A major route for Asn biosynthesis is via the ATP-dependent transfer of the amide group of glutamine to the β-carboxyl group of aspartate by the action of asparagine synthetase[AS]. So far, two types of ASs, AsnA and AsnB, have been identified. While prokaryotes utilize AsnA type ASs that require ammonia as an amide donor as well as AsnB type ASs that can catalyze the reaction using either ammonium or glutamine as an amide donor, most eukaryotes only use AsnB type ASs
[10, 11]. AS is notably difficult to assay from plant tissues
[12]. AsnB-type ASs are members of the N-terminal nucleophile hydrolase[Ntn] group of glutamine amidotransferases
[13, 14]. They are characterized by an N-terminal cysteine nucleophilic residue producing a cysteinyl-glutamine tetrahedral intermediate from which ammonium is abstracted. Glutamate is released by hydrolysis of the resulting γ-glutamyl thioester intermediate. The ammonia is tunneled to a C-terminal transferase domain. This domain activates aspartate through ATP hydrolysis as a β-aspartyl AMP intermediate. Nucleophilic attack by the ammonia results in cleavage and release of Asn. There are two groups of AS enzymes in higher plants designated as class I and II. Results of kinetic analyses of recombinant maize AS enzymes indicated that class I enzymes may have specialized functions as they can have higher affinity for glutamine and their expression is restricted to specific tissues
[15]. The detailed schematic pathways of asparagine metabolic pathways are shown in Figure 2
[16].
Figure 2 Asparagine metabolic pathways
Ammonium is assimilated into the glutamine-amide group for glutamine synthesis by the reaction of glutamine synthetase[GS]. Glutamate synthase[GOGAT] transfers the amide group of glutamine to the 2-position of 2-oxoglutarate, generating glutamate. Asparagine synthetase[AS] converts either the glutamine-amide group or ammonium into aspartate, yielding asparagine. Transamination of glutamate with oxaloacetate by aspartate aminotransferase[AspAT] generates aspartate, which serves as a substrate of asparagine synthesis. The asparagine amide group can be degraded by asparaginase[ASPG], yielding ammonium and aspartate. The asparagine amino group is hydrolyzed by asparagine aminotransferase[AsnAT], producing ammonium and 2-oxosucinamate. AsnAT catalyzes the transamination reaction of asparagine with glyoxylate, pyruvate, 4-hydroxypyruvate and 4-hydroxy 2-oxobutyrate as amino acceptors, producing glycine, alanine, serine and homoserine, respectively. 2-Oxosuccinamate is then converted to ammonium and oxaloacetate by ω-amidase.
Asparagine synthetaseAsparagine synthetase[l-aspartate: ammonia ligase[AMP-forming], EC 6.3.1.1] catalyzes the reversible conversion of l-aspartate, NH4+, and ATP to l-asparagine, AMP, and PPi. The enzyme is distributed widely in nature, but its enzymological properties have not been studied in detail. Pioneering studies have been made on the enzymes from lactic acid bacteria. The enzyme from Lactobacillus arabinosus can be stored at 4 ℃ for 3 weeks but not at–20℃
[17]. The optimum pH is 8.2, and the optimum temperature is about 40℃. The enzyme is specific for l-aspartate and does not act on l-glutamate. β-l-Aspartyl hydroxamate is synthesized when hydroxamate is added to the reaction mixture instead of NH4+. The enzyme requires Mg2+ and is activated by Mn2+. No activation of Mg2+ was observed for the E. coli
[18] and Streptococcus bovis enzymes.
AsparaginaseAsparaginase[l-asparagine amidohydrolase, EC 3.5.1.1] catalyzes the hydrolysis of the amido bond of l-asparagine and irreversibly produces l-aspartate and ammonia. The enzyme is widely distributed in microorganisms, animals, and plants. The bacterial enzymes from Acinetobacter calcoaceticus
[19], Bacillus coagulans[20], E. coli
[21], and Vibrio succinogenes
[22] also show enzymatic activity on d-asparagine. The enzyme from E. coli has been used for the industrial production of l-asparagine. Saccharomyces cerevisiae produces the enzyme both intracellularly and extracellularly
[23]. The synthesis of the enzyme is stimulated by nitrogen starvation, requires an available energy source, and is prevented by cycloheximide. The intracellular enzyme appears to be constitutive. The extracellular activity is relatively insensitive to p-hydroxymercuribenzoate inhibition, whereas the intracellular activity is highly inhibited by this compound.
