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

摘要

pSite-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from iAspergillus niger/i to gain insight into the reaction mechanism for the pectin lyase-catalysed β-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 b5/b, 677—689] and the modelled enzyme—substrate complex of PL1B [Herron, Benen, Scavetta, Visser and Jurnak (2000) Proc. Natl. Acad. Sci. U.S.A. b97/b, 8762—8769], Aspsup154/sup, Argsup176/sup, Argsup236/sup and Lyssup239/sup were mutagenized. Substituting Argsup236/sup with alanine or lysine rendered the enzyme completely inactive, and mutagenesis of Argsup176/sup and Lyssup239/sup severely affected catalysis. The Aspsup154/sup→Arg and Aspsup154/sup→Glu mutant enzymes were only moderately impaired in respect of catalysis. The results strongly indicate that Argsup236/sup, which is sandwiched between Argsup176/sup and Lyssup239/sup, would initiate the reaction upon enzyme—substrate interaction, through the abstraction of the proton at Csup5/sup of the galacturonopyranose ring. The positively charged residues Argsup176/sup and Lyssup239/sup are responsible for lowering the piK/isuba/sub of Argsup236/sup. Argsup176/sup and Lyssup239/sup are maintained in a charged state by interacting with Aspsup154/sup or bulk solvent respectively. The deprotonation of the Aspsup186/sup—Aspsup221/sup 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 b5/b, 677—689]. Substitution of Aspsup186/sup and Aspsup221/sup by Asnsup186/sup and Asnsup221/sup was expected to stabilize the enzyme. However, the Aspsup186/sup→Asn/Aspsup221/sup→Asn enzyme appeared less stable than the wild-type enzyme, even at pH6.0, as evidenced by fluorescence studies. This demonstrates that the pH-dependent conformational change is not driven by deprotonation of the Aspsup186/sup—Aspsup221/sup pair./p