UDP-N-acetylmuramyl-L-alanine functions as an activator in the regulation of the Escherichia coli glutamate racemase activity

Biochemistry. 1995 Feb 28;34(8):2464-70. doi: 10.1021/bi00008a009.

Abstract

D-Glutamate is an essential component of the bacterial peptidoglycan. In Escherichia coli, the biosynthesis of D-glutamate is catalyzed by a glutamate racemase (encoded by the dga gene) and is regulated by UDP-N-acetylmuramyl-L-alanine [Doublet et al. (1994) Biochemistry 33, 5285], a bacterial peptidoglycan subunit precursor. Investigation was conducted to elucidate the interaction between the enzyme and its regulator. Whole and N-terminal truncated enzymes, encoded by individual constructs containing either a full-length or an N-terminal truncated dga gene, were evaluated. In the absence of the regulator, the purified whole enzyme showed a low-level basal racemase activity for which a Km value of 18.9 mM and a Vmax of 0.4 mumol/(min.mg) were determined, using D-glutamate as the substrate. Using the same substrate, in the presence of 6.5 microM UDP-N-acetylmuramyl-L-alanine, a Km value of 4.2 mM and a Vmax of 34 mumol/(min.mg) were measured. Similar kinetic parameters for the activated enzyme were obtained using L-glutamate as the substrate. The N-terminal truncated E. coli enzyme, with a 21 amino acid region removed, is similar in size to the Pediococcus pentosaceus glutamate racemase. Effects of the regulator on the full-length and the N-terminal truncated enzyme in the dialyzed cell lysate were compared. A host cell line, E. coli WM335 delta recA, containing a nonfunctional chromosomal dga gene was used to minimize the background interference. With 6.5 microM regulator added, the N-terminal truncated enzyme displayed a loss of more than 80% of the activity compared to the full-length enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Isomerases / genetics
  • Amino Acid Isomerases / isolation & purification
  • Amino Acid Isomerases / metabolism*
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA Primers / genetics
  • DNA, Bacterial / genetics
  • Enzyme Activation / drug effects
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Genes, Bacterial
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Pediococcus / enzymology
  • Pediococcus / genetics
  • Peptidoglycan / biosynthesis
  • Peptidoglycan / chemistry
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Uridine Diphosphate N-Acetylmuramic Acid / analogs & derivatives*
  • Uridine Diphosphate N-Acetylmuramic Acid / biosynthesis
  • Uridine Diphosphate N-Acetylmuramic Acid / chemistry
  • Uridine Diphosphate N-Acetylmuramic Acid / metabolism
  • Uridine Diphosphate N-Acetylmuramic Acid / pharmacology

Substances

  • DNA Primers
  • DNA, Bacterial
  • Peptidoglycan
  • Uridine Diphosphate N-Acetylmuramic Acid
  • UDP-N-acetylmuramylalanine
  • Amino Acid Isomerases
  • glutamate racemase