Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 326963
Title Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A
Author(s) Sanchez-Torres, P.; Visser, J.; Benen, J.A.E.
Source Biochemical Journal 370 (2003). - ISSN 0264-6021 - p. 331 - 337.
DOI https://doi.org/10.1042/BJ20021071
Department(s) AFSG Staff Departments (WUATV)
Microbiology
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2003
Keyword(s) site-directed mutagenesis - plant virulence factor - pectate lyase - endopolygalacturonase ii - 3-dimensional structure - gene family - enzymes - pathogenesis - expression - erwinia
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.
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