Adenylosuccinate synthetase from bacterium Helicobacter pylori strain 26695: purification and characterization

I. Leščić Ašler1, N. Mrnjavac1, Igor Stuparević2, Z. Štefanić1, M. Luić1

1Ruđer Bošković Institute, Department of Physical Chemistry, Bijenička c. 54, 10000 Zagreb, Croatia

2University of Zagreb, Faculty of Food Technology and Biotechnology, Department of Chemistry and Biochemistry, Pierottijeva 6, 10000 Zagreb, Croatia

Helicobacter pylori is a Gram-negative, microaerophilic bacterium, known for its ability to colonize human stomach and to participate in development of many diseases as gastric ulcers and stomach cancer [1]. Study of the H. pylori, due to the ever growing infection rate and increase of H. pylori antibiotic resistance, is centred on understanding pathogenesis and finding a way to attack and eradicate H. pylori.

Adenylosuccinate synthetase (AdSS) is one of the key enzymes in purine salvage pathway. It catalyses a reversible reaction utilizing IMP, GTP and aspartate in the presence of Mg2+ to form adenylosuccinate, GDP and inorganic phosphate. H. pylori AdSS represents potential drug target as this bacterium cannot synthesize purine rings through de novo pathway and has to rely on purine production through purine salvage pathway [2].  

Adenylosuccinate synthetase gene purA was isolated from genomic DNA of Helicobacter pylori (strain 26695) and amplified using Phusion High-Fidelity PCR kit with the set of specific DNA primers for both 5' and 3' ends of the gene. Plasmid pET21b-HP26695purA was constructed by cloning H. pylori purA gene into pET21b expression vector, with ampicillin resistance and without purification tag. This plasmid was transformed into E. coli strain BL21-CodonPlus(DE3)RIL. Standard IPTG-induction conditions were used for AdSS expression in E. coli.

Purification of overexpressed AdSS from the bacterial protein extract was performed by cation exchange chromatography on SP-Sepharose FF column, followed by size-exclusion chromatography (SEC) on Sephacryl S-200 column. Final step, which gave single protein band on SDS-PAGE was fast protein liquid chromatography (FPLC), performed on anion exchange MonoQ 5/50 GL column.

Enzyme’s molecular weight was estimated by SDS-PAGE and elution volume from SEC. It was concluded that in applied conditions enzyme exists in solution as a dimer. Kinetic studies involving all three substrates (IMP, GTP and aspartate) were conducted. Protein crystals appeared in several conditions of used commercial crystallization screens. Structure solving of adenylosuccinate synthetase from H. pylori is under way.

1. D. Makola, J. Krenitsky, C. R. Parrish, Gastrointest. Endosc. Clin. N. Am., 17, (2007), 747–764.

2. G. Liechti, J. B. Goldberg, J. Bacteriol., 194, (2011), 839–854.

This research has been supported by Croatian Science Foundation (project no. 7423).