title: Recombinant production of Yersinia enterocolitica pyruvate kinase isoenzymes PykA and PykF.
abstract: The glycolytic enzyme pyruvate kinase (PK) generates ATP from ADP through substrate-level phosphorylation powered by the conversion of phosphoenolpyruvate to pyruvate. In contrast to other bacteria, Enterobacteriaceae, such as pathogenic yersiniae, harbour two pyruvate kinases encoded by pykA and pykF. The individual roles of these isoenzymes are poorly understood. In an attempt to make the Yersinia enterocolitica pyruvate kinases PykA and PykF amenable to structural and functional characterisation, we produced them untagged in Escherichia coli and purified them to near homogeneity through a combination of ion exchange and size exclusion chromatography, yielding more than 180 mg per litre of batch culture. The solution structure of PykA and PykF was analysed through small angle X-ray scattering which revealed the formation of PykA and PykF tetramers and confirmed the binding of the allosteric effector fructose-1,6-bisphosphate (FBP) to PykF but not to PykA.
authors: Robert Koch-Institute, Wernigerode Branch, Burgstr. 37, D-38855 Wernigerode, Germany.
authors: Julia
authors: J
authors: Hofmann
authors: Christine
authors: C
authors: Heider
authors: Wei
authors: W
authors: Li
authors: Joern
authors: J
authors: Krausze
authors: Manfred
authors: M
authors: Roessle
authors: Gottfried
authors: G
authors: Wilharm
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D055503:Protein Multimerization; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction; D015008:Yersinia enterocolitica / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D011770:Pyruvate Kinase