D-alanine: D-alanine ligase of Escherichia coli. Expression, purification and inhibitory studies on the cloned enzyme.
A 1.2 kb BamHI fragment from pDK30 [Robinson, Kenan, Sweeney & Donachie (1986) J. Bacteriol. 167, 809-817] was cloned in pDOC55 [O'Connor & Timmis (1987) J. Bacteriol. 169, 4457-4482] to give two constructs, pDOC89 and pDOC87, in which the Escherichia coli D-alanine:D-alanine ligase (EC 6.3.2.4) gene (ddl) was placed under the control of the lac and lambda PL promoters respectively. Both constructs, when used to transform E. coli M72, gave similar levels of expression of the ddl gene. The expressed enzyme was purified to homogeneity and the amino acid sequence of its N-terminal region was found to be consistent with that predicted from the gene sequence, except that the N-terminal methionine was not present in the mature protein. [1(S)-Aminoethyl][(2RS)2-carboxy-1-octyl]phosphinic acid (I), previously shown to bind tightly to Enterococcus faecalis and Salmonella typhimurium D-alanine:D-alanine ligases following phosphorylation Parsons, Patchett, Bull, Schoen, Taub, Davidson, Combs, Springer, Gadebusch, Weissberger, Valiant, Mellin & Busch (1988) J. Med. Chem. 31, 1772-1778; Duncan & Walsh (1988) Biochemistry 27, 3709-3714], was found to be a classical slow-binding inhibitor of the E. coli ligase.
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Department of Biochemistry, University of Southampton, Bassett Crescent East, U.K.
O'Connor
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Giles
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Akhtar
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005796:Genes / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics
D001426:Bacterial Proteins; D009943:Organophosphorus Compounds; C073690:(1-aminoethyl)(2-carboxy-1-octyl)phosphinic acid; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase