalytic conserved residues in their respective domains. Superimposition of the N-terminal ATP binding domain superposition showed that these residues forming this binding site are well conserved. Understanding H. pylori metabolism may provide directions for the development of therapeutics.



Moody, Peter; Schmid, Ralf

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Department of Molecular and Cell Biology

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University of Leicester

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