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Mutational analysis of pea lectin: Substitution of Asn125 for Asp in the monosaccharide-binding site eliminates mannose/glucose-binding activity

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Author: Eijsden, R.R. van · Hoedemaeker, F.J. · Díaz, C.L. · Lugtenberg, B.J.J. · Sylvia Pater, B. de · Kijne, J.W.
Type:article
Date:1992
Source:Plant Molecular Biology, 6, 20, 1049-1058
Identifier: 280629
doi: doi:10.1007/BF00028892
Keywords: mutagenesis · pea lectin · sugar binding · mannose · oligodeoxyribonucleotide · pea lectin · phytohemagglutinin · amino acid sequence · binding site · chemistry · comparative study · Escherichia coli · genetics · legume · medicinal plant · metabolism · molecular genetics · nucleotide sequence · site directed mutagenesis · structure activity relation · Amino Acid Sequence · Asparagine · Aspartic Acid · Base Sequence · Binding Sites · Calcium · Comparative Study · DNA Mutational Analysis · Escherichia coli · Fabaceae · Glucose · Lectins · Manganese · Mannose · Molecular Sequence Data · Mutagenesis, Site-Directed · Oligodeoxyribonucleotides · Phytohemagglutinins · Plants, Medicinal · Sequence Alignment · Structure-Activity Relationship

Abstract

As part of a strategy to determine the precise role of pea (Pisum sativum) lectin, Psl, in nodulation of pea by Rhizobium leguminosarum, mutations were introduced into the genetic determinant for pea lectin by site-directed mutagenesis using PCR. Introduction of a specific mutation, N125D, into a central area of the sugar-binding site resulted in complete loss of binding of Psl to dextran as well as of mannose/glucose-sensitive haemagglutination activity. As a control, substitution of an adjacent residue, A126V, did not have any detectable influence on sugar-binding activity. Both mutants appeared to represent normal Psl dimers with a molecular mass of about 55 kDa, in which binding of Ca<sup>2+</sup> and Mn<sup>2+</sup> ions was not affected. These results demonstrate that the NHD2 group of Asn<sup>125</sup> is essential in sugar binding by Psl. To our knowledge, Psl N125D is the first mutant legume lectin which is unable to bind sugar residues. This mutant could be useful in the identification of the potential role of the lectin in the recognition of homologous symbionts. © 1992 Kluwer Academic Publishers. Chemicals/CAS: asparagine, 70-47-3, 7006-34-0; aspartic acid, 56-84-8, 6899-03-2; calcium, 7440-70-2; glucose, 50-99-7, 84778-64-3; manganese, 16397-91-4, 7439-96-5; mannose, 31103-86-3, 3458-28-4; phytohemagglutinin, 9008-97-3; Asparagine, 7006-34-0; Aspartic Acid, 56-84-8; Calcium, 7440-70-2; Glucose, 50-99-7; Lectins; Manganese, 7439-96-5; Mannose, 31103-86-3; Oligodeoxyribonucleotides; pea lectin; Phytohemagglutinins