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X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
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Structure and function of 2,3-dimethylmalate lyase, a PEP mutase/isocitrate lyase superfamily member

Buvaneswari Narayanan1, Weiling Niu, Henk-Jan Joosten

  • 1Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, 20850, USA.

Journal of Molecular Biology
|January 10, 2009
PubMed

Insights

Aspergillus niger contains a gene for dimethylmalate lyase (DMML) with dual oxaloacetate acetyl hydrolase (OAH) activity. This enzyme

Area of Science:

  • Biochemistry
  • Enzymology
  • Fungal Metabolism

Background:

  • Aspergillus niger possesses four genes related to oxaloacetate acetyl hydrolase (OAH).
  • A previously studied A. niger OAH gene mutant lacks oxalate production.
  • The function of the most similar OAH protein (An07g08390) required investigation.

Purpose of the Study:

  • To determine the catalytic function of the A. niger An07g08390 protein.
  • To elucidate the structural basis of substrate recognition and catalysis for An07g08390.
  • To understand the evolutionary context of this enzyme within the phosphoenolpyruvate mutase/isocitrate lyase superfamily.

Main Methods:

  • Protein expression in E. coli and purification.
  • Enzyme kinetics assays for substrate screening.
  • DNA array analysis for gene regulation studies.
  • X-ray crystallography for structure determination.
  • Site-directed mutagenesis to analyze active-site residues.

Main Results:

  • An07g08390 was identified as (2R,3S)-dimethylmalate lyase (DMML) with significant OAH activity.
  • DMML gene expression is subject to catabolite repression, unlike the A. niger oah gene.
  • The crystal structure of A. niger DMML was determined, revealing key catalytic and substrate-binding residues.
  • Mutagenesis studies confirmed the roles of active-site residues in catalysis.

Conclusions:

  • A. niger DMML functions as a dimethylmalate lyase with dual OAH activity, likely in an unknown metabolic pathway.
  • The enzyme's regulation and substrate specificity differ from known bacterial counterparts.
  • Structural insights provide a foundation for understanding enzyme evolution and functional diversity within the lyase superfamily.

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