Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A.
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.
Sánchez-Torres
Paloma
P
Section Molecular Genetics of Industrial Microorganisms, Wageningen University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.
Visser
Jaap
J
Benen
Jacques A E
JA
D000596:Amino Acids / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D017931:DNA Primers; D011133:Polysaccharide-Lyases; C027349:pectin lyase