A String of Enzymes, Purification and Characterization of a Fusion Protein Comprising the Four Subunits of the Glucose Phosphotransferase System of Escherichia coli

Abstract

A multidomain protein comprising the four subunits of the glucose phosphotransferase system of Escherichia coli was constructed by fusion of the transmembrane subunit IICBGlc and the three cytoplasmic proteins, IIAGlc, HPr, and enzyme I. The subunits were linked in the above order with Ala-Pro-rich linkers; the fusion protein was overexpressed in E. coli and purified by Ni2+ chelate affinity chromatography. Approximately 3 mg of the fusion protein could be purified from 1 liter of culture. The phosphotransferase activity of the purified fusion protein was 3-4 times higher than that of an equimolar mixture of the isolated subunits. The mannose transporter, which also requires enzyme I and HPr, was not an effective competitor in the overall phosphoryltransfer reaction when the fusion protein was used, whereas it was a competitor when an equimolar mixture of the separate subunits was employed. Transphosphorylation activity of the fusion protein was almost indistinguishable from the wild-type IICBglc. Addition of extra IICBGlc subunit could significantly stimulate the phosphotransferase activity of the fusion protein, addition of extra IIAGlc subunit and enzyme I, in contrast, was slightly inhibitory, and HPr had almost no effect. An optimal detergent-lipid ratio is required for maximum activity of the fusion protein. Our results suggest that Ala-Pro-rich linker sequences may be of general use for the construction of catalytically active fusion proteins with novel properties.

Crystal Structure of the Citrobacter freundii Dihydroxyacetone Kinase Reveals an Eight-stranded α-Helical Barrel ATP-binding Domain

“…Protein Purification and Activity assay-The Dha kinase of C. freundii was overexpressed in E. coli K12 WA2127⌬ HIC (⌬ manXYZ ⌬ ptsHIcrr) (36). Cells (3 liters) were grown at 37°C and induced with 200 M isopropyl-1-thio-␤-D-galactopyranoside at A 600 ϭ 0.8, and incubation continued for 5 h. Cells were collected by centrifugation, and the sediment was resuspended in buffer A (25 ml, 50 mM NaP i , pH 8.0, 10 mM ␤-mercaptoethanol, 500 mM NaCl), broken by two passages in a French pressure cell, and fractionated by differential centrifugation into cell debris (10 min, 3000 ϫ g), membrane fraction (60 min, 150,000 ϫ g), and cytoplasmic fraction (supernatant), which was mixed with 10 ml of Ni 2ϩ -nitrilotriacetic acid resin (Qiagen) and allowed to adsorb for 30 min.…”

Implications of macromolecular crowding for signal transduction and metabolite channeling

“…For ␣ϭ 1, crowding effects are assumed to be absent. Mao et al (28) reported the isolation, purification and reconstitution of a fusion protein comprising the four glucose PTS enzymes of E. coli, in which the different enzymes were joined by flexible linkers. In agreement with our results, the phosphorylation activity of an equimolar mixture of the individual isolated enzymes varied more than linearly but less than quadratically with the total enzyme concentration (figure 2A in ref.…”

Glucose and trehalose PTS permeases of Spiroplasma citri probably share a single IIA domain, enabling the spiroplasma to adapt quickly to carbohydrate changes in its environment

“…From our studies, the gene organization of the glucose permeases in Gram-positive bacteria did not correlate with a specific phylogenetic branch, but instead seemed to be more related to the ecological niches of the bacteria. In vitro analyses of the E. coli glucose PTS showed that a fusion protein made of IIC Glc , IIB Glc and IIA Glc domains linked together exhibited higher phosphotransferase activity than a mixture of the isolated subunits (Mao et al, 1995). These data suggested that the linking of functional domains could be an advantage regarding the activity of the permease.…”