Molecular and biochemical characterisation of a Teladorsagia circumcincta glutamate dehydrogenase |
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| Authors: | Umair S Knight J S Patchett M L Bland R J Simpson H V |
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| Affiliation: | aInstitute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand;bHopkirk Research Institute, AgResearch Ltd., Private Bag 11-008, Palmerston North, New Zealand;cInstitute of Molecular Biosciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand |
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| Abstract: | A full length cDNA encoding glutamate dehydrogenase was cloned from Teladorsagia circumcincta (TcGDH). The TcGDH cDNA (1614 bp) encoded a 538 amino acid protein. The predicted amino acid sequence showed 96% and 93% similarity with Haemonchus contortus and Caenorhabditis elegans GDH, respectively. A soluble N-terminal 6xHis-tagged GDH protein was expressed in the recombinant Escherichia coli strain BL21 (DE3) pGroESL, purified and characterised. The recombinant TcGDH had similar kinetic properties to those of the enzyme in homogenates of T. circumcincta, including greater activity in the aminating than deaminating reaction. Addition of 1 mM ADP and ATP increased activity about 3-fold in the deaminating reaction, but had no effect in the reverse direction. TcGDH was a dual co-factor enzyme that operated both with NAD+ and NADP+, GDH activity was greater in the deaminating reaction with NADP+ as co-factor and more with NADH in the aminating reaction. |
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| Keywords: | Teladorsagia (Ostertagia) circumcincta Glutamate dehydrogenase (E.C. 1.4.1.3) Enzyme activity Gene sequence Kinetic properties |
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