Studying a cell division amidase using defined peptidoglycan substrates

Article abstract of DOI:10.1021/ja908916z

(Figure Presented) Three periplasmic N-acetylmuramoyl-L-alanine amidases are critical for hydrolysis of septal peptidoglycan, which enables cell separation. The amidases cleave the amide bond between the lactyl group of muramic acid and the amino group of

Full text of DOI:10.1021/ja908916z

Published on Web 12/03/2009
Studying a Cell Division Amidase Using Defined Peptidoglycan Substrates
Tania J. Lupoli,† Tohru Taniguchi,† Tsung-Shing Wang,† Deborah L. Perlstein,‡ Suzanne Walker,*,‡
and Daniel E. Kahne*,†,§
Department of Chemistry and Chemical Biology, HarVard UniVersity, Cambridge, Massachusetts 02138, and
Department of Microbiology and Molecular Genetics and Department of Biological Chemistry and Molecular
Pharmacology, HarVard Medical School, Boston, Massachusetts 02115
Received October 19, 2009; E-mail: suzanne_walker@hms.harvard.edu; kahne@chemistry.harvard.edu
The bacterial cell wall (murein sacculus) is an essential cellular
component that maintains cell shape and protects against lysis due
to high internal pressure. It is composed of cross-linked strands of
peptidoglycan (PG) that form a meshlike structure around the inner
membrane (Figure 1a).1 Although it provides stability, the cell wall
is a dynamic structure; it is continuously modified by a number of
synthetic and degradative enzymes that are finely tuned to allow
the sacculus to grow and divide without lysing. The action of
degradative enzymes known as murein hydrolases is especially
critical during the late stages of cell division. In Escherichia coli,
three periplasmic N-acetylmuramoyl-L-alanine amidases, AmiA,
AmiB, and AmiC, have been shown to be particularly important
for hydrolysis of septal PG, which enables cell separation.2 The
amidases cleave the amide bond between the lactyl group of
muramic acid and the amino group of L-alanine to release a peptide
moiety (Figure 1b). Cell division amidases remain largely unchar-
acterized because substrates suitable for studying them have not
been available.3 Existing assays for monitoring amidase activity
are mainly based on in-gel degradation of isolated murein sacculi,
which are heterogeneous, insoluble, and contain peptide chains of
varying length, composition, and degree of cross-linking.1a,b This
variability complicates any attempts to analyze amidase substrate
preferences and possible modes of activation.
Figure 1. E. coli amidases cleave peptide cross-links from peptidoglycan
(PG). (a) Schematic representation of an E. coli cell. Amidase activity is
required for cell separation to occur during division. (b) Chemical structures
of synthetic labeled PG fragments Lipid II (1) and Lipid IV (2) and non-
cross-linked (nascent) PG (3).1c
We have developed methods to synthesize the PG precursors
Lipid II (1)4a,b and Lipid IV (2)4c and established enzymatic
methods to convert these substrates to longer glycan strands (nPG,
3) that vary only in chain length.4c-e Here, we use these defined
PG substrates to characterize the E. coli cell division amidase
AmiA.5 The approach described, which is applicable to many other
amidases and cell wall hydrolases, enables a more detailed
understanding of the activity and regulation of this important class
of enzymes.
AmiA is a member of the zinc-dependent amidase 3 family, of
which one member, Bacillus polymyxa var. colistinus CwlV, has
been crystallized (PDB entry 1JWQ), allowing us to predict the
active-site residues involved in metal binding (Figure 2a).6 We
overexpressed and purified C-terminally His-tagged AmiA7 and
incubated it with nPG 3 containing a radiolabel on the pentapeptide
in the presence and absence of zinc. Reactions were analyzed using
paper chromatography.8 Unreacted nPG remained at the baseline,
cleaved peptides migrated, and conversion was quantified by
scintillation counting. The peptide product was verified using an
authentic standard. Control experiments using lysozyme confirmed
that 3 could be cleaved by a PG hydrolase and that the reaction
could be analyzed by paper chromatography (Figure S1 in the
Supporting Information). Without added zinc, AmiA exhibited no
initial activity; in the presence of Zn2+, the radiolabeled peptide
was cleaved from 3 in a concentration-dependent manner (Figure
2a, right, wt rates). Adding zinc chelators such as EDTA and 1,10-
phenanthroline prevented hydrolysis, but the reaction could be
rescued by supplying an excess of zinc (Figure S2).
We also examined the initial rates of several mutants made on
the basis of comparisons of amidase family 3 catalytic domains.
E242 is predicted to act as a general base catalyst, and H65, E80,
and H133 are proposed to coordinate zinc to form the active site
(Figure 2a). Mutation of any of these residues results in a full loss
of activity, except for E80A, which suffered a 100-fold decrease
in rate compared to the wild-type (wt) (Figure S3). Altering residues
D69 and D109, which are also highly conserved but are not
predicted to be essential for activity, produced more modest
decreases in reaction rate (∼10-fold less than the wt). We have
concluded that AmiA is a zinc-dependent metalloprotease with an
active-site configuration similar to that of CwlV.
The substrate requirements of CwlV, AmiA, and related autolytic
amidases have not been determined: to date, the murein sacculus
has been the only reported substrate for these enzymes. In order to
assess the substrate requirements of AmiA, we tested defined PG
fragments of different glycan chain lengths that represent various
stages of PG synthesis in the periplasm (Figure 2b). The substrates
† Harvard University.
‡ Department of Microbiology and Molecular Genetics, Harvard Medical School.
§ Department of Biological Chemistry and Molecular Pharmacology, Harvard
Medical School.
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18230 J. AM. CHEM. SOC. 2009, 131, 18230–18231
10.1021/ja908916z  2009 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Analysis of AmiA substrate preferences and catalytic features. (a) (left) Predicted zinc-binding site of AmiA based on alignment with B. polymyxa
var. colistinus CwlV; (right) comparison of relative cleavage rates of 3 (7.2 µM) by mutants of AmiA (4.0 µM). (b) Reaction scheme for AmiA cleavage
to produce [14C]-pentapeptide 4 from potential PG substrates that differ in length. (c) Gel electrophoresis of 3 (lanes 1 and 2), 2 (lanes 3 and 4), and 1 (lanes
5 and 6) without and with AmiA addition, respectively, under similar reaction conditions. AmiA cleaves 3 and 2 (but not 1) to produce a new band that
represents 4.
were incubated with purified enzyme under similar conditions, and
the reactions were analyzed by gel electrophoresis (Figure 2c).9
AmiA cleaves substrates 2 and 3 to produce a common low-
molecular-weight band, which was identified as the released
pentapeptide 4 by correlation to an authentic standard using HPLC
and electrophoretic mobility (Figure S4). AmiA does not cleave
the peptide from 1 (compare lanes 2 and 4 with lane 6), and a
maximum of 50% of the radiolabeled peptide can be cleaved from
2 (data not shown). These results show that Lipid II (1) is not a
substrate and suggest that AmiA contains an extended binding
pocket that recognizes sugars on either side of the glycopeptide
substrate. Consistent with this idea, disaccharide-peptide fragments
obtained by treating 3 with lytic transglycosylases also were not
cleaved by AmiA (Figure S5). Hence, our results show that AmiA
requires at least a tetrasaccharide as a substrate.
The use of compositionally well-defined PG polymer substrates
has allowed us to characterize a cell wall amidase, E. coli AmiA,
involved in PG degradation during division. The turnover number
for cleavage of the polymer is 0.05 min-1 (Figure S3). Since
cleavage rates from sacculi by similar purified enzymes have not
been reported, there are no in vitro data for comparison.3d Although
this rate is lower than estimates required to support bacterial
growth,10 low rates are typically observed in vitro for the enzymes
that synthesize glycan strands.11 A possible explanation is that cell
wall synthetic and degradative enzymes are proposed to operate as
components of multiprotein machines with tightly coordinated
activities. Higher in vitro rates may be observed when other
important components of the system are reconstituted. Some protein
candidates for amidase regulation have been suggested, but it is
unclear whether they simply recruit amidases to the appropriate
cellular location or influence activity through direct interaction with
the amidase or its substrate.1a,b,12 Access to homogeneous substrates
rather than crude cell wall fractions provides the capability to
evaluate amidase kinetics in response to prospective activators and
could, in turn, help illuminate the nature of these complex
interactions.
to S.W. and D.E.K., Training Grant GM007598 to T.J.L., and a
postdoctoral fellowship for D.L.P.).
Supporting Information Available: Experimental procedures,
including synthesis of peptide standard, cloning and purification of
proteins, and rate and gel electrophoresis analysis. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Acknowledgment. This research was supported by the National
Institutes of Health (Grant GM066174 to D.E.K., Grant GM076710
JA908916Z
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