Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and N-acetylglucosamine metabolism in Bifidobacterium.
Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial D: -fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the D: -fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism.
Foley
Sophie
S
School of Life Sciences, Napier University, Edinburgh, EH10 5DT, UK.
Stolarczyk
Emilie
E
Mouni
Fadoua
F
Brassart
Colette
C
Vidal
Olivier
O
Aïssi
Eliane
E
Bouquelet
Stéphane
S
Krzewinski
Frédéric
F
D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005636:Fructosephosphates / Q000378:metabolism; D015870:Gene Expression; D005944:Glucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D005972:Glutaminase / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
D000117:Acetylglucosamine; D001426:Bacterial Proteins; D005636:Fructosephosphates; D005944:Glucosamine; D019298:Glucose-6-Phosphate; D018698:Glutamic Acid; D005972:Glutaminase; D005973:Glutamine; C001293:glucosamine 6-phosphate; C027618:fructose-6-phosphate; C000731191:component S, glutamate mutase protein, Bacteria; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)