Cloning, expression and characterization of the Streptococcus pyogenes murE gene encoding a UDP-MurNAc- l-alanyl- d-glutamate: l-lysine ligase

Cloning, expression and characterization of the Streptococcus pyogenes murE gene encoding a UDP-MurNAc- l-alanyl- d-glutamate: l-lysine ligase

The l-lysine-adding enzyme encoded by the murE gene catalyzes the ATP-dependent formation of UDP-MurNAc- l-alanyl- d-glutamyl- l-lysine (UDP-MurNAc-tripeptide). MurE has been cloned from Streptococcus pyogenes ( Spy) and expressed as a glutathione- S-transferase (GST)/polyhistidine (His 12) fusion i...

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Bibliographic Details
Published in:Enzyme and microbial technology Vol. 35; no. 4; pp. 300 - 308
Main Authors: Triolo, Thomas A, Chabin, Renee M, Pompliano, David L
Format: Journal Article
Language:English
Published: Amsterdam Elsevier Inc 01-09-2004
Elsevier Science
Subjects:
Biological and medical sciences
Biology of microorganisms of confirmed or potential industrial interest
Biotechnology
Escherichia coli
Fundamental and applied biological sciences. Psychology
l-lysine
Miscellaneous
Mission oriented research
Streptococcus pyogenes
UDP-MurNAc- l-alanyl- d-glutamate
Escherichia coli
l-lysine
UDP-MurNAc- l-alanyl- d-glutamate
Enzyme
UDP-MurNAc-L-alanyl-D-glutamate
L-lysine
Gene expression
Glutamate
Characterization
Streptococcaceae
Ligases
Lysine
Bacteria
Micrococcales
Streptococcus pyogenes
Molecular cloning
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Summary:The l-lysine-adding enzyme encoded by the murE gene catalyzes the ATP-dependent formation of UDP-MurNAc- l-alanyl- d-glutamyl- l-lysine (UDP-MurNAc-tripeptide). MurE has been cloned from Streptococcus pyogenes ( Spy) and expressed as a glutathione- S-transferase (GST)/polyhistidine (His 12) fusion in Escherichia coli ( Eco). Initial velocity studies show that the fusion enzyme has values of k cat=9 s −1 and of K m (ATP) = 125 μM, K m ( l-lysine) = 122 μM and K m (UDP-MurNAc-dipeptide) = 20.5 μM, at 23 °C. Spy murE is expressed using a new plasmid developed for the expression of GST-HIS 12 tagged proteins in Eco. Identification and purification of the UDP-MurNAc- l-alanyl- d-glutamyl- l-lysine product of the GST-HIS 12- Spy MurE enzyme is also described. Surprisingly, this product is a substrate for Eco MurF, an enzyme that normally handles a UDP-MurNAc-tripeptide that contains a diaminopimelic acid residue instead of l-lysine.
Bibliography:ObjectType-Article-2
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ISSN:0141-0229
1879-0909
DOI:10.1016/j.enzmictec.2004.03.020