PLANT PHYSIOLOGY , Vol 108, Issue 1 203-210, Copyright © 1995 by American Society of Plant Biologists
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GENE REGULATION AND MOLECULAR GENETICS |
The Choice of Reducing Substrate Is Altered by Replacement of an Alanine by a Proline in the FAD Domain of a Bispecific NAD(P)H-Nitrate Reductase from Birch
T. Schondorf and W. Hachtel
Botanisches Institut, Universitat Bonn, Kirchallee 1, 53115 Bonn, Germany
Differences in the amino acid sequence between the bispecific
NAD(P)H-nitrate reductase of birch (Betula pendula Roth) and the
monospecific NADH-nitrate reductases of a variety of other higher plants
have been found at the dinucleotide-binding site in the FAD domain. To
pinpoint amino acid residues that determine the choice of reducing
substrate, we introduced mutations into the cDNA coding for birch nitrate
reductase. These mutations were aimed at replacing certain amino acids of
the NAD(P)H-binding site by conserved amino acids located at identical
positions in NADH-mono-specific enzymes. The mutated cDNAs were integrated
into the genome of tobacco by Agrobacterium-mediated transformation.
Transgenic tobacco (Nicotiana tabacum) plants were grown on a medium
containing ammonium as the sole nitrogen source to keep endogenous tobacco
nitrate reductase activity low. Whereas some of the mutated enzymes showed
a slight preference for NADPH, as does the nonmutated birch enzyme, the
activity of some others greatly depended on the availability of NADH and
was low with NADPH alone. Comparison of the mutations reveals that
replacement of a single amino acid in the birch sequence (alanine871 by
proline) is critical for the use of reducing substrate.
