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

• Published in: Biochemical journal 2003-02, Vol.370 (Pt 1), p.331-337
• Main Authors: Sánchez-Torres, Paloma, Visser, Jaap, Benen, Jacques A E
• Format: Article
• Language: English
• Subjects: 3-dimensional structure; Amino Acids - metabolism; Aspergillus niger - enzymology; Base Sequence; Catalysis; Catalytic Domain; Circular Dichroism; Cloning, Molecular; DNA Primers; endopolygalacturonase ii; Enzyme Stability; enzymes; erwinia; expression; gene family; Hydrogen-Ion Concentration; Kinetics; Mutagenesis, Site-Directed; pathogenesis; pectate lyase; plant virulence factor; Polysaccharide-Lyases - chemistry; Polysaccharide-Lyases - genetics; Polysaccharide-Lyases - metabolism; Protein Conformation; site-directed mutagenesis; Spectrometry, Fluorescence
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20021071

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. Natl. Acad. Sci. U.S.A. 97, 8762-8769], Asp154, Arg176, Arg236 and Lys239 were mutagenized. Substituting Arg236 with alanine or lysine rendered the enzyme completely inactive, and mutagenesis of Arg176 and Lys239 severely affected catalysis. The Asp154-->Arg and Asp154-->Glu mutant enzymes were only moderately impaired in respect of catalysis. The results strongly indicate that Arg236, which is sandwiched between Arg176 and Lys239, would initiate the reaction upon enzyme-substrate interaction, through the abstraction of the proton at C5 of the galacturonopyranose ring. The positively charged residues Arg176 and Lys239 are responsible for lowering the p K a of Arg236. Arg176 and Lys239 are maintained in a charged state by interacting with Asp154 or bulk solvent respectively. The deprotonation of the Asp186-Asp221 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 Asp186 and Asp221 by Asn186 and Asn221 was expected to stabilize the enzyme. However, the Asp186-->Asn/Asp221-->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 Asp186-Asp221 pair.