Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A

P. Sanchez-Torres, J. Visser, J.A.E. Benen

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21 Citations (Scopus)

Abstract

Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed beta-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH. On the basis of the three-dimensional structures of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677-689] and the modelled enzyme-substrate complex of PL1B [Herron, Benen, Scavetta, Visser and Jurnak (2000) Proc. Nail. Acad. Sci. U.S.A. 97, 8762-8769], Asp(154), Arg(176), Arg(236) and Lys(239) were mutagenized. Substituting Arg(236) with alanine or lysine rendered the enzyme completely inactive, and mutagenesis of Arg(176) and Lys(239) severely affected catalysis. The Asp(154) --> Arg and Asp(154) --> Glu mutant enzymes were only moderately impaired in respect of catalysis. The results strongly indicate that Arg(236), which is sandwiched between Arg(176) and Lys(239), would initiate the reaction upon enzyme-substrate interaction, through the abstraction of the proton at C-5 of the galacturonopyranose ring. The positively charged residues Arg(176) and Lys(239) are responsible for lowering the pK(a) of Arg(236). Arg(176) and Lys(239) are maintained in a charged state by interacting with Asp(154) or bulk solvent respectively. The deprotonation of the Asp(186)-Asp(221) pair was proposed to be responsible for a pH-driven conformational change of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677-689]. Substitution of Asp(186) and Asp(221) by Asn(186) and Asti(221) was expected to stabilize the enzyme. However, the Asp(186) --> Asn/Asp(221) --> Asn enzyme appeared less stable than the wild-type enzyme, even at pH 6.0, as evidenced by fluorescence studies. This demonstrates that the pH-dependent conformational change is not driven by deprotonation of the Asp(186) --> Asp(221) pair.
Original languageEnglish
Pages (from-to)331-337
JournalBiochemical Journal
Volume370
DOIs
Publication statusPublished - 2003

Keywords

  • site-directed mutagenesis
  • plant virulence factor
  • pectate lyase
  • endopolygalacturonase ii
  • 3-dimensional structure
  • gene family
  • enzymes
  • pathogenesis
  • expression
  • erwinia

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