title: Characterization of the pyrophosphate-dependent 6-phosphofructokinase from Xanthomonas campestris pv. campestris.
abstract: Xanthomonads are plant pathogenic proteobacteria that produce the polysaccharide xanthan. They are assumed to catabolize glucose mainly via the Entner-Doudoroff pathway. Whereas previous studies have demonstrated no phosphofructokinase (PFK) activity in xanthomonads, detailed genome analysis revealed in Xanthomonas campestris pathovar campestris (Xcc) genes for all Embden-Meyerhof-Parnas pathway (glycolysis) enzymes, including a conserved pfkA gene similar to 6-phosphofructokinase genes. To address this discrepancy between genetic and physiological properties, the pfkA gene of Xcc strain B100 was cloned into the expression vector pET28a+. The 45-kDa pfkA gene product exhibited no conventional PFK activity. Bioinformatic analysis of the Xcc PfkA amino acid sequence suggested utilization of pyrophosphate as an alternative cosubstrate. Pyrophosphate-dependent PFK activity was shown in an in vitro enzyme assay for purified Xcc PfkA, as well as in the Xcc B100 crude protein extract. Kinetic constants were determined for the forward and reverse reactions. Primary structure conservation indicates the global presence of similar enzymes among Xanthomonadaceae.
authors: Abteilung für Proteom- und Metabolomforschung, Fakultät für Biologie, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors: Marcel
authors: M
authors: Frese
authors: Abteilung für Proteom- und Metabolomforschung, Fakultät für Biologie, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors: Sarah
authors: S
authors: Schatschneider
authors: Abteilung für Proteom- und Metabolomforschung, Fakultät für Biologie, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors: Julia
authors: J
authors: Voss
authors: Abteilung für Proteom- und Metabolomforschung, Fakultät für Biologie, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors: Frank-Jörg
authors: FJ
authors: Vorhölter
authors: Abteilung für Proteom- und Metabolomforschung, Fakultät für Biologie, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany. Electronic address: kniehaus@CeBiTec.Uni-Bielefeld.DE.
authors: Karsten
authors: K
authors: Niehaus
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D019295:Computational Biology; D011756:Diphosphates / Q000378:metabolism*; D016680:Genome, Bacterial / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016959:Xanthomonas campestris / Q000201:enzymology* / Q000235:genetics
chemical_list: D011756:Diphosphates; D010732:Phosphofructokinase-1
keywords: Embden–Meyerhof–Parnas pathway; Glucose metabolism; Glycolysis; Pyrophosphate-dependent phosphofructokinase; Two-substrate enzyme kinetics