title: Identification of pyruvate carboxylase genes in Pseudomonas aeruginosa PAO1 and development of a P. aeruginosa-based overexpression system for alpha4- and alpha4beta4-type pyruvate carboxylases.
abstract: Pyruvate carboxylase (PYC) is an ecologically, medically, and industrially important enzyme. It is widespread in all three domains of life, the archaea, bacteria, and eukarya. PYC catalyzes ATP-dependent carboxylation of pyruvate to oxaloacetate. Detailed structure-function studies of this enzyme have been hampered due to the unavailability of a facile recombinant overexpression system. Except for the alpha4 enzyme from a thermophilic Bacillus species, Escherichia coli has been unsuitable for overexpression of PYCs. We show that a Pseudomonas aeruginosa strain carrying the T7 polymerase gene can serve as a host for the overexpression of Mycobacterium smegmatis alpha4 PYC and Pseudomonas aeruginosa alpha4beta4 PYC under the control of the T7 promoter from a broad-host-range conjugative plasmid. Overexpression occurred both in aerobic (LB medium) and nitrate-respiring anaerobic (LB medium plus glucose and nitrate) cultures. The latter system presented a simpler option because it involved room temperature cultures in stationary screw-cap bottles. We also developed a P. aeruginosa Deltapyc strain that allowed the expression of recombinant PYCs in the absence of the native enzyme. Since P. aeruginosa can be transformed genetically and lysed for cell extract preparation rather easily, our system will facilitate site-directed mutagenesis, kinetics, X-ray crystallographic, and nuclear magnetic resonance-based structure-function analysis of PYCs. During this work we also determined that, contrary to a previous report (C. K. Stover et al., Nature 406:959-964, 2000), the open reading frame (ORF) PA1400 does not encode a PYC in P. aeruginosa. The alpha4beta4 PYC of this organism was encoded by the ORFs PA5436 and PA5435.
authors.affiliation: Virginia Bioinformatics Institute, Department of Biochemistry, Virginia Polytechnic Institute and State University, Washington Street, MC 0477, Blacksburg, VA 24061, USA.
authors.forename: Huafang
authors.initials: H
authors.lastname: Lai
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Kraszewski
authors.forename: Endang
authors.initials: E
authors.lastname: Purwantini
authors.forename: Biswarup
authors.initials: B
authors.lastname: Mukhopadhyay
mesh_terms: D001709:Biotechnology / Q000379:methods; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics*; D011766:Pyruvate Carboxylase / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D014764:Viral Proteins / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase; D011766:Pyruvate Carboxylase