In plants, L-asparagine is the most abundant metabolite for the storage and transport of nitrogen that is utilized in protein biosynthesis. Asparagine hydrolysis is catalyzed by the enzyme asparaginase. Kinetic studies of asparaginase from Lupinus luteus (LlA) show that the enzyme is predominantly active as isoaspartyl aminopeptidase. In this light, LlA gain in relevance as enzyme responsible for the degradation of misformed proteins, in which a peptide bond occurs at the side chain of asparagine or aspartic acid. The crystal structure of LlA demonstrates that it is composed of two subunits, a and b, generated during autoproteolytic cleavage of the precursor molecule at Gly192-Thr193 peptide bond. The threonine residue that becomes an N-terminus of subunit b plays crucial role in both, enzymatic reaction and autoactivation. We have designed, cloned and expressed an active-site mutant of LlA with the catalytic threonine residue replaced by an alanine in order to prevent autocleavage and preserve precursor molecule. We intend to make biochemical tests on LlA mutant to confirm decline enzymatic activity as well as determine its crystal structure to analyze structural basis of maturation mechanism.

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