UDP-N-acetylmuramyl-L-alanine functions as an activator in the regulation of the Escherichia coli glutamate racemase activity.
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)
Ho
H T
HT
Department of Microbiology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492.
Falk
P J
PJ
Ervin
K M
KM
Krishnan
B S
BS
Discotto
L F
LF
Dougherty
T J
TJ
Pucci
M J
MJ
D000591:Amino Acid Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010376:Pediococcus / Q000201:enzymology / Q000235:genetics; D010457:Peptidoglycan / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D017931:DNA Primers; D004269:DNA, Bacterial; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C076606:UDP-N-acetylmuramylalanine; D000591:Amino Acid Isomerases; C062292:glutamate racemase