title: Expression, purification and enzymatic characterization of undecaprenyl pyrophosphate phosphatase from Vibrio vulnificus.
abstract: 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 (<1.3% of the wild type) or no protein expression at all (G163R and G168A), whereas S23A and S167A retain enzyme activity (65% and 34%). Kinetic analysis indicates that S23A and S167A result in 1.4- and 5-fold decreases in kcat, whereas the substrate Kmvalue exhibits only minor changes compared with wild-type UppP, implying that they are involved in enzyme catalysis. The structural modeling and molecular dynamics simulation analyses also provide a plausible structure of the catalytic core, centered on a conserved histidine (His-26) that initiates the hydrolysis of phosphate esters, rationalizing the mutagenesis data. This conclusion can be applied generally to all bacterial UppP enzymes.
authors.affiliation: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan, ROC; Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, ROC; The Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan, ROC; Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, ROC.
authors.forename: Hsin-Yang
authors.initials: HY
authors.lastname: Chang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, ROC; Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan, ROC; National Center for High Performance Computing, Hsinchu, Taiwan, ROC.
authors.forename: Chia-Cheng
authors.initials: CC
authors.lastname: Chou
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, ROC; Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: Mao-Lun
authors.initials: ML
authors.lastname: Wu
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, ROC; Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan, ROC; PhD Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan, ROC. Electronic address: ahjwang@gate.sinica.edu.tw.
authors.forename: Andrew H J
authors.initials: AHJ
authors.lastname: Wang
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018360:Crystallography, X-Ray; D015870:Gene Expression; D056004:Molecular Dynamics Simulation; D000072417:Protein Domains; D011755:Pyrophosphatases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D041261:Vibrio vulnificus / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D011755:Pyrophosphatases; C503879:undecaprenyl pyrophosphate phosphatase
keywords: BacA; Undecaprenyl pyrophosphate phosphatase; UppP; Vibrio vulnificus