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The catalytic intermediate stabilized by a "down" active site loop for diaminopimelate decarboxylase from Helicobacter pylori. Enzymatic characterization with crystal structure analysis.

ABSTRACT: The meso-diaminopimelate decarboxylase (DAPDC, EC 4.1.1.20) catalyzes the final step of L-lysine biosynthesis in bacteria and is regarded as a target for the discovery of antibiotics. Here we report the 2.3A crystal structure of DAPDC from Helicobacter pylori (HpDAPDC). The structure, in which the product L-lysine forms a Schiff base with the cofactor pyridoxal 5'-phosphate, provides structural insight into the substrate specificity and catalytic mechanism of the enzyme, and implies that the carboxyl to be cleaved locates at the si face of the cofactor. To our knowledge, this might be the first reported external aldimine of DAPDC. Moreover, the active site loop of HpDAPDC is in a "down" conformation and shields the ligand from solvent. Mutations of Ile(148) from the loop greatly impaired the catalytic efficiency. Combining the structural analysis of the I148L mutant, we hypothesize that HpDAPDC adopts an induced-fit catalytic mechanism in which this loop cycles through "down" and "up" conformations to stabilize intermediates and release product, respectively. Our work is expected to provide clues for designing specific inhibitors of DAPDC.

SUBMITTER: Hu T

PROVIDER: S-EPMC3258949 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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The catalytic intermediate stabilized by a "down" active site loop for diaminopimelate decarboxylase from Helicobacter pylori. Enzymatic characterization with crystal structure analysis.

Hu Tiancen T Wu Dalei D Chen Jing J Ding Jianping J Jiang Hualiang H Shen Xu X

The Journal of biological chemistry 20080528 30

The meso-diaminopimelate decarboxylase (DAPDC, EC 4.1.1.20) catalyzes the final step of L-lysine biosynthesis in bacteria and is regarded as a target for the discovery of antibiotics. Here we report the 2.3A crystal structure of DAPDC from Helicobacter pylori (HpDAPDC). The structure, in which the product L-lysine forms a Schiff base with the cofactor pyridoxal 5'-phosphate, provides structural insight into the substrate specificity and catalytic mechanism of the enzyme, and implies that the car ...[more]

PMID: 18508763

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Project description:Effective eradication therapy for Helicobacter pylori is a worldwide demand. Aspartate α-decarboxylase (ADC) was reported as a drug target in H. pylori, in an in silico study, with malonic acid (MA) as its inhibitor. We evaluated eradicating H. pylori infection through ADC inhibition and the possibility of resistance development. MA binding to ADC was modeled via molecular docking. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of MA were determined against H. pylori ATCC 43504, and a clinical H. pylori isolate. To confirm selective ADC inhibition, we redetermined the MIC in the presence of products of the inhibited enzymatic pathway: β-alanine and pantothenate. HPLC was used to assay the enzymatic activity of H. pylori 6x-his tagged ADC in the presence of different MA concentrations. H. pylori strains were serially exposed to MA for 14 passages, and the MICs were determined. Cytotoxicity in different cell lines was tested. The efficiency of ADC inhibition in treating H. pylori infections was evaluated using a Sprague–Dawley (SD) rat infection model. MA spectrum of activity was determined in different pathogens. MA binds to H. pylori ADC active site with a good docking score. The MIC of MA against H. pylori ranged from 0.5 to 0.75 mg/mL with MBC of 1.5 mg/mL. Increasing β-alanine and pantothenate concentrations proportionally increased MA MIC. The 6x-his tagged ADC activity decreased by increasing MA concentration. No resistance to ADC inhibition was recorded after 14 passages; MA lacked cytotoxicity in all tested cell lines. ADC inhibition effectively eradicated H. pylori infection in SD rats. MA had MIC between 0.625 to 1.25 mg/mL against the tested bacterial pathogens. In conclusion, ADC is a promising target for effectively eradicating H. pylori infection that is not affected by resistance development, besides being of broad-spectrum presence in different pathogens. MA provides a lead molecule for the development of an anti-helicobacter ADC inhibitor. This provides hope for saving the lives of those at high risk of infection with the carcinogenic H. pylori.

Dataset Information

The catalytic intermediate stabilized by a "down" active site loop for diaminopimelate decarboxylase from Helicobacter pylori. Enzymatic characterization with crystal structure analysis.

Publications

The catalytic intermediate stabilized by a "down" active site loop for diaminopimelate decarboxylase from Helicobacter pylori. Enzymatic characterization with crystal structure analysis.

Similar Datasets

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