Expression, purification and enzymatic characterization of undecaprenyl pyrophosphate phosphatase from Vibrio vulnificus
| Autor: | Chia-Cheng Chou, Andrew H.-J. Wang, Mao Lun Wu, Hsin Yang Chang |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Gene Expression Molecular Dynamics Simulation Crystallography X-Ray Enzyme catalysis Dephosphorylation Serine 03 medical and health sciences Bacterial Proteins Protein Domains Enzyme kinetics Pyrophosphatases Vibrio vulnificus Histidine chemistry.chemical_classification 030102 biochemistry & molecular biology biology Periplasmic space Enzyme assay Recombinant Proteins 030104 developmental biology Enzyme chemistry Biochemistry biology.protein Biotechnology |
| Zdroj: | Protein expression and purification. 133 |
| ISSN: | 1096-0279 |
| Popis: | Undecaprenyl pyrophosphate phosphatase (UppP), a cell membrane integral enzyme, catalyzes the dephosphorylation of undecaprenyl pyrophosphate to undecaprenyl phosphate, which is an essential carrier lipid in bacterial cell wall synthesis. We previously purified E. coli UppP and concluded that its catalytic site is likely located in the periplasm. To search for additional natural UppP homologs to elucidate what constitutes a common catalytic mechanism and to gain a better chance of obtaining high-resolution crystal structural information, we expressed and purified recombinant Vibrio vulnificus UppP using E. coli as a host. Mutagenesis analysis demonstrates that the proposed catalytic residues Gln-13, Glu-17, His-26 and Arg-166 are directly involved in enzyme catalysis. Additionally, mutations of most of the conserved serine and glycine residues within the proposed catalytic site (S22A, G163A and S165A) lead to complete inactivity, very low activity ( |
| Databáze: | OpenAIRE |
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