The aspartate aminotransferase family in conifers: biochemical analysis of a prokaryotic-type enzyme from maritime pine.
Plant aspartate aminotransferase (AAT, EC 2.6.1.1) plays a key role in primary nitrogen assimilation, the transfer of reducing equivalents and the interchanges of carbon and nitrogen pools between subcellular compartments. We investigated the AAT family in conifers using maritime pine as the experimental model. Genes for cytosolic, mitochondrial and two plastidic isoenzymes (eukaryotic- and prokaryotic-types) were identified and their deduced amino acid sequences compared. The primary structure of the eukaryotic-type enzymes is quite well conserved, whereas the prokaryotic-type AAT is highly divergent (15% of identity). These molecular data were confirmed by the absence of immunological cross-reactivity between the two types of native AATs. The mature prokaryotic-type polypeptide was overexpressed in Escherichia coli, and the native enzyme was purified to apparent homogeneity and its molecular properties determined. The fully active recombinant holoenzyme showed highest catalytic activity at 50-60 degrees C and was moderately thermostable, retaining about 50% of its activity after incubation at 70 degrees C for 5-10 min. The presence of pyridoxal 5'-phosphate significantly increased the thermostability of the enzyme. These molecular characteristics were exploited to develop a rapid protocol for the purification of this prokaryotic-type enzyme from pine cotyledons. The results will be useful for studying aspartate and amino acid metabolism in trees.
de la Torre
Fernando
F
Departamento de Biología Molecular y Bioquímica, Instituto Andaluz de Biotecnología, Unidad Asociada UMA-CSIC, Campus Universitario de Teatinos, Universidad de Málaga, 29071-Málaga, Spain.
Suárez
María Fernanda
MF
Santis
Laura de
Ld
Cánovas
Francisco M
FM
D000595:Amino Acid Sequence; D001219:Aspartate Aminotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018548:Cotyledon / Q000201:enzymology; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D028223:Pinus / Q000201:enzymology* / Q000235:genetics; D018087:Plastids / Q000201:enzymology*; D011387:Prokaryotic Cells / Q000201:enzymology
D007527:Isoenzymes; D001219:Aspartate Aminotransferases