Asparagine catabolism in bryophytes: Purification and characterization of two L-asparaginase isoforms from Sphagnum fallax

Nitrogen represents a critical nutrient in raised bogs. In Sphagna , dominating this habitat, the prevalent storage amino acid asparagine is catabolized predominantly by the enzyme L-asparaginase (EC 3.5.1.1). L-asparaginase activity has been detected in each of 10 Sphagnum species investigated. In Sphagnum fallax Klinggr. (Klinggr. clone 1) cultivated under axenie conditions in continuous feed bioreactors, the enzyme displayed a light dependent increase in activity. We separated two isoforms, designated L-asparaginase 1 and 2, characterized by their different elution patterns from an anion-exchange column. In stem segments only L-asparaginase 2 could be detected, whereas in capitulae L-asparaginase 1 represented the dominating isoform. Purified chloroplasts displayed no L-asparaginase activity. Almost the entire activity was located in the cytosohc fraction. L-asparaginase 1 and 2 have been purified 82-fold and 188-fold, respectively, by ion-exchange, size-exclusion and hydrophobic interaction chrornatography. Identical pH optima (8.2) and molecular weights (126 000) were determined. The K_m values for asparagine (7.4 m M for L-asparaginase 1 and 6.2 m M for L-asparaginase 2) were in the range of those described for higher plants. On the other hand Sphagnum L-asparaginase is comprised of four subunits as are the L-asparaginases of microorganisms. So, the characteristics of the bryophyte enzyme appear to be intermediate between those from higher plants and those from microorganisms.