Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport.
Zymomonas mobilis is known to transport glucose by a facilitated diffusion process. A putative glucose facilitator gene (glf), closely related to a large family of glucose transporters, is located in a cluster of genes that code for enzymes of glucose metabolism. The Z. mobilis glf gene is able to complement glucose transport in an Escherichia coli strain that is defective in native glucose transport and glucokinase. In this study, the recombinant E. coli was shown to be capable of influx counterflow when preloaded with glucose and had an apparent Km for glucose of approximately 1.1-2.9 mM, consistent with the function of Glf as a low-affinity glucose facilitator. The ability of glucokinase mutants expressing glf to transport glucose made it clear that glucokinase activity was not required for Glf-dependent glucose transport. The possibility that glucokinase can interact with Glf to improve the affinity for glucose was not supported since expression of the Z. mobilis glucokinase gene, in addition to glf, did not affect the Km of Glf for glucose in recombinant E. coli. The inability of various sugars to compete with glucose during glucose transport by recombinant E. coli expressing glf indicated that Glf is specific for glucose. While the results of fructose transport assays did not completely rule out the possibility of very low affinity for fructose, the apparent specificity of Glf for glucose makes it possible that Z. mobilis utilizes a different transporter(s) for fructose.
Parker
C
C
School of Biological Sciences, University of Nebraska, Lincoln 68588-0118, USA.
Barnell
W O
WO
Snoep
J L
JL
Ingram
L O
LO
Conway
T
T
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005838:Genotype; D005941:Glucokinase / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside / Q000235:genetics / Q000494:pharmacology; D007700:Kinetics; D009005:Monosaccharides / Q000494:pharmacology; D013379:Substrate Specificity; D017355:Zymomonas / Q000235:genetics*
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D009005:Monosaccharides; C086512:glucose facilitator protein, Zymomonas; D007544:Isopropyl Thiogalactoside; D005941:Glucokinase; D005947:Glucose