Molecular cloning, sequencing, and expression of a L -glutamine D-fructose 6-phosphate amidotransferase gene from Volvariella volvacea.
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.
Luo
Chuping
C
Institute of Plant Protection, Jiangsu Academy of Agriculture Sciences, Nanjing, Jiangsu, 210014, China. Chupingluo@sina.com
Shao
Weilan
W
Li
Xun
X
Chen
Zhiyi
Z
Liu
Yongfeng
Y
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D055367:Volvariella / Q000201:enzymology*
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D004247:DNA; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)