Purification and properties of a membrane-bound lytic transglycosylase from Escherichia coli.
A membrane-bound lytic transglycosylase (Mlt) has been solubilized in the presence of 2% Triton X-100 containing 0.5 M NaCl from membranes of an Escherichia coli mutant that carries a deletion in the slt gene coding for a 70-kDa soluble lytic transglycosylase (Slt70). The enzyme was purified by a four-step procedure including anion-exchange (HiLoad SP-Sepharose and MonoS), heparin-Sepharose, and poly(U)-Sepharose 4B column chromatography. The purified protein that migrated during denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band corresponding to an apparent molecular mass of about 38 kDa is referred to as Mlt38. Optimal activity was found in buffers with a pH between 4.0 and 4.5. The enzyme is stimulated by a factor of 2.5 in the presence of Mg2+ at a concentration of 10 mM and loses its activity rapidly at temperatures above 30 degrees C. Besides insoluble murein sacculi, the enzyme was able to degrade glycan strands isolated from murein by amidase treatment. The enzymatic reaction occurred with a maximal velocity of about 2.2 mg/liter/min with murein sacculi as a substrate. The amino acid sequences of four proteolytic peptides showed no identity with known sequences in the data bank. With Mlt38, the number of proteins in E. coli showing lytic transglycosylase activity rises to three.
Ursinus
A
A
Abteilung Biochemie, Max-Planck-Institut für Entwicklungsbiologie, Tübingen, Germany.
Höltje
J V
JV
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D006026:Glycoside Hydrolases; D016695:Glycosyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010457:Peptidoglycan / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010457:Peptidoglycan; D016695:Glycosyltransferases; C085441:MltA protein, bacteria; D006026:Glycoside Hydrolases; C085428:slt protein, E coli