Schöndorf T, Hachtel W
Botanisches Institut, Universität Bonn, Germany.
Plant Physiol. 1995 May;108(1):203-10. doi: 10.1104/pp.108.1.203.
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-monospecific 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.
已发现白桦（Betula pendula Roth）的双特异性NAD(P)H-硝酸还原酶与多种其他高等植物的单特异性NADH-硝酸还原酶在FAD结构域的二核苷酸结合位点存在氨基酸序列差异。为了确定决定还原底物选择的氨基酸残基，我们对白桦硝酸还原酶的编码cDNA进行了突变。这些突变旨在用NADH单特异性酶中相同位置的保守氨基酸替换NAD(P)H结合位点的某些氨基酸。通过农杆菌介导的转化将突变的cDNA整合到烟草基因组中。转基因烟草（Nicotiana tabacum）植株在以铵作为唯一氮源的培养基上生长，以使内源性烟草硝酸还原酶活性保持较低水平。尽管一些突变酶与未突变的白桦酶一样，对NADPH表现出轻微偏好，但其他一些酶的活性很大程度上取决于NADH的可用性，单独使用NADPH时活性较低。对这些突变的比较表明，白桦序列中的单个氨基酸替换（丙氨酸871替换为脯氨酸）对于还原底物的使用至关重要。
