In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa.
Bacterial peptidoglycan is the cell wall component responsible for maintaining cell integrity against osmotic pressure. Biosynthesis of the cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is catalyzed by the Mur enzymes. Genomic analysis of the three regions encoding Mur proteins was achieved. We have cloned and over-expressed the murA, -B, -D, -E and -F genes of Pseudomonas aeruginosa in pET expression system by adding a His-Tag to the C-termini of the proteins. Mur proteins were purified to homogeneity by a single chromatographic step on affinity nickel columns. Protein identities were verified through N-terminal sequencing. Enzyme activity was proved by the identification of the pathway's final product.
El Zoeiby
A
A
Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Faculté de Médecine, Ste-Foy, QC, Canada.
Sanschagrin
F
F
Havugimana
P C
PC
Garnier
A
A
Levesque
R C
RC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D010457:Peptidoglycan / Q000096:biosynthesis* / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D001426:Bacterial Proteins; D010457:Peptidoglycan; D011993:Recombinant Fusion Proteins