© 2007 Heron Publishing—Victoria, Canada
The aspartate aminotransferase family in conifers: biochemical analysis of a prokaryotic-type enzyme from maritime pine
Fernando de la Torre (1), María Fernanda Suárez (1), Laura de Santis (1) and Francisco M. Cánovas (1, 2)
1. Departamento de Biología Molecular y Bioquímica, Instituto Andaluz de Biotecnología, Unidad Asociada UMA-CSIC, Campus Universitario
de Teatinos, Universidad de Málaga, 29071-Málaga, Spain / 2. Corresponding author (canovas@uma.es) / Received June 22, 2006; accepted January 24, 2007; published online June 1, 2007
Summary
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 °C and was moderately thermostable, retaining about 50% of its activity
after incubation at 70 °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.
Keywords:
amino acids, aspartate metabolism, nitrogen assimilation, peptide fingerprinting, recombinant expression.
