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L-alanine:2-oxoglutarate aminotransferase isoenzymes from Arabidopsis thaliana leaves

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Abstract

The occurrence of four l-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proteins): alanine aminotransferase 1 and 2 (AlaAT1 and AlaAT2, EC 2.6.1.2) and l-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approximately 60 kDa. The kinetic parameters: Km (Ala)=1.53 mM, Km (2-oxoglutarate)=0.18 mM, kcat=124.6 s−1, kcat/Km=8.1 × 104 M−1·s−1 of AlaAT1 were comparable to those determined for other AlaATs isolated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3–70 kDa. The estimated Km values for l-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.

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Abbreviations

AlaAT:

alanine aminotransferase

AOAT:

l-alanine:2-oxoglutarate aminotransferase

GGAT:

l-glutamate:glyoxylate aminotransferase

PLP:

pyridoxal phosphate

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Agriculture and Biology, Warsaw Agricultural University, Nowoursynowska 159, 02-776, Warszawa, Poland

    Paweł Wiśniewski, Justyna Szklarczyk, Marcin Maciaga & Andrzej Paszkowski

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  1. Paweł Wiśniewski
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  2. Justyna Szklarczyk
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  3. Marcin Maciaga
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  4. Andrzej Paszkowski
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Correspondence to Andrzej Paszkowski.

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Wiśniewski, P., Szklarczyk, J., Maciaga, M. et al. L-alanine:2-oxoglutarate aminotransferase isoenzymes from Arabidopsis thaliana leaves. Acta Physiol Plant 28, 577–588 (2006). https://doi.org/10.1007/s11738-006-0053-7

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  • DOI: https://doi.org/10.1007/s11738-006-0053-7

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