An anhydro-N-acetylmuramyl-L-alanine amidase with broad specificity tethered to the outer membrane of Escherichia coli

Abstract

From its amino acid sequence homology with AmpD, we recognized YbjR, now renamed AmiD, as a possible second 1,6-anhydro-N-acetylmuramic acid (anhMurNAc)-l-alanine amidase in Escherichia coli. We have now confirmed that AmiD is an anhMurNAc-l-Ala amidase and demonstrated that AmpD and AmiD are the only enzymes present in E. coli that are able to cleave the anhMurNAc-l-Ala bond. The activity was present only in the outer membrane fraction obtained from an ampD mutant. In contrast to AmpD, which is specific for the anhMurNAc-l-alanine bond, AmiD also cleaved the bond between MurNAc and l-alanine in both muropeptides and murein sacculi. Unlike the periplasmic murein amidases, AmiD did not participate in cell separation. ampG mutants, which are unable to import GlcNAc-anhMurNAc-peptides into the cytoplasm, released mainly peptides into the medium due to AmiD activity, whereas an ampG amiD double mutant released a large amount of intact GlcNAc-anhMurNAc-peptides into the medium.

FIG. 1.

Alignment of the amino acid sequences of the AmpD amidase and YbjR (AmiD) from Escherichia coli by the T-Coffee program (25). Identical amino acids and conserved substitutions are indicated by asterisks and colons, respectively. Dashes indicate a gap in the sequence. Amino acid numbers for both proteins are given on the right. The residues of the zinc-binding triad His³⁴-His¹⁵⁴-Asp¹⁶⁴ of AmpD are shaded, and the lipobox sequence (L¹⁴AGC¹⁷) of YbjR is underlined.

FIG. 2.

Effect of AmiD on the GlcNAc-anhMurNAc content of ampD nagZ cells. Shaded bars, the amiD⁺ strain TP78B (nagB nagZ ampD); solid bars, the amiD deletion mutant TP78BD (nagB nagZ ampD amiD). Cells were labeled with [6-³H]GlcN in 4 ml of M9 glycerol minimal medium, and hot-water extracts were analyzed by HPLC. The amounts of radioactivity in the fractions corresponding to GlcNAc-anhMurNAc, UDP-MurNAc-pentapeptide, and GlcNAc-anhMurNAc-tripepide were measured and adjusted to equal cell populations. Values are averages from two separate experiments.

FIG. 3.

Subcellular location of AmiD. Membranes of TP71/pTrcAmiD cells before (lanes 1, 3, and 5) and after (lanes 2, 4, and 6) induction of AmiD for 1 h following the addition of 50 μM IPTG were separated by sucrose gradient centrifugation into inner membrane (lanes 1 and 2), intermediate membrane (lanes 3 and 4), and outer membrane (lanes 5 and 6) fractions. The samples were fractionated by SDS-PAGE and stained with CBB. Arrow indicates AmiD protein. Each value below the gel is the percentage of [³H]GlcN-labeled GlcNAc-anhMurNAc-tripeptide (1,000 cpm) cleaved by 1/200 of the fraction loaded on the SDS-PAGE gel and was determined by incubation with the substrate at 37°C for 30 min, followed by heat inactivation and thin-layer chromatographic analysis.

FIG. 4.

Overexpression of AmiD inhibits growth. TP71 carrying pTrcAmiD was grown at 37°C in LB broth containing 100 μg ampicillin/ml and 0.2% glucose. IPTG (100 μM) was added at a turbidity of 52 Klett units (filled triangles) or 100 Klett units (filled circles), as indicated by the arrows. Open squares, control culture.

FIG. 5.

HPLC analysis of GlcNAc-anhMurNAc-l-Ala-d-Glu-Dap before (dashed line) and after (solid line) digestion with AmiD. A mixture of 11 kcpm of [³H]GlcNAc-[³H]anhMurNAc-tripeptide and 2.5 kcpm of GlcNAc-anhMurNAc-l-Ala-d-Glu-[³H]Dap was incubated with 0.25 μg of purified AmiD at 37°C for 30 min and separated by HPLC with the formic acid solvent system. Fraction A, l-Ala-d-Glu-Dap; fraction B, GlcNAc-anhMurNAc; fraction C, GlcNAc-anhMurNAc-l-Ala-d-Glu-Dap.

FIG. 6.

Cleavage of muropeptides by AmiD. Purified sacculi were first digested with 10 U of mutanolysin (Sigma, St. Louis, MO) at 37°C overnight in 10 mM morpholineethanesulfonic acid-NaOH (pH 6.0), followed by incubation with (black line) or without (gray line) 5 μg of AmiD in 50 mM HEPES-NaOH (pH 7.0) overnight. The samples were reduced with sodium borohydride and separated by HPLC with the trifluoroacetic acid solvent system. Based on the elution time, peak A corresponds to cross-linked tetra-tetrapeptide; peak B, GlcNAc-MurNAc tetrapeptide; peak C, cross-linked GlcNAc-MurNAc tetrapeptide-tetrapeptide-MurNAc-GlcNAc.

FIG. 7.

Release of peptides from murein by AmiD. A 0.2-mg portion of purified murein digested by purified AmiD was separated by HPLC with the trifluoroacetic acid solvent system as described in Materials and Methods. Compounds in the three main peaks (A, B, and C) were collected, lyophilized, and analyzed by MALDI-TOF mass spectrometry. Fraction A, l-Ala-d-Glu-Dap (m/z 391) tripeptide; fraction B, l-Ala-d-Glu-Dap-d-Ala (m/z 462) tetrapeptide; fraction C, tetra-tetrapeptide (m/z 905).

FIG. 8.

Amounts of Dap-containing compounds in the spent medium from cultures growing for about 5 generations in 2 ml of M9 minimal medium containing 1 μCi of [³H]Dap/ml. The medium was lyophilized and analyzed by HPLC to measure the amount of each compound. wt, wild type; anhD, GlcNAc-anhMurNAc.