A kinetic comparison of asparagine synthetase isozymes from higher plants

Duff, Stephen M. G.; Qi, Qungang; Reich, Timothy; Wu, Xia; Brown, Todd J.; Crowley, James H.; Fabbri, Brad

doi:10.1016/j.plaphy.2010.12.006

Cited by 28 publications

(31 citation statements)

References 25 publications

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“…Such hybrid activity was only previously demonstrated for type B enzymes, which prefer glutamine to ammonia [15]–[19]. The small differences in K m of Tb AS-A and Tc AS-A for ammonia and glutamine (1.5 and 2 fold, respectively) are lower than the difference found in most AS-B enzymes, with the exception of the human enzyme, which has similar affinities for both [16]–[20]. Purified E. coli AS-A used only ammonia as the nitrogen source, and results from Klebsiella aerogenes also suggested that AS-A preferentially uses ammonia as substrate [5], [21], [22].…”

Section: Discussionmentioning

confidence: 71%

Knockdown of Asparagine Synthetase A Renders Trypanosoma brucei Auxotrophic to Asparagine

et al. 2013

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Asparagine synthetase (AS) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A), known as strictly ammonia-dependent, and asparagine synthetase B (AS-B), which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A) and Trypanosoma brucei (TbAS-A): the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg2+. TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn't affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.

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Section: Discussionmentioning

confidence: 71%

Knockdown of Asparagine Synthetase A Renders Trypanosoma brucei Auxotrophic to Asparagine

et al. 2013

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“…The effect of any of these enzymes on nitrogen uptake or metabolism can only be speculated upon and would require whole plants studies and in vivo analysis which are currently underway. Analysis of the kinetics of aspartate aminotransferase (AspAT) from higher plants has also been carried out recently with similar intentions of crop improvement [30].…”

Section: Resultsmentioning

confidence: 99%

Analysis of the Enzymatic Properties of a Broad Family of Alanine Aminotransferases

et al. 2013

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Alanine aminotransferase (AlaAT) has been studied in a variety of organisms due to the involvement of this enzyme in mammalian processes such as non-alcoholic hepatocellular damage, and in plant processes such as C4 photosynthesis, post-hypoxic stress response and nitrogen use efficiency. To date, very few studies have made direct comparisons of AlaAT enzymes and fewer still have made direct comparisons of this enzyme across a broad spectrum of organisms. In this study we present a direct kinetic comparison of glutamate:pyruvate aminotransferase (GPAT) activity for seven AlaATs and two glutamate:glyoxylate aminotransferases (GGAT), measuring the KM values for the enzymes analyzed. We also demonstrate that recombinant expression of AlaAT enzymes in Eschericia coli results in differences in bacterial growth inhibition, supporting previous reports of AlaAT possessing bactericidal properties, attributed to lipopolysaccharide endotoxin recognition and binding. A probable lipopolysaccharide binding region within the AlaAT enzymes, homologous to a region of a lipopolysaccharide binding protein (LBP) in humans, was also identified in this study. The AlaAT enzyme differences identified here indicate that AlaAT homologues have differentiated significantly and the roles these homologues play in vivo may also have diverged significantly. Specifically, the differing kinetics of AlaAT enzymes and how this may alter the nitrogen use efficiency in plants is discussed.

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“…Enzymes were extracted according to Gibon et al (2004), except where differently indicated. Asparagine synthetase (AS; EC 6.3.5.4) was extracted in roots of 20-day-old plants and immediately desalted and assayed in a solution containing 1 mM aspartate semialdehyde (an inhibitor of asparaginase) and 1 mM amino(oxy)acetic acid (an inhibitor of aspartate aminotransferase) according to Duff et al (2011). NADH-dependent glutamate synthase (Fd-GOGAT; EC 1.4.1.14), glutamine synthetase (GS; EC 6.3.1.2), and nitrate reductase (NR; EC 1.6.6.1) were assayed according to Gibon et al (2004).…”

Section: Methodsmentioning

confidence: 99%

Durum Wheat Roots Adapt to Salinity Remodeling the Cellular Content of Nitrogen Metabolites and Sucrose

et al. 2017

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Plants are currently experiencing increasing salinity problems due to irrigation with brackish water. Moreover, in fields, roots can grow in soils which show spatial variation in water content and salt concentration, also because of the type of irrigation. Salinity impairs crop growth and productivity by inhibiting many physiological and metabolic processes, in particular nitrate uptake, translocation, and assimilation. Salinity determines an increase of sap osmolality from about 305 mOsmol kg−1 in control roots to about 530 mOsmol kg−1 in roots under salinity. Root cells adapt to salinity by sequestering sodium in the vacuole, as a cheap osmoticum, and showing a rearrangement of few nitrogen-containing metabolites and sucrose in the cytosol, both for osmotic adjustment and oxidative stress protection, thus providing plant viability even at low nitrate levels. Mainly glycine betaine and sucrose at low nitrate concentration, and glycine betaine, asparagine and proline at high nitrate levels can be assumed responsible for the osmotic adjustment of the cytosol, the assimilation of the excess of ammonium and the scavenging of ROS under salinity. High nitrate plants with half of the root system under salinity accumulate proline and glutamine in both control and salt stressed split roots, revealing that osmotic adjustment is not a regional effect in plants. The expression level and enzymatic activities of asparagine synthetase and Δ1-pyrroline-5-carboxylate synthetase, as well as other enzymatic activities of nitrogen and carbon metabolism, are analyzed.

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A kinetic comparison of asparagine synthetase isozymes from higher plants

Cited by 28 publications

References 25 publications

Knockdown of Asparagine Synthetase A Renders Trypanosoma brucei Auxotrophic to Asparagine

Knockdown of Asparagine Synthetase A Renders Trypanosoma brucei Auxotrophic to Asparagine

Analysis of the Enzymatic Properties of a Broad Family of Alanine Aminotransferases

Durum Wheat Roots Adapt to Salinity Remodeling the Cellular Content of Nitrogen Metabolites and Sucrose

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