Reactions of the three AmpD enzymes of Pseudomonas aeruginosa

Article abstract of DOI:10.1021/ja400970n

A group of Gram-negative bacteria, including the problematic pathogen Pseudomonas aeruginosa, has linked the steps in cell-wall recycling with the ability to manifest resistance to β-lactam antibiotics. A key step at the crossroads of the two events is performed by the protease AmpD, which hydrolyzes the peptide in the metabolite that influences these events. In contrast to other organisms that harbor this elaborate system, the genomic sequences of P. aeruginosa reveal it to have three paralogous genes for this protease, designated as ampD, ampDh2, and ampDh3. The recombinant gene products were purified to homogeneity, and their functions were assessed by the use of synthetic samples of three bacterial metabolites in cell-wall recycling and of three surrogates of cell-wall peptidoglycan. The results unequivocally identify AmpD as the bona fide recycling enzyme and AmpDh2 and AmpDh3 as enzymes involved in turnover of the bacterial cell wall itself. These findings define for the first time the events mediated by these three enzymes that lead to turnover of a key cell-wall recycling metabolite as well as the cell wall itself in its maturation.

Full text of DOI:10.1021/ja400970n

Communication
pubs.acs.org/JACS
Reactions of the Three AmpD Enzymes of Pseudomonas aeruginosa
Weilie Zhang, Mijoon Lee, Dusan Hesek, Elena Lastochkin, Bill Boggess, and Shahriar Mobashery*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
S
* Supporting Information
ABSTRACT: A group of Gram-negative bacteria, includ-
ing the problematic pathogen Pseudomonas aeruginosa,
has linked the steps in cell-wall recycling with the ability
to manifest resistance to β-lactam antibiotics. A key step
at the crossroads of the two events is performed by the
protease AmpD, which hydrolyzes the peptide in the
metabolite that influences these events. In contrast to
other organisms that harbor this elaborate system, the
genomic sequences of P. aeruginosa reveal it to have three
paralogous genes for this protease, designated as ampD,
ampDh2, and ampDh3. The recombinant gene products
were purified to homogeneity, and their functions were
assessed by the use of synthetic samples of three bacterial
metabolites in cell-wall recycling and of three surrogates of
cell-wall peptidoglycan. The results unequivocally identify
AmpD as the bona fide recycling enzyme and AmpDh2
and AmpDh3 as enzymes involved in turnover of the
bacterial cell wall itself. These findings define for the first
time the events mediated by these three enzymes that lead
to turnover of a key cell-wall recycling metabolite as well as
the cell wall itself in its maturation.
Figure 1. Early events of bacterial cell-wall recycling and their link to
antibiotic resistance. Lytic transglycosylases degrade the cell wall in the
periplasm, located between the outer membrane (OM) and the inner
membrane (IM).
embers of Enterobacteriaceae and Pseudomonas aeruginosa
Mhave evolved an elaborate sequence of events that re- membrane to be used in assembly of the nascent peptidoglycan
(Figure 1). As a branching point in these events, metabolite 2
stimulates gene transcription to result in the production of an
antibiotic-resistance enzyme, the AmpC β-lactamase (Figure 1).
Hence, the action of AmpD, at the crossroads of these events,
shuts down the production of the AmpC β-lactamase and
commits to recycling of the cell wall.3,5,11
In contrast to other organisms that possess this system, ana-
lyses of the genomic sequences of P. aeruginosa have led to the
annotation of three paralogous genes for the protease AmpD.
These are designated as AmpD, AmpDh2, and AmpDh3.12
AmpDh2 has a signal peptide that targets it to the periplasm
and an anchor that is believed to insert it into the outer
membrane.13 On the other hand, AmpD is believed to be
cytoplasmic, and the cellular location of AmpDh3 is currently
unknown.13 Abrogation of the respective activities for these
enzymes by mutational inactivation or by gene ablation led to a
stepwise upregulation of antibiotic resistance, with the full effect
achieved only by the loss of all three enzyme activities at the
whole-organismal level.12−14 Interestingly, inactivation of ampD
is not sufficient to affect fitness or virulence of P. aeruginosa in
murine models of infection, but double or triple mutants with
cycles more than 50% of the bacterial cell wall during normal
growth for reasons that are not fully understood.1−5 The re-
cycling also takes place when damage to the cell wall occurs. As
damage to cell wall is also inflicted by exposure of bacteria to
β-lactam antibiotics, these organisms have evolved a link between
the biochemical steps of recycling and repair to unleashing of
an inducible antibiotic-resistance mechanism involving the
AmpC β-lactamase (Figure 1).1,5−8
Bacterial cell-wall recycling commences by degradation of
the peptidoglycan, the major constituent of the cell wall, by the
family of lytic transglycosylases.3,4,9,10 Whereas these enzymes
turn over their polymeric substrates with some variations on the
non-hydrolytic reaction that generates the fragmentation prod-
ucts, the major end product is N-acetyl-β-D-glucosamine-
(1→4)-1,6-anhydro-N-acetyl-β-D-muramyl-peptide (1), with the
full-length peptide being L-Ala-D-γ-Glu-meso-DAP-D-Ala-D-Ala
(DAP = diaminopimelate).9
Metabolite 1 is internalized by the permease AmpG. Once
in the cytoplasm, the glycosidase NagZ converts compound 1
to 2, which in turn serves as the substrate for the protease
AmpD, which removes the peptide stem from the saccharide.
The product of this reaction, compound 3, enters a sequence
of biochemical events that synthesizes the building unit Lipid II,
which is transported to the periplasmic side of the inner
Received: January 28, 2013
Published: March 19, 2013
© 2013 American Chemical Society
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Communication
losses of additional activities of the other two enzymes
(AmpDh2 and AmpDh3) would appear to be defective in
both.13
Scheme 1
Notwithstanding the careful analyses of the mutational effects
at the whole-organismal level, the activities of these three
AmpD enzymes of P. aeruginosa have not been studied with
suitable substrates by enzymological analysis. This is largely due
to the lack of availability of these substrates. We report herein
our cloning of the genes ampD, ampDh2, and ampDh3 from the
strain P. aeruginosa PA01. The respective proteins (AmpD,
AmpDh2, and AmpDh3) were expressed and purified to
homogeneity. We synthesized compounds 4−9 (Chart 1)
Chart 1. Chemical Structures of Substrates for AmpD
Enzymes Used in This Study
NHS-ester 1517 or muramic NHS-ester 16.15,16 Global deprotec-
tion of 17 or 18 by catalytic hydrogenation (18 required acid
treatment prior to hydrogenation) gave the final anhydromur-
amyl tetrapeptide 5 or muramyl tetrapeptide 8, respectively.
With the availability of all three enzymes and the six com-
pounds as potential substrates for these enzymes, we performed
HPLC analyses of the kinetics. The kinetics were monitored
quantitatively from consumption of the substrate and the
formation of products. The products were individually analyzed
by mass spectrometry to delineate the nature of the individual
reactions. This type of analysis is exemplified by turnover of
4 by the protease AmpD, as shown in Figure 2. The peak
by the methodology that we reported earlier.15−17 Compounds
5 and 8 had not been made previously and were prepared for
the first time for this study (described below), along with the
other four known compounds.16,17 As indicated in Figure 1,
1,6-anhydromuramyl derivatives 2 are bona fide substrates for
AmpD. The variation among compounds 2 is in the length of
the peptide stem, which is traced back to what is found in the
cell wall. This is a uniquely bacterial peptide, whose full-length
sequence is L-Ala-D-γ-Glu-meso-DAP-D-Ala-D-Ala. Maturation
of cell wall generates variants of the peptide stem with loss
of amino acids from the C-terminal end.3,18,19 As such, the
tripeptide and tetrapeptide stems are more common, and
the pentapeptide, which is biosynthetically introduced into the
nascent peptidoglycan, is less so.3,18 We have synthesized all
three versions, 4−6. We used 7−9 as mimetics of the standard
N-acetylglucosamine-based muropeptides from the cell wall
(Chart 1). These compounds would allow detection of turn-
over of standard cell-wall peptidoglycan (which exists only in the
periplasm), as opposed to that of the 1,6-anhydromuramyl deriv-
atives that are formed by the action of lytic transglycosylases.
Syntheses of the tetrapeptide-containing derivatives 5 and 8
are shown in Scheme 1. The key tetrapeptide intermediate 13
was prepared by the same methodology developed for the syn-
thesis of pentapeptide in our laboratory.16 The synthesis started
Figure 2. HPLC analysis of the AmpD reaction. (A) Reaction mixture
containing 100 nM purified AmpD and 3 mM 4 was incubated at
25 °C. (B) Partial transformation identifies 19 and 3 as products, with
m/z values of 391 and 276, respectively.
with the acetonide 10, which was prepared in 13 steps accord-
dez and Martin.20 The carboxylate
corresponding to 4 diminished as the reaction proceeded, with
two additional peaks appearing, for which the chemical structures
were assigned by mass spectrometry as 3 and 19. Hence, the
nature of the enzymatic reaction of AmpD with this compound is
hydrolysis of the amide bond to the lactyl moiety. Quantification
of the turnover with the use of an internal standard in each case
was performed, and the kinetic parameters were determined
(Table 1). The very same type of analysis was performed for
the reactions of each substrate with each enzyme, revealing that
we were monitoring hydrolysis of the amide bond at the lactyl
moiety in every case. The results are given in Table 1.
́
ing to the method of Hernan
́
moiety in 10 was activated by NHS/EDCI, and the resulting
NHS ester was treated with D-alanyl benzyl ester to give 11.
The three-step transformation of the acetonide functionality to
benzyl ester went smoothly using these conditions, which were
used initially for the synthesis of pentapeptide.16
The Boc group in 12 was removed in the presence of
trifluoroacetic acid, and the resultant product was readily
coupled to Boc-Ala-D-Glu(ONHS)-OBn to give the key tetra-
peptide 13. The Boc group in 13 was removed, and the resultant
amine 14 was allowed to react with either 1,6-anhydromuramic
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abundant components in the peptidoglycan analyses of E. coli
and P. aeruginosa.3,18 It is the reaction of DD-carboxypeptidases
which generates the tetrapeptide version.3,15,23,24
Table 1. Steady-State Kinetic Data for Turnover of
Compounds by AmpD, AmpDh2, and AmpDh3
a
enzyme/compd
Km (mM)
kcat (s−1
)
kcat/Km (M−1 s−1
)
If AmpDh2 and AmpDh3 were periplasmic enzymes, as
proposed in this report, then they will never come in contact
with 4−6, the cytoplasmic metabolites, notwithstanding their
marginal ability to turn them over. This is a curiosity, as we
have noted that inactivation of the genes for these two enzymes
results in a stepwise and incremental increase in antibiotic
resistance.12,13 However, AmpDh2 and AmpDh3 do encounter
1 in the periplasm. Could the effect be manifested by AmpDh2
and AmpDh3 removing the peptide from metabolite 1, at least
for a fraction of its total concentration? Indeed, this is the case.
Compound 1, which we synthesized by a reported method,25 is
a poor substrate for both AmpDh2 and AmpDh3: kcat = 0.45
0.03 s−1, Km = 1.0 0.1 mM, and kcat/Km = 440 53 M−1 s−1
for AmpDh2; kcat = 2.2 0.2 s−1, Km = 1.2 0.3 mM, and
AmpD
4
2.0 0.1
1.2 0.3
1.9 0.2
−
24.2 3.5
39.8 3.1
47.5 5.5
−
12100 1800
33200 7500
25000 4600
−
5
6
b
7−9
4
AmpDh2
AmpDh3
1.5 0.2
4.6 0.5
1.9 0.2
1.5 0.1
1.8 0.3
1.4 0.2
3.2 0.5
4.5 0.6
1.4 0.1
2.2 0.3
1.4 0.2
2.4 0.3
0.20 0.02
130 17
5
20 1.9
4300 790
95 14
6
0.18 0.02
51.8 8.9
73.6 5.3
1.6 0.2
17.9 1.3
70.6 6.6
20.6 1.9
195 24
212 19.3
85.3 10.2
7
34500 4300
40900 6800
1100 150
5590 610
15700 1900
14700 1600
88600 13600
8
9
4
5
6
7
kcat/Km = 1800
480 M−1 s−1 for AmpDh3. Hence, the
8
151000 20500
35500 4800
primary reaction of AmpDh2 and AmpDh3 is turnover of the
normal cell-wall peptidoglycan. However, these two enzymes
commence removal of the peptide from a fraction of 1 in the
periplasm, prior to the completion of the process by the action
of AmpD within the cytoplasm.
9
a
The activities of all three enzymes decreased in the presence of
EDTA, indicating that they are zinc proteases. Kinetic measurements
were performed in the presence of 100 μM ZnCl2 as a supplement for
AmpDh2 and AmpDh3. No measurable activity was detected for
b
That the cell-wall surrogates 7−9 were turned over by
AmpDh2 and AmpDh3 was intriguing. However, do these en-
zymes actually turn over the cell wall itself? To address this
issue, we prepared the P. aeruginosa sacculus according to a
reported method.9,26 Overnight incubation of the polymeric
sacculus with AmpDh2 produced products with oligomeric cell-
wall sugars without the peptide stem. Mass spectrometric detec-
tion identified (m/zz) values of 479+, 957+, 1435+, 9572+, 11962+,
14362+, 11173+, and 12763+, corresponding to the general
formula, (NAG-NAM)n-NAG-anhMur (structure 20, n = 0−7).
The reaction with AmpDh3 also produced products 20, but its
yield was three times less than that with AmpDh2.
AmpD with these compounds.
The important observation from these data is that AmpD
is the true protease for the recycling process in P. aeruginosa, as
its function is exclusive for turnover of 1,6-anhydromuramyl
variants 4−6. This enzyme does not turn over variants 7−9,
which are mimetics of the standard peptidoglycan. The AmpD
enzyme has evolved for turnover of 1,6-anhydromuramyl
compounds, akin to the reaction that we documented earlier
for the enzyme from Citrobacter freundii.17
On the other hand, AmpDh2 and AmpDh3 exhibit activities
with both types of substrates. However, the activities of AmpDh2
and AmpDh3 with the 1,6-anhydromuramyl compounds 4−6 are
a mere 6% and 12%, respectively, of the total activity (as assessed
by the kcat/Km values). For these two enzymes the overwhelming
activity is documented for derivatives 7−9 as substrates. Hence,
it is clear that AmpDh2 and AmpDh3 have evolved as enzymes
for processing of the peptidoglycan components of the cell wall
in the periplasmic space. The mere 6−12% hydrolytic activities
with 4−6 that we measure for these two enzymes are none-
theless real, a subject to which we return below. However, this
marginal activity is likely to be adventitious. Since these
enzymes clearly prefer the forms of the peptidoglycan found in
the cell wall, they are likely counterparts to the cell-wall
amidases such as AmiA, B, C, and D, which have been found in
Escherichia coli.3
The AmpD enzyme, the bona fide recycling protease, reaches
saturation (Km) at the low millimolar range with its substrates
(Table 1), indicating that the intracellular concentrations of
metabolites 4−6 during active cell-wall recycling must be in
that range. The rather high concentration is not uncommon for
other important bacterial metabolites as well.15,21,22 This ob-
servation regarding the Km of metabolites 4−6 holds true for the
other two enzymes as well, although their true function, per our
findings, is processing of cell wall in the periplasm. We also note that
all three enzymes exhibit modest preferences for the tetrapeptide
stems in the substrates (kcat/Km effect). Whereas the importance
of this observation is not immediately apparent, we are aware
that muropeptides with the tetrapeptide stem are among the most
In summary, we have provided the first enzymological ana-
lyses of reactions of AmpD, AmpDh2, and AmpDh3 from
P. aeruginosa with unique synthetic substrates (three bacterial
metabolites and three structural surrogates for the cell wall).
This analysis identifies AmpD of P. aeruginosa as the cytoplasmic
protease for commitment to the cell-wall recycling events and for
the reversal of the activation of the antibiotic-resistance pathway. The
enzymes AmpDh2 and AmpDh3 exhibited marginal activities
with the 1,6-anhydromurmyl compounds 4−6 as substrates.
Their function is turnover of cell wall within the periplasmic
space. Nonetheless, the marginal activity in turnover of metab-
olite 1 with AmpDh2 and AmpDh3 explains the stepwise effect
observed in augmenting derepression of AmpC β-lactamase by
eliminations of these enzymes one by one.12,13
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for syntheses of the new compounds,
cloning, enzyme purification, kinetics, MS analyses of products,
and NMR spectra of the new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
mobashery@nd.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant from the NIH
(GM61629). The Mass Spectrometry & Proteomics Facility
of the University of Notre Dame is supported by grant
CHE0741793 from the NSF.
REFERENCES
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