Changing a single amino acid residue switches processive and non-processive behavior of Aspergillus niger endopolygalacturonase I and II.
Processivity, also known as multiple attack on a single chain, is a feature commonly encountered only in enzymes in which the substrate binds in a tunnel. However, of the seven Aspergillus niger endopolygalacturonases, which have an open substrate binding cleft, four enzymes show processive behavior, whereas the other endopolygalacturonases are randomly acting enzymes. In a previous study (Benen, J.A.E., Kester, H.C.M., and Visser, J. (1999) Eur. J. Biochem. 259, 577-585) we proposed that the high affinity for the substrate of subsite -5 of processive endopolygalacturonase I constitutes the origin of the multiple attack behavior. Based on primary sequence alignments of A. niger endopolygalacturonases and three-dimensional structure analysis of endopolygalacturonase II, an arginine residue was identified in the processive enzymes at a position commensurate with subsite -5, whereas a serine residue was present at this position in the non-processive enzymes. In endopolygalacturonase I mutation R95S was introduced, and in endopolygalacturonase II mutation S91R was introduced. Product progression analysis on polymer substrate and bond cleavage frequency studies using oligogalacturonides of defined chain length for the mutant enzymes revealed that processive/non-processive behavior is indeed interchangeable by one single amino acid substitution at subsite -5, Arg-->Ser or Ser-->Arg.
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Section Molecular Genetics of Industrial Microorganisms, Wageningen University, Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.
Kester
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HC
Visser
J
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Benen
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JA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry; D001234:Aspergillus niger / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011096:Polygalacturonase / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013997:Time Factors
D000596:Amino Acids; D012694:Serine; D001120:Arginine; C400187:endopolygalacturonase II; D011096:Polygalacturonase