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PMID:22001512

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Citation

González-Leiza, SM, de Pedro, MA and Ayala, JA (2011) AmpH, a bifunctional DD-endopeptidase and DD-carboxypeptidase of Escherichia coli. J. Bacteriol. 193:6887-94

Abstract

In Escherichia coli, low-molecular-mass penicillin-binding proteins (LMM PBPs) are important for correct cell morphogenesis. These enzymes display DD-carboxypeptidase and/or dd-endopeptidase activities associated with maturation and remodeling of peptidoglycan (PG). AmpH has been classified as an AmpH-type class C LMM PBP, a group closely related to AmpC β-lactamases. AmpH has been associated with PG recycling, although its enzymatic activity remained uncharacterized until now. Construction and purification of His-tagged AmpH from E. coli permitted a detailed study of its enzymatic properties. The N-terminal export signal of AmpH is processed, but the protein remains membrane associated. The PBP nature of AmpH was demonstrated by its ability to bind the β-lactams Bocillin FL (a fluorescent penicillin) and cefmetazole. In vitro assays with AmpH and specific muropeptides demonstrated that AmpH is a bifunctional DD-endopeptidase and DD-carboxypeptidase. Indeed, the enzyme cleaved the cross-linked dimers tetrapentapeptide (D45) and tetratetrapeptide (D44) with efficiencies (k(cat)/K(m)) of 1,200 M(-1) s(-1) and 670 M(-1) s(-1), respectively, and removed the terminal D-alanine from muropeptides with a C-terminal D-Ala-D-Ala dipeptide. Both DD-peptidase activities were inhibited by 40 μM cefmetazole. AmpH also displayed a weak β-lactamase activity for nitrocefin of 1.4 × 10(-3) nmol/μg protein/min, 1/1,000 the rate obtained for AmpC under the same conditions. AmpH was also active on purified sacculi, exhibiting the bifunctional character that was seen with pure muropeptides. The wide substrate spectrum of the DD-peptidase activities associated with AmpH supports a role for this protein in PG remodeling or recycling.

Links

PubMed PMC3232839 Online version:10.1128/JB.05764-11

Keywords

Dipeptidases/chemistry; Dipeptidases/genetics; Dipeptidases/metabolism; Escherichia coli/chemistry; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Kinetics; Penicillin-Binding Proteins/chemistry; Penicillin-Binding Proteins/genetics; Penicillin-Binding Proteins/metabolism; Protein Transport; Substrate Specificity; beta-Lactamases/chemistry; beta-Lactamases/genetics; beta-Lactamases/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:AMPH

involved_in

GO:0009253: peptidoglycan catabolic process

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

ECOLI:AMPH

enables

GO:0004180: carboxypeptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:AMPH

enables

GO:0004175: endopeptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

See also

References

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