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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.

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Abbreviations

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

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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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Authors and Affiliations

  1. Institute of Plant Protection, Jiangsu Academy of Agriculture Sciences, Nanjing, Jiangsu, 210014, China

    Chuping Luo, Zhiyi Chen & Yongfeng Liu

  2. Jiangsu Key Laboratory of Biodiversity and Biotechnology, Nanjing Normal University, Nanjing, Jiangsu, 210097, China

    Chuping Luo, Weilan Shao & Xun Li

  3. Department of Bioengineering, Nanjing Forestry University, Nanjing, Jiangsu, 210017, China

    Xun Li

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  1. Chuping Luo
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  2. Weilan Shao
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Correspondence to Chuping Luo.

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Luo, C., Shao, W., Li, X. et al. Molecular Cloning, Sequencing, and Expression of a l-Glutamine d-Fructose 6-Phosphate Amidotransferase Gene from Volvariella volvacea . Protein J 28, 34–43 (2009). https://doi.org/10.1007/s10930-009-9163-6

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