Overproduction and one-step purification of Escherichia coli UDP-N-acetylglucosamine enolpyruvyl reductase.
The Escherichia coli gene murB, encoding the enzyme uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucosenicoti namide adenine dinucleotide phosphate oxidoreductase (EC 1.1.1.158) (EP-reductase), the second enzyme in the peptidoglycan biosynthetic pathway, has been amplified using PCR technology with the Kohara recombinant lambda phage E11C11 (534) as template. The synthetic gene was subcloned into the NdeI and BamHI restriction sites of the expression vector pT7-7, designed to utilize T7 RNA polymerase to direct transcription of the target gene, in a two-step procedure. The first step involved the directional insertion of the 590-bp NdeI to BamHI restriction fragment of murB into the pT7-7 vector to give the plasmid pT7-7-murB-590. The construction of the desired overproducing plasmid was completed by the bidirectional insertion of the 442-bp BamHI to BamHI restriction fragment of murB into a similarly restricted pT7-7-murB-590 plasmid followed by restriction digestion to select the properly oriented insert, pT7-7-murB. Overexpression of EP-reductase from the E. coli strain BL 21 (DE 3) containing the pT7-7-murB gene, after induction, allowed the production of 36 mg of target protein per 3 wet grams of E. coli cells. The EP-reductase was purified in a single step utilizing dye-ligand chromatography to yield 30 mg of pure protein. The availability of these levels of reductase will allow the mechanism of this pivotal enzyme to be thoroughly studied as a potential target for the design of a new generation of antibiotics. In addition, the EP-reductase generated in this study has been utilized as a coupling enzyme to assay the first enzyme in the peptidoglycan biosynthetic pathway, UDP-N-acetylglucosamine enolpyruvyl transferase, and these results are also presented.
Tayeh
M A
MA
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA.
Dotson
G D
GD
Clemens
J C
JC
Woodard
R W
RW
D001483:Base Sequence; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D002237:Carbohydrate Dehydrogenases; C023215:UDP-N-acetylmuramate dehydrogenase