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. 1992 Jan;174(2):441-6.
doi: 10.1128/jb.174.2.441-446.1992.

Purification of a nocardicin A-sensitive LD-carboxypeptidase from Escherichia coli by affinity chromatography

A Ursinus  1 , H Steinhaus, J V Höltje
Affiliations

Purification of a nocardicin A-sensitive LD-carboxypeptidase from Escherichia coli by affinity chromatography

A Ursinus et al. J Bacteriol. 1992 Jan.

Abstract

An LD-carboxypeptidase releasing the terminal D-Ala from UDP-MurNAc-L-Ala-D-Glu-m-A2pm-D-Ala (UDP-MurNAc-tetrapeptide) was purified from Escherichia coli to biochemical homogeneity and characterized biochemically. Final purification was achieved by nocardicin A-Sepharose affinity chromatography. An apparent molecular weight of 32,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme, which seems to be a monomeric protein as indicated by gel filtration. The optimum pH of the enzyme was 8.4, and the pI was 5.5. The Km for UDP-MurNAc-tetrapeptide was 1.5 x 10(-4) M, and the Vmax was 0.4 nmol/min. Nocardicin A inhibited the enzyme competitively, with a Ki of 5 x 10(-5) M. Benzylpenicillin, cephalosporin C, thienamycin, and D-alanyl-D-alanine did not affect the enzyme activity. Possible functions of the enzyme for growth and division of the murein sacculus are discussed.

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References

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