A new robust kinetic assay for DAP epimerase activity

Biochimie. 2013 Oct;95(10):1949-53. doi: 10.1016/j.biochi.2013.06.026. Epub 2013 Jul 6.

Abstract

DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase-DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP(+) to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.

Keywords: DAP; DAP decarboxylase; DAP dehydrogenase; DAP epimerase; DAPDC; DAPDH; Diaminopimelate biosynthesis; Kinetic assay; PLP; THDP; diaminopimelate; diaminopimelate decarboxylase; diaminopimelate dehydrogenase; ll-DAP; pyridoxal-5′-phosphate; tetrahydrodipicolinate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Isomerases / chemistry
  • Amino Acid Isomerases / genetics
  • Amino Acid Isomerases / metabolism*
  • Amino Acid Oxidoreductases / chemistry
  • Amino Acid Oxidoreductases / genetics
  • Amino Acid Oxidoreductases / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Carboxy-Lyases / chemistry
  • Carboxy-Lyases / genetics
  • Carboxy-Lyases / metabolism
  • Corynebacterium glutamicum / enzymology*
  • Corynebacterium glutamicum / genetics
  • Diaminopimelic Acid / metabolism
  • Enzyme Assays / methods
  • Enzyme Assays / standards*
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Kinetics
  • Lysine / biosynthesis
  • NADP / chemistry
  • NADP / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Reproducibility of Results

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • Recombinant Proteins
  • NADP
  • Diaminopimelic Acid
  • Amino Acid Oxidoreductases
  • diaminopimelate dehydrogenase
  • Carboxy-Lyases
  • LysA protein, E coli
  • Amino Acid Isomerases
  • diaminopimelate epimerase
  • Lysine