The MurE synthetase from Thermotoga maritima is endowed with an unusual D-lysine adding activity.
The peptidoglycan of Thermotoga maritima, an extremely thermophilic eubacterium, was shown to contain no diaminopimelic acid and approximate amounts of both enantiomers of lysine (Huber, R., Langworthy, T. A., König, H., Thomm, M., Woese, C. R., Sleytr, U. B., and Stetter, K. O. (1986) Arch. Microbiol. 144, 324-333). To assess the possible involvement of the MurE activity in the incorporation of D-lysine, the murE gene from this organism was cloned in Escherichia coli, and the corresponding protein was purified as the C-terminal His6-tagged form. In vitro assays showed that D-lysine and meso-diaminopimelic acid were added to UDP-N-acetylmuramoyl-dipeptide with 25 and 10% efficiencies, respectively, relative to L-lysine. The purified enzyme was used to synthesize the L- and D-lysine-containing UDP-N-acetylmuramoyl-tripeptides; chemical analysis revealed an unusual structure for the D-lysine-containing nucleotide, namely acylation of the epsilon-amino function of D-lysine by the D-glutamyl residue. In vitro assays with MurF and MraY enzymes from T. maritima showed that this novel nucleotide was not a substrate for MurF but that it could be directly processed into tripeptide lipid I by MraY, thereby substantiating the role of MurE in the incorporation of D-lysine into peptidoglycan.
Boniface
Audrey
A
Laboratory of Bacterial Envelopes and Antibiotics, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Université Paris-Sud, 91405 Orsay, France.
Bouhss
Ahmed
A
Mengin-Lecreulx
Dominique
D
Blanot
Didier
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D005798:Genes, Bacterial; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D008025:Ligases; D008239:Lysine