Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Feb 15;370(Pt 1):331–337. doi: 10.1042/BJ20021071

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

Paloma Sánchez-Torres 1, Jaap Visser 1, Jacques A E Benen 1
PMCID: PMC1223150  PMID: 12418964

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. 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.

Full Text

The Full Text of this article is available as a PDF (171.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Armand S., Wagemaker M. J., Sánchez-Torres P., Kester H. C., van Santen Y., Dijkstra B. W., Visser J., Benen J. A. The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis. J Biol Chem. 2000 Jan 7;275(1):691–696. doi: 10.1074/jbc.275.1.691. [DOI] [PubMed] [Google Scholar]
  2. Brown I. E., Mallen M. H., Charnock S. J., Davies G. J., Black G. W. Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module. Biochem J. 2001 Apr 1;355(Pt 1):155–165. doi: 10.1042/0264-6021:3550155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. DEUEL H., STUTZ E. Pectic substances and pectic enzymes. Adv Enzymol Relat Subj Biochem. 1958;20:341–382. doi: 10.1002/9780470122655.ch11. [DOI] [PubMed] [Google Scholar]
  4. Edelhoch H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 1967 Jul;6(7):1948–1954. doi: 10.1021/bi00859a010. [DOI] [PubMed] [Google Scholar]
  5. Harmsen J. A., Kusters-van Someren M. A., Visser J. Cloning and expression of a second Aspergillus niger pectin lyase gene (pelA): indications of a pectin lyase gene family in A. niger. Curr Genet. 1990 Aug;18(2):161–166. doi: 10.1007/BF00312604. [DOI] [PubMed] [Google Scholar]
  6. Herron S. R., Benen J. A., Scavetta R. D., Visser J., Jurnak F. Structure and function of pectic enzymes: virulence factors of plant pathogens. Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):8762–8769. doi: 10.1073/pnas.97.16.8762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kusters-van Someren M., Flipphi M., de Graaff L., van den Broeck H., Kester H., Hinnen A., Visser J. Characterization of the Aspergillus niger pelB gene: structure and regulation of expression. Mol Gen Genet. 1992 Jul;234(1):113–120. doi: 10.1007/BF00272352. [DOI] [PubMed] [Google Scholar]
  8. Lietzke S. E., Yoder M. D., Keen N. T., Jurnak F. The Three-Dimensional Structure of Pectate Lyase E, a Plant Virulence Factor from Erwinia chrysanthemi. Plant Physiol. 1994 Nov;106(3):849–862. doi: 10.1104/pp.106.3.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mayans O., Scott M., Connerton I., Gravesen T., Benen J., Visser J., Pickersgill R., Jenkins J. Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases. Structure. 1997 May 15;5(5):677–689. doi: 10.1016/s0969-2126(97)00222-0. [DOI] [PubMed] [Google Scholar]
  10. Pagès S., Heijne W. H., Kester H. C., Visser J., Benen J. A. Subsite mapping of Aspergillus niger endopolygalacturonase II by site-directed mutagenesis. J Biol Chem. 2000 Sep 22;275(38):29348–29353. doi: 10.1074/jbc.M910112199. [DOI] [PubMed] [Google Scholar]
  11. Parenicová L., Benen J. A., Kester H. C., Visser J. pgaE encodes a fourth member of the endopolygalacturonase gene family from Aspergillus niger. Eur J Biochem. 1998 Jan 15;251(1-2):72–80. doi: 10.1046/j.1432-1327.1998.2510072.x. [DOI] [PubMed] [Google Scholar]
  12. Pickersgill R., Jenkins J., Harris G., Nasser W., Robert-Baudouy J. The structure of Bacillus subtilis pectate lyase in complex with calcium. Nat Struct Biol. 1994 Oct;1(10):717–723. doi: 10.1038/nsb1094-717. [DOI] [PubMed] [Google Scholar]
  13. Scavetta R. D., Herron S. R., Hotchkiss A. T., Kita N., Keen N. T., Benen J. A., Kester H. C., Visser J., Jurnak F. Structure of a plant cell wall fragment complexed to pectate lyase C. Plant Cell. 1999 Jun;11(6):1081–1092. doi: 10.1105/tpc.11.6.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Vitali J., Schick B., Kester H. C., Visser J., Jurnak F. The tree-dimensional structure of aspergillus niger pectin lyase B at 1.7-A resolution. Plant Physiol. 1998 Jan;116(1):69–80. doi: 10.1104/pp.116.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Woodcock D. M., Crowther P. J., Doherty J., Jefferson S., DeCruz E., Noyer-Weidner M., Smith S. S., Michael M. Z., Graham M. W. Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 1989 May 11;17(9):3469–3478. doi: 10.1093/nar/17.9.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Yoder M. D., Keen N. T., Jurnak F. New domain motif: the structure of pectate lyase C, a secreted plant virulence factor. Science. 1993 Jun 4;260(5113):1503–1507. doi: 10.1126/science.8502994. [DOI] [PubMed] [Google Scholar]
  17. de Graaff L., van den Broeck H., Visser J. Isolation and characterization of the Aspergillus niger pyruvate kinase gene. Curr Genet. 1992 Jul;22(1):21–27. doi: 10.1007/BF00351737. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES