Molecular Cloning, Sequencing, and Expression of a l-Glutamine d-Fructose 6-Phosphate Amidotransferase Gene from Volvariella volvacea
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Abstract
Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N 3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.
Keywords
l-Glutamine:d-fructose-6-phosphate amidotransferase Volvariella volvacea cDNA cloning Heterologous expressionAbbreviations
- GFAT
l-Glutamine d-fructose 6-phosphate amidotransferase
- GATase
Glutamine amidotransferase
- SDS–PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- RACE
Rapid amplification of cDNA ends
- GLcN-6-P
d-Glucosamine-6-phosphate
- UDP-GlcNAc
Uridine 5′-diphospho-N-acetylglucosamine
- FMDP
N 3-(4-Methoxyfumaroyl)-l-2,3-diaminopropanoic acid
- ADGP
2-Amino-2-deoxy-d-glucitol-6-phosphate
- CMC
Carboxymethyl cellulose
- DTT
Dithiothreitol
- EDTA
Ethylenediamine tetraacetic acid disodium salt
- cAMP
cyclic Adenosine monophosphate
- PKA
cAMP-dependent protein kinase
- l-Gln
l-Glutamine
- Fru-6-P
d-Fructose-6-phosphate
- PMSF
Phenylmethylsulfonyl fluoride
Notes
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (Grant No. 30370034) and the 211 Foundation of Ministry of Light Industry of China (2001). We thank Dr John Buswell (Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences) for linguistic revision of the manuscript.
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