From Genome to Proteome to Elucidation of Reactions for All Eleven Known Lytic Transglycosylases from Pseudomonas aeruginosa

Article abstract of DOI:10.1002/anie.201611279

An enzyme superfamily, the lytic transglycosylases (LTs), occupies the space between the two membranes of Gram-negative bacteria. LTs catalyze the non-hydrolytic cleavage of the bacterial peptidoglycan cell-wall polymer. This reaction is central to the growth of the cell wall, for excavating the cell wall for protein insertion, and for monitoring the cell wall so as to initiate resistance responses to cell-wall-acting antibiotics. The nefarious Gram-negative pathogen Pseudomonas aeruginosa encodes eleven LTs. With few exceptions, their substrates and functions are unknown. Each P. aeruginosa LT was expressed as a soluble protein and evaluated with a panel of substrates (both simple and complex mimetics of their natural substrates). Thirty-one distinct products distinguish these LTs with respect to substrate recognition, catalytic activity, and relative exolytic or endolytic ability. These properties are foundational to an understanding of the LTs as catalysts and as antibiotic targets.

Full text of DOI:10.1002/anie.201611279

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International Edition: DOI: 10.1002/anie.201611279
German Edition: DOI: 10.1002/ange.201611279
Lytic Transglycosylases
From Genome to Proteome to Elucidation of Reactions for All Eleven
Known Lytic Transglycosylases from Pseudomonas aeruginosa
Mijoon Lee, Dusan Hesek, David A. Dik, Jennifer Fishovitz, Elena Lastochkin, Bill Boggess,
Jed F. Fisher, and Shahriar Mobashery*
Abstract: An enzyme superfamily, the lytic transglycosylases
LTs), occupies the space between the two membranes of
muramic acid (NAM), crosslinked to a neighboring glycan
strand through the peptide stems of the NAM saccharide. The
full-length uncrosslinked stem structure for P. aeruginosa is l-
Ala–g-d-Glu–m-DAP–d-Ala–d-Ala, where DAP is diamino-
pimelate (Figure 1). Neighboring strands of the peptidoglycan
(
Gram-negative bacteria. LTs catalyze the non-hydrolytic
cleavage of the bacterial peptidoglycan cell-wall polymer.
This reaction is central to the growth of the cell wall, for
excavating the cell wall for protein insertion, and for monitor-
ing the cell wall so as to initiate resistance responses to cell-
wall-acting antibiotics. The nefarious Gram-negative pathogen
Pseudomonas aeruginosa encodes eleven LTs. With few
exceptions, their substrates and functions are unknown. Each
P. aeruginosa LT was expressed as a soluble protein and
evaluated with a panel of substrates (both simple and complex
mimetics of their natural substrates). Thirty-one distinct
products distinguish these LTs with respect to substrate
recognition, catalytic activity, and relative exolytic or endolytic
ability. These properties are foundational to an understanding
of the LTs as catalysts and as antibiotic targets.
P
seudomonas aeruginosa is an opportunistic bacterial
pathogen. Its nearly 5700 open reading frames—a genetic
complexity approaching the simple eukaryote Saccharomyces
cerevisiae—encode a diversity of antibiotic-resistance mech-
[1]
anisms, few of which are fully understood. Many of these
resistance mechanisms focus on antibiotics that compromise
cell-wall integrity. Although a polymer, the peptidoglycan of
the bacterial cell wall is a dynamic structure that is in
Figure 1. Cell-wall turnover by LTs within the periplasm of Gram-
negative bacteria.
[
2]
perpetual states of assembly and disassembly. This dyna-
mism requires numerous enzymes. A key process is the
recycling of the cell-wall components that are liberated
concurrent with cell-wall growth. This recycling not only
conserves biosynthetic intermediates, but signals (by its
crosslink by acyl transfer of the penultimate d-Ala of one
strand to the m-DAP of an adjacent strand. LTs cleave the
glycosidic bond between NAM and NAG, through a NAM
oxocarbenium-like intermediate that is trapped by the C6
hydroxy of NAM. Muropeptides having the 1,6-anhydro-N-
acetylmuramic acid (anhNAM) terminus, which results from
LT activity, are transported from the periplasm through the
AmpG transporter of the inner membrane, into the cytoplasm
for subsequent transformations.
The 11 LTs of P. aeruginosa are MltA, MltB, MltD, MltF,
MltF2, MltG, RlpA, Slt, SltB1, SltB2, and SltB3 (Table 1);
Mlt stands for “membrane-bound LT”, Rlp for “rare lip-
oprotein” and Slt for “soluble LT”. The genes were cloned
and each LT was expressed as a soluble protein (by exclusion
of the signal peptide, and where applicable also the mem-
brane-anchoring lipobox site) and purified to > 95% purity
[3]
perturbation) the presence of cell-wall active antibiotics.
Cell-wall recycling in P. aeruginosa PAO1 generates over 20
[4]
different cell-wall components (called muropeptides).
The lytic transglycosylases (LTs) are essential catalysts of
[5]
bacterial cell-wall function. Genome analysis of P. aerugi-
[
6,7]
nosa PAO1 reveals at least 11 LTenzymes.
The function of
the LTs is to truncate the glycan component of the cell wall.
This polymer consists of glycan strands showing a repeating
disaccharide motif, N-acetylglucosamine (NAG)–N-acetyl-
[
*] Dr. M. Lee, Dr. D. Hesek, D. A. Dik, Dr. J. Fishovitz, E. Lastochkin,
Dr. B. Boggess, Dr. J. F. Fisher, Prof. S. Mobashery
Department of Chemistry and Biochemistry
University of Notre Dame
(Table S1 and Figure S1 in the Supporting Information).
Functional studies of five LTs were reported. Three
[8]
[9]
P. aeruginosa LTs (MltB and SltB1, RlpA ) were studied by
Notre Dame, IN 46556 (USA)
E-mail: mobashery@nd.edu
[
10]
[11]
others, and two LTs (SltB3 and MltF ) by ourselves. As
these studies used different substrates and different analytical
methodologies, we report here the first systematic and
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201611279.
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quantitative comparison of the 11 known LT ensemble of P.
aeruginosa. Each LT was evaluated using four synthetic
substrates as well as the intact cell-wall polymer—the
sacculus—as substrates.
herein reported for the first time. While syntheses of large (8
saccharide) muropeptides having a NAM-terminus are
[
13]
known, those of muropeptides with an anhNAM-terminus
are rarer and heretofore were limited to the NAG–anhNAM
[
14,15]
Our first synthetic substrate, NAG–NAM(pentapeptide)–
NAG–NAM(pentapeptide) 1, presents the minimal motif for
an LT substrate (Scheme 1). We reported previously its 63
disaccharide.
As outlined in Scheme 2B, our synthesis of
2 used dimethylmaleoyl (DMM) amine protection to both
direct b-glycosidic bond formation and to provide improved
solubility, O-TBDMS protection at C-1 of the NAG, trichloro-
acetimidate glycoside activation, and catalytic hydrogenation
for the final global deprotection. Key tetrasaccharide 15 was
prepared by consecutive glycosylations of disaccharide 13.
The DMM groups in 15 were removed by sequential base/acid
treatment. Acetylation of the amine gave 16. Deprotection at
C-3, lactyl attachment, and catalytic hydrogenation yielded
substrate 2. Two additional substrates (3 and 4), each devoid
of peptide stems, were prepared by catalytic hydrogenation of
[
12]
step convergent synthesis (Scheme 2A). Surprisingly, only
MltB and its subfamily (SltB1, SltB2, and SltB3) recognized
1
0 and 17, respectively. Synthetic standards for the LT
products (5–9) also were synthesized (Scheme 2). The syn-
theses of 5 and 6 were reported previously by our labora-
[
12,14]
tory.
We note that 7 (“tracheal cytotoxin”) is not only the
turnover product of 2, but is also a major turnover product of
the sacculus by LTs. Although its synthesis was reported
[
15]
earlier, we now provide its full spectral characterization.
All four LTs that turned over 1—MltB, SltB1, SltB2, and
SltB3—also turned over 2 (100% conversion to 7). Whereas
MltA fully converted 2 to 7. The rest of LTs did so to a lesser
extent (4–41%: Table 1 and Figure S3). Nonetheless, 2 is
a more general LT substrate than 1. Fewer LTs turned over
the two tetrasaccharide substrates lacking peptide stems, 3
Scheme 1. Turnover of four synthetic substrates (1–4) by LTs.
1
as a substrate, to yield 5 and 6 as products (Table 1 and
Figure S2). The possibility that an anhNAM-terminus at the
2 position (two sugars to the right of the scissile bond) was
(NAG–NAM–NAG–NAM) and
4
(NAG–NAM–NAG–
anhNAM), as compared to 1 and 2 (Figures S4 and S5,
Table 1). Comparison of the four structurally distinct sub-
strates gave these conclusions. MltA requires a + 2 anhNAM
(prior product of an LT reaction). MltB requires a peptidyl
stem. SltB1, SltB2, and SltB3 accept all tetrasaccharide
+
required for substrate recognition by the other seven LTs was
evaluated with our second synthetic substrate, NAG—NAM-
(
tetrapeptide)–NAG–anhNAM(tetrapeptide) 2 (Scheme 1),
Scheme 2. Syntheses of substrates (1–4) and of products of LT catalysis (5–9).
2
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[
a,b,c]
Table 1: Reactions of 11 LTs with synthetic substrates 1–4.
À1
À1
[
a] Percentage of product is given. This was converted to specific activity in nmol productmin mg protein in Table S2 in Supporting Information.
Reactions were carried out in 20 mm HEPES, 0.1m NaCl, 0.1% Triton X, pH 7.0 for 2 h at 378C. [b] Gray highlights mean avid turnover of a given
substrate. [c] “NR” means no reaction.
[
17]
substrates. The rest of the LTs appear to have additional
requirements for substrate recognition, such as a glycan
strand length of greater than four saccharides. A limitation of
the tetrasaccharide substrates is their inability to differentiate
between intrinsic exolytic (degradation from a terminus) or
endolytic activity. This preference was examined by the
reaction of the LTs with the purified P. aeruginosa sacculus.
LT degradation of the sacculus releases a set of soluble
muropeptides (Figure 2, Tables 2 and S3).
amidase (hydrolysis at the lactyl moiety of NAM)
remove both crosslinked and uncrosslinked stem peptides.
to
We further examined oligomeric substrates, (NAG–
NAM) –NAG–anhNAM (22, where n = 2–9, and lacking
n
peptide stems) which interrogates the requirement for the
presence of the stem peptide for recognition, as well as the
exolytic or endolytic preference. The results are summarized
in Table 2. Figure 2 shows chromatograms for the reactions of
MltA and RlpA (representing two LT families) with the
sacculus. A detailed summary of each reaction is given in the
Supporting Information
Unexpectedly, all 11 enzymes have endolytic and exolytic
capabilities. The LTs distinguished for endolytic capability
(MltD, MltF2, RlpA, and Slt) are highlighted in dark gray.
The LTs distinguished for exolytic activity (MltA, SltB1,
SltB2, and SltB3) are highlighted in light gray (Table 2). MltB,
A total of 31 muropeptides were identified, at picomole
[
16]
sensitivity by LC/MS analysis, per earlier methodology. The
complete list of LT products is given in Table S3, and the 12
most abundant products are shown in Figure 2A. In a second
assay, the sacculus was first allowed to react with the LT, and
the products of this reaction then reacted with the AmpDh3
Figure 2. A) Reactions of LTs with various substrates used in this study. B–H) LC/MS extracted-ion chromatograms of reactions of MltA and RlpA.
Reactions with the sacculus (B, C), with the sacculus, followed by AmpDh3 (D, E), with 22 (F, G, H).
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[
a,b]
Table 2: Summary of reactions of 11 LTs of P. aeruginosa with sacculus.
[
a] Average of two runs with standard deviations for reactions with the sacculus. [b] Data indicating endolytic activity are highlighted in dark gray and
those for exolytic activity are in light gray. [c] These numbers are for saccharides with stem peptides and those in parentheses are for total saccharides.
d] Activities are normalized to the value for SltB1.
[
MltF, and MltG have dual-activity. The total muropeptides
released from the sacculus gave this order of overall activity:
stem presence, and exo/endolytic preference. This subtlety is
substantiated by the unperturbed planktonic growth of multi-
[
5,7]
(
most active) SltB1, SltB3 > MltA, SltB2 > MltB > MltF,
ple LT gene knockouts,
implicating not only a redundancy
RlpA, MltG > Slt > MltD, MltF2 (least active).
of function, but a redundancy arising in response to critical
biological function.
While more uncrosslinked (7a and 7) than crosslinked
(
21a, 21b, 21c) products were seen for each LT, all LTs
Although breadth of LT function is now proven, the
complete functional assignment within the LT superfamily is
the outstanding challenge. P. aeruginosa SltB1 and MltF have
primary roles in the muropeptide-recycling pathway that
connects, through PBP4 inhibition by b-lactam antibiotics, to
AmpC b-lactamase induction (whereas, seemingly paradoxi-
cally, loss of either Slt or MltF decreases b-lactam resist-
turnover both. We also observed minor products having
a reducing muramyl terminus, arising from partitioning of the
transient oxocarbenium species of LT catalysis between
interception by the C hydroxy (major product) and water.
6
The amount of water-derived product is greater for the two
enzymes with the lowest specific activities (MltD and MltF2).
MltB is unique in its requirement for the presence of the
peptide stem for substrate recognition (22 is unreactive). In
contrast MltA, MltF, MltG, RlpA, SltB1, SltB2, and SltB3
show up to 100% conversion of 22. RlpA prefers muropep-
tide substrates lacking the peptide stem, as first observed by
Jorgenson et al., yielding the di- (8), tetra- (4), hexa- (4a),
and octasaccharide (4b) product ensemble. With the sole
exception of MltB, all other LTs produced NAG–anhNAM
[
7]
ance). The SPOR domain of RlpA directs this LT to the
septum for roles in both the rod shaping and daughter-cell
[
9]
separation of P. aeruginosa. MltG is assigned as the glycan-
[
20]
sizing LT of peptidoglycan glycan strand elongation. The
robust syntheses that yielded the four key tetrasaccharide
substrates used in this study have the power for further
probing LT substrate identity. This foundational study of the
intricacies of substrate recognition and catalysis within the LT
family of P. aeruginosa is a prelude to further design of LT
substrates for ultimate correlation with LT critical function.
[
9]
(8) to the extent of 1–13% (Table S3), but not the larger
tetra-, hexa- and octasaccharides (4, 4a and 4b).
These data confirm the presence of a superfamily of at
least 11 LTs in the periplasm of P. aeruginosa PAO1. The LTs
[
5,18]
of E. coli were classified on the basis of sequence,
but Acknowledgements
continuing study of the P. aeruginosa and Escherichia coli
genomes and proteomes has increased the number and the
This work is supported by grants from NIH (GM61629 and
T32GM075762). Hualiang Pi contributed to gene cloning.
[5,7,19]
diversity of the LT superfamily.
This study—the first
comprehensive comparison of substrate recognition and
processing by the individual members of the P. aeruginosa
LT superfamily—provides important insight toward func- Conflict of interest
tional LT classification. A key discovery is the requirement of
some LTs for a + 2 anhNAM residue for substrate recog-
The authors declare no conflict of interest.
nition. All LTs show a subtle interplay among glycan length,
4
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Keywords: anhydromuramic acid · antibiotic resistance ·
muropeptide · sacculus · b-lactam
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Final Article published: && &&, &&&&
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Lytic Transglycosylases
The lytic transglycosylases (LTs) are gly-
coside-cleaving enzymes found in Gram-
negative bacteria. Their diversity of
structure contrasts with a common sub-
strate: the peptidoglycan of the bacterial
cell wall. By the systematic evaluation of
synthetic peptidoglycans and the poly-
meric peptidoglycan, 11 LTs of Pseudo-
monas aeruginosa are now characterized
with respect to their substrate recogni-
tion, catalytic activity, and exolytic or
endolytic preference.
M. Lee, D. Hesek, D. A. Dik, J. Fishovitz,
E. Lastochkin, B. Boggess, J. F. Fisher,
S. Mobashery*
&&& — &&&
From Genome to Proteome to
Elucidation of Reactions for All Eleven
Known Lytic Transglycosylases from
Pseudomonas aeruginosa
6
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