Bacterial AmpD at the crossroads of peptidoglycan recycling and manifestation of antibiotic resistance

Article abstract of DOI:10.1021/ja9025566

(Chemical Equation Presented) The bacterial enzyme AmpD is an early catalyst in commitment of cell wall metabolites to the recycling events within the cytoplasm. The key internalized metabolite of cell wall recycling, β-D-N-acetylglucosamine-(1→4)-1,6-anh

Full text of DOI:10.1021/ja9025566

Published on Web 06/04/2009
Bacterial AmpD at the Crossroads of Peptidoglycan Recycling and
Manifestation of Antibiotic Resistance
Mijoon Lee, Weilie Zhang, Dusan Hesek, Bruce C. Noll, Bill Boggess, and Shahriar Mobashery*
Department of Chemistry and Biochemistry, UniVersity of Notre Dame, Notre Dame, Indiana 46556
Received March 31, 2009; E-mail: mobashery@nd.edu
Bacterial peptidoglycan, the major constituent of the cell wall, is a
polymer comprised of repeating units of N-acetylglucosamine (NAG)
and N-acetylmuramic acid (NAM). An unusual peptide stem is
appended to the NAM unit, which is the site of cross-linking among
neighboring peptidoglycan strands in the mature cell wall.1 In the
course of the normal bacterial growth, more than 50% of the parental
peptidoglycan is recycled.2,3 The recycling is initiated by the action
of lytic transglycosylases, which convert the NAM unit to the 1,6-
anhydromuramyl moiety, a process that results in liberation of the novel
disaccharide 1 from the polymeric cell wall (Figure 1).4 The peptide
in the cell wall is processed by a number of enzymes, and it is typically
comprised of three to five amino acids in the mature cell wall.5 On
liberation of 1, it is internalized by the membrane protein AmpG. Once
in the cytoplasm, compound 1 serves as the substrate for NagZ, which
converts it to 2. Compound 2, in its various peptide forms, is believed
to be an important player both in entry into the ensuing peptidoglycan
recycling events and in an induction event that leads to the expression
of ꢀ-lactamase, a key ꢀ-lactam antibiotic resistance enzyme.6,7
The tripeptide and tetrapeptide forms of the peptide stem are
prevalent in the mature peptidoglycan, because of the action of
penicillin-binding proteins (PBPs) and certain bacterial cell wall
endopeptidases. Hence, when 1 is internalized and processed by NagZ,
the variants 2a and 2b predominate. On the other hand, when bacteria
are exposed to ꢀ-lactam antibiotics, which inhibit PBPs, the result is
the predominance of the form 2c.3,8 The enzyme AmpD would appear
to be central to the fate of compounds 2. It has been proposed to be
a peptidase that removes the peptide stem from the saccharide
scaffolding of 2, initiating the recycling process.9 The enzyme is
specifically implicated in processing of the tripeptide variant 2a, which
makes it a player in the early stages of commitment to recycling.3,9
Binding of 2a to the dimer of the gene regulator AmpR is proposed
to induce transcription of the gene for ꢀ-lactamase, unleashing
resistance against ꢀ-lactam antibiotics. The notion is that exposure of
the bacterium to the antibiotic would influence cell wall fragmentation
and the recycling events, which down the line lead to induction of the
resistance gene to counter the challenge by the antibiotic.3,10
The studies of these biochemical events have been held back by
the lack of availability of authentic substrate(s) for AmpD. We describe
herein the synthesis of a set of metabolites involved in cell wall
recycling. Furthermore, we document that AmpD, as a gatekeeper for
many of these biochemical events, is competent to turn over not merely
the tripeptidyl analogue 2a but also the full-length peptide derivative
2c. Hence, it is also the key catalyst in the reversal of induction of
resistance, once the challenge of the antibiotic is no longer present.
We cloned the ampD gene from Citrobacter freundii (ATCC6879)
into the pET24a(+) vector under the T7 promoter by PCR amplifica-
tion of the chromosomal gene. Escherichia coli BL21(DE3) cells were
transformed with the vector for expression of the protein. The protein
was purified to homogeneity in three chromatographic steps (see
Supporting Information).
As indicated earlier, the major difficulty in studies of AmpD has
been the lack of authentic substrate(s) for characterization of the
enzyme.9,11 This has been an obstacle, since the putative substrates
are inherently complex12,13 and they have not been available for
characterization of the AmpD reaction. We have addressed this paucity
of information in this study. We synthesized compounds 1 (pentapep-
tide), 2a, 2c, 4a, and 4b for characterization of the reaction of AmpD.
We prepared 1, 2a, and 2c to explore the specificity of AmpD for its
proposed 1,6-anhydromuramyl-containing substrate(s). Whereas the
cytoplasmic AmpD would not come in contact with species 4a and
4b, we prepared these two compounds to explore the selectivity or
specificity of AmpD for the intermediates containing the 1,6-anhy-
dromuramyl moiety found in 1, 2a, and 2c.
The synthesis of compound 1 is reported elsewhere.14 Compound
6, an important intermediate in the syntheses of compounds 2a and
2c, was prepared from 4-benzyl D-glucal (5) (Scheme 1).14,15
Compound 6 was poised to receive the requisite protected peptide at
its carboxylic acid, which then was put through deprotection to furnish
the desired target compounds 2a and 2c. We also confirmed the
structure of 1,6-anhydromuramic acid derivative by determination of
the X-ray crystal structure of 11. The methyl ester of anhydromuramic
acid 3 (compound 11) keeps a typical anhydropyranose structure, where
all substituents are in axial positions. The details of synthetic steps
leading to compounds 2a and 2c are given in the Supporting
Information. The approach to synthesis of 4a and 4b, and related
compounds, has been described earlier.16
Figure 1. Peptidoglycan fragmentation leads to the formation of 1, which is
transported across plasma membrane to initiate its recycling and the gene
induction for production of ꢀ-lactamase.
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8742 J. AM. CHEM. SOC. 2009, 131, 8742–8743
10.1021/ja9025566 CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1
Table 1. Kinetic Parameters for Turnover of Substrates by AmpD
at pH 7.0
kcat (s-1
)
Km (µM)
kcat/Km (M-1 s-1
)
114
2a
2c
0.4 ( 0.1
25 ( 2.0
11 ( 1
1760 ( 210
360 ( 10
500 ( 60
(2.3 ( 0.1) × 102
(6.9 ( 0.4) × 104
(2.2 ( 0.3) × 104
moiety is comprised of the sterically encumbered bicyclo system, with
all its substituents in the axial positions, which is in sharp contrast to
the muramyl ring found in the peptidoglycan (and in 4a and 4b) with
its all-equatorial substituents.17
In summary, AmpD is capable of turning over 1,6-anhydromuramyl
species 2a and 2c equally well. The importance of this finding is two-
fold. AmpD is the gatekeeper for entry into the peptidoglycan recycling
events by its turnover of 2a. Equally importantly, since it has the ability
to turn over 2csthe resultant predominant species after exposure of
bacteria to the ꢀ-lactam antibiotics involved in antibiotic resistance
gene expression3,8sAmpD is the catalyst that reverses the recruitment
of bacterial resources in induction of ꢀ-lactamase, the antibiotic
resistance enzyme. It is likely that 1, the immediate product of
fragmentation of peptidoglycan, does not experience effective turnover
in ViVo, if at all. Furthermore, AmpD has evolved to recognize both
the atypical peptidoglycan peptide stem and the 1,6-anhydromuramyl
moiety; hence it is a peptidase with a unique function in bacterial
physiology.
Scheme 2
Supporting Information Available: Experimental procedures of
cloning, enzyme purification, kinetics, mass spectrometry, syntheses of the
new compounds, and crystallographic information file (CIF). This material
is available free of charge via the Internet at http://pubs.acs.org.
We subsequently investigated whether 2a and 2c would be processed
by AmpD. The analysis by LC/MS revealed that AmpD hydrolyzed
both compounds at the lactyl amide bond to generate a peptide 12a
(or 12b) and the corresponding 1,6-anhydromuramyl moiety 3 (Scheme
2). Authentic synthetic samples of the peptides and of the 1,6-
anhydromuramyl moiety 3 confirmed the structure assignments for
the products of the AmpD reaction (see Supporting Information).
The quantitative analysis by nonlinear regression of the data for
either the consumption of the substrate or the formation of the products
reveals that compounds 2a and 2c were turned over by AmpD with
kcat/Km values of (6.9 ( 0.4) × 104 and (2.2 ( 0.3) × 104 M-1 s-1,
respectively (Table 1). In essence, the enzyme does not discriminate
between the two substrates (a mere difference of 3-fold on kcat/Km). It
is interesting to note that 1 (R ) pentapeptide) is also turned over by
AmpD, but in terms of kcat/Km, it is 300-fold worse as a substrate than
2a. But, more importantly since its Km is in the millimolar range, it is
likely that AmpD would not experience saturation with this compound
in ViVo. This observation, compounded by the fact that the kcat value
is also considerably attenuated for this substrate, indicates that it is
likely that 1 is not turned over in ViVo.
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A more striking finding is that compounds 4a and 4b are not
substrates for AmpD. Evolution of the AmpD function as a peptidase
has clearly been driven by the atypical structure of the peptide, which
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