Loss of a Functionally and Structurally Distinct ld-Transpeptidase, Ldt_Mt5, Compromises Cell Wall Integrity in Mycobacterium tuberculosis

Background: M. tuberculosis Ldt_Mt5 is an Ldt_Mt2 paralog that cross-links peptidoglycan stem peptides.

Results: Ldt_Mt5 is structurally divergent, strains lacking Ldt_Mt5 are more susceptible to chemical and environmental stresses, and Ldt_Mt2 cannot compensate for its loss.

Conclusion: Ldt_Mt2 and Ldt_Mt5 serve non-redundant roles in peptidoglycan maintenance.

Significance: Ldt_Mt5 is necessary for properly maintaining cell wall integrity and should be pursued as a drug target.

Abstract

The final step of peptidoglycan (PG) biosynthesis in bacteria involves cross-linking of peptide side chains. This step in Mycobacterium tuberculosis is catalyzed by ld- and dd-transpeptidases that generate 3→3 and 4→3 transpeptide linkages, respectively. M. tuberculosis PG is predominantly 3→3 cross-linked, and Ldt_Mt2 is the dominant ld-transpeptidase. There are four additional sequence paralogs of Ldt_Mt2 encoded by the genome of this pathogen, and the reason for this apparent redundancy is unknown. Here, we studied one of the paralogs, Ldt_Mt5, and found it to be structurally and functionally distinct. The structures of apo-Ldt_Mt5 and its meropenem adduct presented here demonstrate that, despite overall architectural similarity to Ldt_Mt2, the Ldt_Mt5 active site has marked differences. The presence of a structurally divergent catalytic site and a proline-rich C-terminal subdomain suggest that this protein may have a distinct role in PG metabolism, perhaps involving other cell wall-anchored proteins. Furthermore, M. tuberculosis lacking a functional copy of Ldt_Mt5 displayed aberrant growth and was more susceptible to killing by crystal violet, osmotic shock, and select carbapenem antibiotics. Therefore, we conclude that Ldt_Mt5 is not a functionally redundant ld-transpeptidase, but rather it serves a unique and important role in maintaining the integrity of the M. tuberculosis cell wall.

Introduction

Virtually all bacteria possess a peptidoglycan (PG)⁴ layer that encapsulates the cytoplasmic membrane and provides shape and rigidity to the cell. Considered as a vital bacterial “organelle,” the PG is essential for cellular growth and viability. β-Lactams, the class of drugs with the highest impact in treating bacterial infections in humans (1), act by inhibiting PG biosynthesis (2).

Mature PG comprises aminosugar strands with alternating N-acetylmuramic acid and N-acetylglucosamine units and peptide moieties that are cross-linked to one another (3). Lipid II, a precursor to PG, is synthesized intracellularly and consists of a single disaccharide-peptide and lipid moiety. In Mycobacterium tuberculosis, the infectious pathogen that causes tuberculosis, the pathway for lipid II biosynthesis comprises more than 20 genes, and all are essential for the viability of this pathogen (4). Lipid II is transported across the membrane, and units are linked together by transglycosylases that extend the aminosugar strand. The final step in PG maturation involves cross-linking the peptide moieties to one another by both dd- and ld-transpeptidases.

Until recently, the PG was considered to be largely cross-linked by dd-transpeptidases that generate 4→3 transpeptide linkages between the fourth amino acid (d-alanine) of one chain and the third amino acid (meso-diaminopimelic acid in M. tuberculosis) of an adjacent chain. Interestingly, the majority of transpeptide linkages in Mycobacterium spp. are between meso-diaminopimelic acid residues of two peptide chains (5). These 3→3 linkages are formed by ld-transpeptidases (6, 7), and the dominant ld-transpeptidase in M. tuberculosis is Ldt_Mt2 (8). Unlike dd-transpeptidases that utilize a catalytic serine and pentapeptide substrates (9), ld-transpeptidases require a catalytic cysteine residue (10, 11) and utilize tetrapeptide substrates. ld-Transpeptidation has now been identified in a range of pathogenic bacteria (11–13), highlighting the importance of this fundamental mechanism across bacterial species.

In M. tuberculosis, loss of Ldt_Mt2 results in altered cell size, growth, and virulence as well as loss of the ability of the organism to secrete low molecular weight proteins and increased susceptibility to amoxicillin (8, 14). The genome of M. tuberculosis encodes four additional paralogs of Ldt_Mt2. On the basis of in vitro cross-linking activity or sequence similarity, they have been annotated as Ldt_Mt1 (Rv0116c), Ldt_Mt3 (Rv1433), Ldt_Mt4 (Rv0192), and Ldt_Mt5 (Rv0483) and share amino acid sequence identity of 36, 34, 35, and 28% with Ldt_Mt2, respectively. It is unclear whether the five sequence paralogs are functionally redundant.

We used a combination of biophysical, biochemical, and genetic approaches to study Ldt_Mt5. Here, we report the first crystal structures of apo- and meropenem-bound Ldt_Mt5 and describe the phenotypic effects on M. tuberculosis lacking this enzyme. Our data indicate that Ldt_Mt5 is structurally divergent compared with other M. tuberculosis ld-transpeptidases and that this protein serves a critical and distinct role in proper maintenance of M. tuberculosis cell wall integrity, highlighting its potential as a novel drug target.

Experimental Procedures

General Methods

All reagents were obtained from commercial sources. Spectrophotometric analyses were performed on a Shimadzu UV-1800 UV-visible spectrophotometer. Primers were purchased from Integrated DNA Technologies. Isothermal titration calorimetry (ITC) experiments were performed using a high precision VP-ITC titration calorimeter system (Microcal Inc.). Ultraperformance liquid chromatography (LC)-high resolution MS samples were analyzed on a Waters Acquity H-Class ultraperformance LC system equipped with a multiwavelength ultraviolet-visible diode array detector in conjunction with a Waters Acquity BEH-300 ultraperformance LC column packed with a C₄ stationary phase (2.1 × 50 mm; 1.7 μm) in tandem with high resolution MS analysis by a Waters Xevo-G2 quadrupole-TOF electrospray ionization mass spectrometer. Molecular graphics and analyses were performed with the UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by National Institutes of Health NIGMS Grant P41-GM103311).

Cloning, Overexpression, and Purification of Ldt_Mt5

A truncated version of ldt_Mt5 (encoding amino acids 55–451) was amplified by PCR (1× New England Biolabs GC reaction buffer, 200 μm dNTPs, 2 ng/μl CDC1551 M. tuberculosis genomic DNA, 500 nm primers (Table 1), 1 unit of Phusion polymerase, and 3% DMSO), digested with NdeI and XhoI, and cloned into a modified pET28a vector that encodes for a TEV-cleavable N-terminal His₆ tag (10). Escherichia coli BL21(DE3) cells harboring the ldt_Mt5-pET28a plasmid were grown to an A₆₀₀ of ∼0.5 at 37 °C. Flasks were then cooled on ice with periodic shaking. Protein overexpression was induced with 100 μm isopropyl 1-thio-β-d-galactopyranoside, and flasks were returned to an incubator shaker at 16 °C for an additional 24 h. Cells were harvested at 4 °C and stored overnight at −20 °C. Thawed cells were resuspended in protein purification buffer (25 mm Tris, pH 8.0, 400 mm NaCl, 10% glycerol, and 1 mm tris(2-carboxyethyl)phosphine (TCEP)) and protease inhibitor mixture (Roche Applied Science) and lysed by ultrasonication, and cell debris was removed by centrifugation at 4 °C. The supernatant was incubated with nickel-nitrilotriacetic acid resin at 4 °C for 90 min, and His₆-tagged Ldt_Mt5 was eluted from the resin over a stepwise gradient of 5–500 mm imidazole. Fractions containing Ldt_Mt5 (as determined by SDS-PAGE) were combined, and protein concentration was determined using the Bio-Rad Protein Assay with bovine serum albumin (BSA) as a standard. The sample was then subjected to dialysis overnight at 4 °C against 1 liter of 25 mm Tris, pH 8.0, 100 mm NaCl, 10% glycerol, and 1 mm TCEP in the presence of TEV protease (1:100 TEV:Ldt_Mt5). Following dialysis, the TEV-treated sample was incubated with fresh nickel-nitrilotriacetic acid resin at 4 °C for 90 min. Cleaved Ldt_Mt5 was collected as flow-through, whereas the His-tagged TEV protease and cleaved His₆ tag remained bound to the resin. Ldt_Mt5 was subjected to a second dialysis against 1 liter of 25 mm Tris, pH 8.0, 100 mm NaCl, and 1 mm TCEP for 4 h at 4 °C. The concentration of Ldt_Mt5 was determined using the Bio-Rad Protein Assay with BSA as a standard, and Ldt_Mt5 was concentrated to 12.8 mg/ml prior to being flash frozen in liquid N₂. Protein was stored at −80 °C.

TABLE 1.

Summary of primers used

Restriction sites are underlined. F, forward primer; R, reverse primer.

Gene/variant	Primers
ldtMt5	F, 5′-TATTGCCATATGAAGCTGGCCGAGAAGAGG-3′
	R, 5′-TATTGCCTCGAGCTACCCACCCGGTCCGTT-3′
H342A LdtMt5	F, 5′-CAACAACGGCGAGTTCATCGCTG CCAACCCTATGAGCGCC-3′
	R, 5′-GGCGCTCATAGGGTTGGCAGCGATGAACTCGCCGTTGTTG-3′
H342Q LdtMt5	F, 5′-CAACAACGGCGAGTTCATCCAGGCCAACCCTATGAGCGCC-3′
	R, 5′-GGCGCTCATAGGGTTGGCCTGGATGAAC TCGCCGTTGTTG-3′
M346W LdtMt5	F, 5′-GAGTTCATCCATGCCAACCCTTGGAGCGCCGGTGCCCAGGGCAAC-3′
	R, 5′-GTTGCCCTGGGCACCGGCGCTCCAAGGGTTGGCATGGATGAACTC-3′
T357V LdtMt5	F, 5′-CAGGGCAACAGCAATGTCGTCAACGGCTGTATCAACCTGTCG-3′
	R, 5′-CGACAGGTTGATACAGCCGTTGACGACATTGCTGTTGCCCTG-3′
N358A LdtMt5	F, 5′-GGCAACAGCAATGTCACCGCCGGCTGTATCAACCTGTCGACG-3′
	R, 5′-CGTCGACAGGTTGATACAGCCGGCGGTGACATTGCTGTTGCC-3′
N358H LdtMt5	F, 5′-CAGGGCAACAGCAATGTCACCCACGGCTGTATCAACCTGTCGACG-3′
	R, 5′-CGTCGACAGGTTGATACAGCCGTGGGTGACATTGCTGTTGCCCTG-3′
C360A LdtMt5	F, 5′-CAGCAATGTCACCAACGGCGCTATCAACCTGTCGACGGAG-3′
	R, 5′-CTCCGTCGACAGGTTGATAGCGCCGTTGGTGACATTGCTG-3′
N362A LdtMt5	F, 5′-GTCACCAACGGCTGTATCGCCCTGTCGACGGAGAACGCC-3′
	R, 5′-GGCGTTCTCCGTCGACAGGGCGATACAGCCGTTGGTGAC-3′

Site-directed Mutagenesis Studies

Site-directed mutagenesis reactions were performed as described previously with minor modifications (15). Briefly, two PCRs (25 μl) containing either the forward or reverse primer were set up in parallel. Each PCR contained 1× New England Biolabs GC reaction buffer, 200 μm dNTPs, 1 ng/μl template, a 500 nm concentration of either the forward or reverse primer, 1 unit of Phusion polymerase, and 3% DMSO. The ldt_Mt5-pET28a plasmid was used as the template to generate each Ldt_Mt5 variant (see Table 1 for primers). Sufficient elongation of primer at 68 °C occurred over 15 min. Forward and reverse PCRs were then combined (new volume of 50 μl), and complementary strands were reannealed following gradual cooling (95 °C, 5 min; 90 °C, 1 min; 80 °C, 1 min; 70 °C, 30 s; 60 °C, 30 s; 50 °C, 30 s; and 40 °C, 30 s). Samples were then incubated at 37 °C, and template DNA was digested with 1 unit of DpnI for 3 h. All constructs were fully sequenced, and competent bacteria were transformed with mutagenic plasmid. All variants were purified as described above.

Kinetic Analyses

The nitrocefin hydrolytic activities of Ldt_Mt2, Ldt_Mt5, and Ldt_Mt5 variants were measured spectrophotometrically as reported previously for Ldt_Mt2 (10) but with modifications. Briefly, reaction mixtures containing 1× tribuffer (100 mm MES, 50 mm N-ethylmorpholine, and 50 mm diethanolamine, pH 10), 0.1 mg/ml BSA, 100 mm NaCl, 1 mm TCEP, 5% DMSO, and 10 μm Ldt_Mt5 or Ldt_Mt5 variant were preincubated at 37 °C for 5 min. Nitrocefin (Calbiochem) was added to initiate each reaction, and the rate of nitrocefin hydrolysis was measured at 486 nm at 37 °C. For each set of reactions, the rate of nitrocefin hydrolysis in the absence of enzyme was observed and was subtracted from the initial rate of nitrocefin hydrolysis in the presence of enzyme at each substrate concentration. Initial rates of nitrocefin hydrolysis were measured over 3 min. An extinction coefficient (ϵ_M) of 20,500 m⁻¹ cm⁻¹ was used in determining the concentration of hydrolyzed nitrocefin as it was experimentally determined that ϵ_M does not change with changing pH under these buffering conditions (data not shown). Non-linear regression analyses of initial velocities were performed using GraphPad Prism (version 5). Reaction mixtures containing nitrocefin (100 μm) and Ldt_Mt5 or Ldt_Mt2 (5 μm) at varying pH values in 1× tribuffer and the conditions described above were monitored spectrophotometrically for determining the optimal pH for each enzyme. To rule out the possibility that residual TEV incompletely removed during protein purification could be contributing to nitrocefin hydrolysis at pH 10, we evaluated nitrocefin as a substrate for TEV at pH 10. Only baseline levels of hydrolysis were observed, indicating that TEV was not significantly contributing to the observed rates of hydrolysis by Ldt_Mt5 and Ldt_Mt5 variants (data not shown). Conversely, when we evaluated nitrocefin as a substrate for His₆-Ldt_Mt5, we observed rates of hydrolysis that were comparable with TEV-cleaved Ldt_Mt5 (data not shown).

Mass Spectrometry Analyses

Ldt_Mt5 or N358H/M346W Ldt_Mt5 (2 μm) in 12.5 mm Tris buffer, pH 8 was incubated in the presence or absence of 50 μm carbapenem for 5 h at room temperature. Reactions were quenched by the addition of trifluoroacetic acid (TFA; final concentration, 0.1%). Samples were filtered through a 0.2-μm filter and analyzed by ultraperformance LC/MS at 60 °C. The mobile phase was as follows: 0–1 min, isocratic 90% water + 10% ACN + 0.1% formic acid; 1–7.5 min, gradient to 20% water + 80% ACN + 0.1% formic acid; 7.5–8.4 min, isocratic 20% water + 80% ACN + 0.1% formic acid; 8.4–8.5 min, gradient to 90% water + 10% ACN + 0.1% formic acid; 8.5–10 min, isocratic 90% water + 10% ACN + 0.1% formic acid. The flow rate was 0.3 ml min⁻¹.

Crystallization Conditions

Crystals of Ldt_Mt5 were obtained by the hanging drop vapor diffusion method at 20 °C. Drops of 2 μl of protein (12.8 mg/ml) and 1 μl of reservoir solution were equilibrated against a reservoir containing 85 mm sodium citrate, pH 5.6, 25.5% polyethylene glycol (PEG) 4,000, 170 mm ammonium acetate, and 15% glycerol. Crystals suitable for data collection grew within 1 week. Crystals of Ldt_Mt5 in complex with meropenem were obtained with protein incubated with the ligand (645 μm) using crystallization conditions identical to that of the apo crystal.

Data Collection, Structure Determination, and Refinement

All diffraction data were collected at beamline X6A of the National Synchrotron Light Source of the Brookhaven National Laboratory (Table 2). X-ray diffraction experiments were carried out with crystals frozen in their respective mother liquor without addition of cryoprotectant. The crystal structure of apo-Ldt_Mt5 was determined by molecular replacement with the program MOLREP (16) using previously determined Ldt_Mt2 structures of individual domains as searching models (Protein Data Bank code 3TUR, catalytic domain (CD) and bacterial Ig-like (BIg) B domains; Protein Data Bank code 4HU2, BIgA domain). After 20 cycles of refinement of the three separate domains as rigid bodies with REFMAC (CCP4), the structure was rebuilt with the molecular modeling program Coot (17) and further refined with the program PHENIX using restrained and translation, libration, and screw anisotropic refinement protocols with four translation, libration, and screw groups for each BIg domain and two for the CD (18). The apo-Ldt_Mt5 structure was refined to a final R_work of 0.21 and an R_free of 0.255 with 96.1% of the residues in favored stereochemistry regions (Table 2) and solved to 1.98 Å.

TABLE 2.

Statistics of x-ray data collection and structure refinement

	Apo-LdtMt5	Meropenem-bound LdtMt5
Wavelength (Å)	1.0	1.0
Resolution range (last shell) (Å)	28.92–1.98 (2.051–1.98)	29.04–2.8 (2.9–2.8)
Space group	P622	P622
Unit cell (Å; °)	101.0, 101.0, 192.7; 90, 90, 120	99.4, 99.4, 193.4; 90, 90, 120
Total reflections	3,529,565	1,838,131
Unique reflections	41,056 (3,985)	14,522 (1,400)
Multiplicity	7.2 (7.0)	22.4 (22.9)
Completeness (%)	99.6 (99.15)	99.4 (98.73)
Mean I/σ(I)	57.4 (3.9)	53.3 (6.3)
Wilson B-factor	37.81	61.89
Rsym	0.048 (0.513)	0.09 (0.9)
Reflections used for Rfree	2,046	734
Rwork	0.23 (0.28)	0.23 (0.29)
Rfree	0.255 (0.36)	0.275 (0.36)
Number of non-hydrogen atoms	2,947	2,750
Protein	2,740	2,669
Ligands	39	32
Water	168	49
Protein residues	355	345
r.m.s.	0.006	0.011
Bonds
Angles	1.07	1.43
Ramachandran favored (%)	96	93
Ramachandran outliers (%)	0.85	2.6
Clashscore	3.5	9.45
Average B-factor	80	77.0
Protein	80.1	79.8
Ligands	114.3	76
Solvent	70.9	76

The crystal structure of meropenem-bound Ldt_Mt5 was determined by molecular replacement and refined using a protocol similar to that used when solving the apo-Ldt_Mt5 structure (Table 2). The partial meropenem adduct was built inside the positive σA (mF_o − DF_c) electron density difference map. Weak electron density for the sulfur atom of meropenem was observed, and no electron density for the pyrrolidine ring extension of meropenem was observed. The meropenem adduct structure was refined to an R_work of 0.23 and an R_free of 0.275 with 93% of the residues in Ramachandran favored regions (Table 2). The structure was solved to 2.8 Å. r.m.s.d. calculations and alignments were performed using the program Molecular Operating Environment (MOE; v.2014.09, GCC Inc.). Additional structural figures were completed using PyMOL (v.1.7.1.3, ©2009–2014 Schrödinger, LLC) and Chimera, v.1.9 (19). Buried surface area calculations were performed using the Protein Interfaces, Surfaces, and Assemblies' Service (PISA) at the European Bioinformatics Institute (20). Coordinates and structure factors were deposited in the Protein Data Bank under the codes 4Z7A (apo-Ldt_Mt5) and 4ZFQ (meropenem-bound Ldt_Mt5).

Calorimetric Studies

Freshly thawed Ldt_Mt5 protein was dialyzed overnight in 1 liter of buffer containing 25 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 1 mm TCEP at 4 °C. Dialyzed protein solution was then filtered through a 0.22-μm filter and diluted to 10 μm. The protein concentration was determined by UV absorption at 280 nm using a calculated extinction coefficient of 78,840 m⁻¹ cm⁻¹. Freshly thawed aliquots of 20 mm carbapenem stock solutions were diluted to 1 mm in protein dialysis buffer. Protein and ligand solutions were degassed for 20 min in a ThermaVac®. Ligand injections (10 μl) into the cell containing Ldt_Mt5 were performed with 240-s equilibrations between injections. Data were analyzed with Origin 7 software (OriginLab). All calorimetry experiments were carried out at 27 °C.

M. tuberculosis Strains and Culture Conditions

M. tuberculosis CDC1551 (21) (herein referred to as “wild type”) was used as the host strain to generate a transposon insertion mutant in the MT0501 (ldt_Mt5::Tn) gene as described previously (22). All strains were grown in Middlebrook 7H9 liquid medium supplemented with 0.2% glycerol, 0.05% Tween 80, 10% (v/v) oleic acid/albumin/dextrose/catalase, 50 μg/ml cycloheximide (herein referred to as 7H9 complete medium), and when indicated 2.0 μg/ml crystal violet. The ldt_Mt5::Tn strain was grown in the presence of 20 μg/ml kanamycin. Middlebrook selective 7H11 solid medium (BD Biosciences) was used for enumerating colony forming units (cfus) in in vitro growth studies. M. tuberculosis H37Rv was used in generating meropenem-resistant strains.

In Vitro Growth and Crystal Violet Studies

All M. tuberculosis strains were grown to midexponential phase with an A₆₀₀ of ∼0.8 in 7H9 complete medium at 37 °C. Cultures were then diluted to an A₆₀₀ of 0.1 in 7H9 complete medium in the presence or absence of 2.0 μg/ml crystal violet, and turbidity of the cultures was determined daily. Appropriate dilutions of each strain were cultured on Middlebrook 7H11 medium to determine surviving bacilli by enumerating the cfus.

Osmotic Tolerance Studies

Wild-type or ldt_Mt5::Tn strains were grown to late exponential phase (A₆₀₀ ∼ 2–3) in 7H9 complete medium. Cultures were diluted to an A₆₀₀ of 0.5, and cells were pelleted at low speed. Cells were resuspended in 150 mm NaCl or double distilled H₂O (0 mm NaCl) solutions containing 0.05% Tween 80. Cells were incubated in these conditions for 1 h with shaking at 37 °C. Viability was determined by culturing and enumerating the cfus on 7H11 selective agar.

Determination of Minimum Inhibitory Concentration

Carbapenem minimum inhibitory concentrations were determined using the standard broth dilution method (23). Briefly, 10⁵ M. tuberculosis bacilli were inoculated into 2.5 ml of 7H9 broth supplemented with 0.2% glycerol, 10% (v/v) oleic acid/albumin/dextrose/catalase, and 50 μg/ml cycloheximide, and the drug was added at different concentrations in the μm–mm range. The cultures were incubated at 37 °C without shaking and evaluated for growth by visual inspection of the broth at 14 and 21 days. Minimum inhibitory concentration values are representative of three independent experiments.

Electron Microscopy Experiments

Field emission scanning electron microscopy and transmission electron microscopy experiments were performed as described previously (14).

Results

Ldt_Mt5 Structure

The apo and meropenem adduct structures of an N-terminally truncated Ldt_Mt5 protein lacking the hydrophobic domain predicted to be a membrane anchor for this protein (amino acids 55–451) were determined using x-ray crystallography (Fig. 1). This truncated protein displays higher sequence identity to Ldt_Mt2 (31%) than the full-length protein does (28%) and includes the proline-rich extension of the C-terminal subdomain (ex-CTSD) comprising residues 417–451 that is absent in all other M. tuberculosis ld-transpeptidases (Fig. 2A).

FIGURE 1.

Crystal structure of Ldt_Mt5. Left, apo-Ldt_Mt5; right, rotated 90°. Ldt_Mt5 is composed of two BIg domains and a CD. The semitransparent volume is surface-accessible by a 3.5-Å radius probe. Residues of the active site (His³⁴², Thr³⁵⁷, Asn³⁵⁸, and Cys³⁶⁰) are represented as sticks within the CD. Tryptophan residues of the CTSD are also represented as sticks and interact with a hydrophobic patch at the interface of the BIgB domain. The prominent outer cavity that is observed in Ldt_Mt2 is absent in apo-Ldt_Mt5 but is indicated as a reference (10). The Ldt_Mt5 secondary structure schematic is colored as a rainbow from blue (N terminus) to red (C-terminus). Orange dashes represent the disordered portion of loop L_C, and red dashes represent the disordered ex-CTSD. This figure was made using Chimera (19).

FIGURE 2.

Comparison of M. tuberculosisld-transpeptidases. A, the sequence alignment based on the structural superposition. The observed secondary structures are noted above the amino acid sequences. Starred residues are those highlighted in the text (blue, coordinating His³⁴²; cyan, BIgA and BIgB interface; green, CTSD-BIgB-CD core interaction; red, catalytic residues; yellow, loop L_D). Named loops are also marked, and the characteristic ld-transpeptidase motif is boxed in blue rectangle. B, overlay of the apo structures of Ldt_Mt2 (Protein Data Bank code 3VYN; in cyan) and Ldt_Mt5 (this work; in red). Observed differences in the CD are concentrated in the β-hairpin and loops L_C–L_E. Disordered regions are represented as dashed lines. C, the Ldt_Mt5 adduct structure displays the largest CD conformational changes among characterized ld-transpeptidases. The EYY-folded CD cores of apo-Ldt_Mt5 (red), holo-Ldt_Mt5 (pink), and the apo and holo crystal structures of Ldt_Mt1 (Protein Data Bank code 4JMN, apo, in light green; Protein Data Bank code 4JMX, imipenem adduct, in dark green), and Ldt_Mt2 (Protein Data Bank code 3VYN, apo, in cyan; Protein Data Bank code 3VYP, meropenem adduct, in blue) are shown as smoothed Cα traces. Structural elements with high r.m.s.d. are shown in full opacity; the remaining structural elements are displayed as transparent Cα traces to demonstrate conservation of the EYY-folded CD cores. The largest changes among structurally characterized ld-transpeptidases are observed in the β-hairpin, and in the case of Ldt_Mt5, there is a dramatic displacement of loop L_C that occurs after adduct formation (indicated with red curved arrows). The C-terminal portion of the CTSDs was excluded for clarity. D, accessible surface map of apo-Ldt_Mt5 colored by the magnitude of the observed atomic temperature factors from low (green) to high (magenta) motility. The flexibility of the β-hairpin as indicated by the high atomic temperature factor correlates with its large displacement upon adduct formation. These images, the sequence alignment, and structural superpositions were performed using the program MOE. The sequence representation was performed using ESPript3 (48).

Apo-Ldt_Mt5 and meropenem adduct crystals belong to the P6₂22 space group with similar cell dimensions and one molecule in the asymmetric unit (Table 2). Electron density for residues 56–346 and 353–416 was observed in the apo crystal form. Residues 347–352 within loop L_C (residues 344–362; nomenclature from Ref. 10) and residues 417 to the C-terminus (residue 451) are disordered; their electron density was not observed in either apo- or meropenem adduct-Ldt_Mt5 crystal structures. Ldt_Mt5 possesses three globular domains (Fig. 1) as observed in its paralog Ldt_Mt2 (Fig. 2B) (10, 24–26). The structure reveals an N terminus span of long-chain hydrophilic residues (residues 56–60) that precede two in-tandem antiparallel β-barrels resembling BIg domains joined by a short three-residue linker (BIgA, residues 61–153; BIgB, residues 156–252) followed by a C-terminal CD (residues 253–417). The CD can be divided into a core (residues 253–385) that contains an ErfK/YbiS/YhnG fold (EYY-fold) (27) consisting of a β-sandwich with two mixed β-sheets and one helix (α₂) and a helically folded C-terminal subdomain (CTSD; residues 386–416). ld-Transpeptidases are characterized by the presence of this EYY-folded domain, which contains the characteristic HXX_14–17(S/T)HGChN motif (h represents a hydrophobic residue) where cysteine is the catalytic residue (Fig. 2A). By extension, we presume Cys³⁶⁰ to be the catalytic residue of Ldt_Mt5.

The overall structural overlap between apo-Ldt_Mt5 and Ldt_Mt2 (Protein Data Bank code 3VYN) has an r.m.s. deviation of 2.2 Å for 225 aligned Cα atoms, including 79 identical residues, highlighting their overall structural similarity (Fig. 2, B and C). There are small differences with regard to the orientation of the domains among other structurally characterized ld-transpeptidases. The equivalent BIg domains of Ldt_Mt5 and Ldt_Mt2 are similar; the BIgA domains display a small r.m.s. deviation of 1.0 Å among 65 pairs of Cα atoms aligned, and the BIgB domains display a r.m.s. deviation of 0.94 Å among 103 pairs of aligned Cα atoms. The EYY-folded CD cores of Ldt_Mt1, Ldt_Mt2, and Ldt_Mt5 overlap well (Fig. 2C). However, differences localized to the β-hairpin flap (residues 312–330), loop L_C, and the C-terminal region of loop L_D increased the pairwise r.m.s.d. An r.m.s.d. of 1.25 Å for the 67 Cα atom pairs within the CD core was observed compared with r.m.s.d. values of 2.3 and 2.6 Å for 115 and 103 Cα atom pairs when the entire CD of Ldt_Mt5 was compared with that of Ldt_Mt2 and Ldt_Mt1 (Protein Data Bank code 4JMN), respectively (the Ldt_Mt1/Ldt_Mt2 overlap is 0.66 Å for 67 Cα atom pairs). Interestingly, the imipenem adduct structure of Ldt_Mt1 (Protein Data Bank code 4JMX) minimally affects placement of the β-hairpin in comparison with its position in apo-Ldt_Mt1, whereas the meropenem adduct-Ldt_Mt5 structure displays the largest changes in the β-hairpin (Fig. 2C).

The Conformation of the Ldt_Mt5 BIg Domains Is Mostly Maintained by Unspecified Hydrophobic Interactions

A small, solvent-accessible area (312 Ų) is buried in the interface between the BIg domains in Ldt_Mt5. A short, three-residue linker (Ala¹⁵³, Pro¹⁵⁴, and Val¹⁵⁵) joins BIgA and BIgB. BIgA is rotated ∼30° around the axis passing through the interdomain linker compared with BIgA of Ldt_Mt2 (Fig. 2B). Two hydrophobic patches comprising Pro⁸⁰, Tyr¹²⁵, and Pro¹⁵⁴ of BIgA and Tyr²³⁹ and Tyr²⁴⁸ from BIgB and main-chain atoms of β-barrel loops from both of the domains make an interdomain contact. These proline and tyrosine residues are well conserved among three-domain ld-transpeptidases (Fig. 2A). Tyr¹²⁵ (from BIgA) and Tyr²³⁹ (from BIgB) exchange hydrogen bonds with main-chain atoms of the opposite domain. This greasy and weak contact may provide flexibility in the orientation of the domains. In the Ldt_Mt5 crystal form, a PEG molecule from the crystallization buffer is bound to an exposed hydrophobic patch (Pro¹²² and Tyr²³⁹) at the interface of the BIgA and BIgB domains, apparently stabilizing the observed relative orientations of the domains.

The Conformations of the BIgB Domain and EYY-fold Are Maintained by the CTSD

The L_E loop of the Ldt_Mt5 CTSD is slightly longer in comparison with that of Ldt_Mt2 (Fig. 2, B and C). This loop wedges between the EYY-fold and the BIgB domain (Fig. 1). The Ldt_Mt5 CTSD is rich in tryptophan residues (Trp³⁹⁸, Trp⁴⁰⁰, Trp⁴⁰⁴, and Trp⁴⁰⁷). Extensive hydrophobic contacts among the CTSD, BIgB, and CD domains increase the rigidity of the BIgB/CD assembly. In addition to regular contacts between Tyr³⁹² and Trp³⁹⁸ in loop L_E with Leu²⁰⁹ and Pro²¹⁰ in BIgB, the aromatic rings of Trp⁴⁰⁰, Trp⁴⁰⁴, and Trp⁴⁰⁷ in the α₃ helix of the CTSD form a “zipper-like” interaction with the aromatic ring of Tyr²²⁵ and aliphatic portions of the side chains of Arg²²³ and Arg²²¹ of the BIgB domain. This structure provides 1336 Ų of additional area buried in the BIgB/EYY-fold assembly, which itself only contributes 433 Ų.

The Ldt_Mt5 CD Displays Large Structural Differences Relative to Ldt_Mt1 and Ldt_Mt2

The CD of Ldt_Mt5 displays marked differences in comparison with those of Ldt_Mt1 and Ldt_Mt2. The largest differences are seen 1) within the fold and placement of a β-hairpin flap that includes loop L_F (the shortest among homologs), 2) in the conformation and partial disorder of loop L_C, and 3) in the size of loop L_D and loop L_E (of the CTSD) (Fig. 2). All of these structural differences are in close proximity to the Ldt_Mt5 active site. The β-hairpin flap covers the active site and is the structural feature that distinguishes some ld-transpeptidases (10, 24, 28) from the first structurally characterized protein containing the EYY-fold (27). This flap displays the largest temperature factors relative to the remainder of Ldt_Mt5 (Fig. 2D), is nine residues shorter in Ldt_Mt5, and displays low homology to other M. tuberculosis ld-transpeptidase β-hairpin flaps (Fig. 2C). In Ldt_Mt5, loop L_C displays considerable disorder. Electron density for residues 347–353 in apo-Ldt_Mt5 and residues 348–356 in the meropenem adduct structure was not observed. However, the residues of loop L_C that are ordered display fold differences relative to Ldt_Mt1 and Ldt_Mt2 (Fig. 2C).

In previously solved ld-transpeptidase structures, the catalytic site is exposed through two connected cavities, the outer and inner cavities (Fig. 2, B and C). Compared with Ldt_Mt1 and Ldt_Mt2, the small footprint and placement of this β-hairpin in Ldt_Mt5 lead to greater exposure of the catalytic site from the inner cavity (Fig. 2). The β-hairpin flap and loop L_C of apo-Ldt_Mt5 are shifted toward the outer cavity, closing it (Figs. 2C and 3A). In our meropenem adduct-Ldt_Mt5 structure, the hairpin and loop are partially disordered; however, the ordered portions appear to shift away from the catalytic site, thereby exposing it (Fig. 3B). Thus, the acylation of Ldt_Mt5 by meropenem appears to “create” an outer cavity reminiscent of that observed in Ldt_Mt1 and Ldt_Mt2 (Fig. 3C).

FIGURE 3.

Comparison of the Ldt_Mt5 and Ldt_Mt2 active sites. A, apo-Ldt_Mt5; B, meropenem-bound Ldt_Mt5 (meropenem adduct shown as purple sticks); C, Ldt_Mt2-PG fragment complex (Protein Data Bank code 3TUR; PG fragment shown as cyan sticks). The left panels show secondary structure schematic representations, and residues within the active site are represented as sticks. The right panels show the probe-accessible surface (3.5-Å radius). Surface zones related to the β-hairpin and loop L_C that display the largest structural differences among apo and holo structures are colored purple. Acylation of Ldt_Mt5 by meropenem causes displacements of these structural elements as indicated by the green arrows (right panel) that “restore” the outer cavity.

Loop L_D (residues 289–302) within the CD core of Ldt_Mt5 is larger compared with Loop L_D of Ldt_Mt1 and Ldt_Mt2 (Fig. 2A-C). The Ldt_Mt5 L_D loop has a three-residue insertion that includes a bulky arginine residue (Arg²⁹⁷) and forms a protruding insertion (Fig. 2C). Although most of this loop fold remains unperturbed, the insertion displaces the adjacent L_C loop (residues 338–358), thereby closing the active site outer cavity and dramatically modifying the fold and placement of the L_C loop.

The Ldt_Mt5 Active Site Is Structurally Divergent Relative to Ldt_Mt1 and Ldt_Mt2

The structural differences within the Ldt_Mt5 CD have dramatic effects on the active site architecture and the readiness of catalytic residues to participate in enzymatic reactions and presumably PG stem recognition. In Ldt_Mt1 and Ldt_Mt2, a conserved methionine residue (Met¹⁷⁵ and Met³⁰³, respectively) on the internal side of the β-hairpin flap limits the space around the catalytic cysteine. The unique placement of this loop in Ldt_Mt5 results in displacement of this methionine (Met³¹⁶ in Ldt_Mt5) by the non-conserved Glu³²⁸ (Fig. 3). Glu³²⁸ is substituted with smaller hydrophobic residues in Ldt_Mt2 (Val³²²) and Ldt_Mt1 (Ala¹⁹⁵). Interestingly, the electron density surrounding this glutamate residue indicates that Glu³²⁸ is present in three alternative conformations in the apo-Ldt_Mt5 structure but shows only one conformation in the meropenem adduct structure (Fig. 3, A and B). The most populated conformation of Glu³²⁸ and the conserved motif Asn³⁶² (implicated in PG stem recognition) form hydrogen bonds with His³⁴², thereby orienting His³⁴² in such a way that it is rotated 180° from the orientation required to deprotonate Cys³⁶⁰ (Fig. 4, A and B). Furthermore, Cys³⁶⁰ and His³⁴² make a strong contact with one another; electron density connecting the sulfur atom to the imidazole ring carbon is visible in the experimental electron density map (Fig. 4A). However, no covalent bond is present: the coordination geometry of the Cϵ1–Sγ–Cβ bond angle is much smaller than 109°, which is expected for a direct bond. It is likely that this strong contact and coordination of the histidine ring by Asn³⁶² and Glu³²⁸ make it difficult for the imidazole ring to rotate into a position that is better poised for catalysis (Fig. 4B). Thus, it is clear that His³⁴² is not optimally poised to act as a catalytic base in Ldt_Mt5 as is the equivalent residue in Ldt_Mt2 (His³³⁶) is (Fig. 3C).

FIGURE 4.

An “active” conformation of Ldt_Mt5 is achieved when meropenem binds. A, a nonproductive orientation of His³⁴² is observed in apo-Ldt_Mt5 (σA-weighted electron density map contoured at 1.0 σ level). Residues of the refined structures are shown as stick representations. His³⁴² is in close proximity to Cys³⁶⁰ as suggested by the electron density connecting these two residues. In addition, His³⁴² hydrogen bonds with the most populated alternative conformation of Glu³²⁸ and Asn³⁶² (dashed lines). Collectively, these interactions fix the imidazole ring in a nonproductive orientation that is not present in the meropenem adduct structure. B, mechanism of acylation of Ldt_Mt5 by meropenem. C, overlay of active sites of apo and meropenem adduct structures. The red arrows indicate the large displacements of Met³⁴⁶ (Sδ–Sδ, 6.6 Å), Thr³⁵⁷ (Cβ–Cβ, 8.2 Å), and Asn³⁵⁸ (Cβ–Cβ, 4.1 Å) relative to the apo structure. The active site of apo-Ldt_Mt5 is colored gray, and the meropenem adduct structure is colored brown. The ∼180° rotation of His³⁴² is noted with a curved red arrow. Meropenem is represented as magenta sticks. Disordered residues are represented as dashed lines. This figure was made using Chimera (19).

The L_C loop of Ldt_Mt5 is fully embedded in the conserved HXX_14–17(S/T)HGChN motif that characterizes this family of transpeptidases. Ldt_Mt5 has two variations in the conserved motif: a motif alternative Thr³⁵⁷ of Ldt_Mt5 replaces the Ldt_Mt2 serine (Ser³⁵¹), and Asn³⁵⁸ replaces the characteristic motif histidine (His³⁵² in Ldt_Mt2). The C-terminus of loop L_C forms the “anion hole” at the catalytic site of Ldt_Mt2. Thr³⁵⁷ occludes the outer entrance to the active site (Figs. 3A and 4C) where the PG stem binds to Ldt_Mt2 (Fig. 3C) (10). The loop, which contains the anion hole that comprises a large quantity of positively charged atoms, is folded differently relative to Ldt_Mt2, and Met³⁴⁶ of loop L_C replaces a tryptophan residue that is conserved in all other M. tuberculosis ld-transpeptidases (Fig. 2A).

Ldt_Mt5 Is Acylated by Meropenem during Crystallization

The crystal form grown in the presence of meropenem shows electron density for Ldt_Mt5 residues 56–317, 327–348, and 356–416. Electron density for most of the β-hairpin flap is missing in this crystal form, and like apo-Ldt_Mt5, most of loop L_C and the ex-CTSD are disordered. The carbapenem core of meropenem was fitted in additional electron density near the catalytic cysteine, which forms an adduct with Cys³⁶⁰ (Fig. 3B); however, no electron density for the 3-[5-(dimethylcarbamoyl)pyrrolidin-2-yl] group of meropenem was observed. Interestingly, the presence of this adduct restores the anion hole and other portions of L_C to a similar fold previously observed in other ld-transpeptidases, placing residues with a probable role in catalysis (Met³⁴⁶, Asn³⁵⁸, and Thr³⁵⁷) in positions equivalent to those observed in active ld-transpeptidases (Fig. 4C). Thus, meropenem binding induces a conformational change that enables access to the catalytic site from the outer cavity as observed in other ld-transpeptidases (Fig. 3). In addition, this change promotes release of His³⁴² from its nonproductive contact such that it now hydrogen bonds with Cys³⁶⁰ (distance of Nϵ–Sγ, 3.2 Å; Figs. 3B and 4C).

The most stable tautomer of the carbapenem core is observed where the ring nitrogen is deprotonated (double bond between C3 and N4) and C2 is sp³ hybridized and is in agreement with previously reported Ldt_Mt2-meropenem adduct structures (24, 25). The meropenem core lies with its most apolar side facing a hydrophobic patch formed by Gly³³⁸, the aliphatic portion of the side chain of Glu³³⁹, and Phe³⁴⁰ at the inner cavity. The C-terminal portion of the main chain of loop L_C (Gly³⁵⁹) provides apolar contacts with the other side of the carbapenem core. Four hydrophilic interactions are also observed between the carbapenem core and Ldt_Mt5: 1) Asn³⁵⁸ and 2) the main chain nitrogen atom of Cys³⁶⁰ hydrogen bond to the carbonyl of the opened penem ring, 3) Glu³²⁸ hydrogen bonds to the meropenem hydroxyethyl group, and 4) a water molecule (W⁶⁰¹) mediates interaction between the meropenem core carboxylate and the carboxylate of Glu³³⁹ (Fig. 3B).

We evaluated a series of β-lactams, including the carbapenems listed in Table 3, and measured the thermodynamics of β-lactam binding to Ldt_Mt5 using ITC. Despite the presence of a meropenem adduct on Ldt_Mt5, no significant heat exchange associated with binding was measured by ITC, and no adduct was detected by mass spectrometry after a 5-h incubation of meropenem and Ldt_Mt5 (Table 3).

TABLE 3.

Summary of mass spectrometry data obtained after preincubation of Ldt_Mt5 and different carbapenems

Enzyme	Carbapenem	Calculated mass of inhibited species	Mass of carbapenem	Observed massa
		Da	Da	Da
LdtMt5	None	42,603		42,605
	Meropenem	42,986.5	383.5	42,605
	Imipenem	42,902.4	299.4	42,605
	Doripenem	43,023.1	420.1	42,604
	Biapenem	42,953.4	350.4	42,606
	Tebipenem	42,986.5	383.5	42,605
N358H/M346W LdtMt5	None	42,681		42,683
	Meropenem	43,064.5	383.5	42,684
	Imipenem	42,980.4	299.4	42,683
	Doripenem	43,101.1	420.1	42,683
	Biapenem	43,031.4	350.4	42,683
	Tebipenem	43,064.5	383.5	42,684

^a Reported masses are those that comprise the largest species in the mass spectrum at 100% abundance.

Ldt_Mt5-catalyzed Nitrocefin Hydrolysis Is Optimal at Basic pH

Ldt_Mt5 was probed for transpeptidase/β-lactamase activity using nitrocefin as a substrate. A pH rate profile analysis revealed that Ldt_Mt5 is optimally active at pH >9 (Fig. 5) even after correcting for spontaneous ring opening at basic pH in the absence of enzyme, although its activity was not significantly different from that observed for Ldt_Mt2, which optimally catalyzes nitrocefin hydrolysis at pH 7 (10) (Fig. 5). We also measured nitrocefin binding at pH 8 where little Ldt_Mt5-catalyzed nitrocefin hydrolysis was observed (Fig. 5), but no detectable heat of exchange was observed using ITC (data not shown).

FIGURE 5.

pH rate profile analysis of Ldt_Mt5 (open circles) and Ldt_Mt2 (closed circles). Error bars represent S.E.

Conserved Active Site Residues Are Not Required for Nitrocefin Hydrolysis

On the basis of the apo-Ldt_Mt5 structure, we rationally designed and purified Ldt_Mt5 putative active site variants in an attempt to identify residues responsible for nitrocefin hydrolysis at pH >9. Surprisingly, all of the Ldt_Mt5 variants evaluated, including C360A Ldt_Mt5, hydrolyzed nitrocefin (Table 4 and Fig. 6) with the rates of hydrolysis ordered as follows: N358H > wild type > H342Q > H342A > T357V > N358A > N362A > C360A. Although the rates of hydrolysis were relatively low, conservative mutations (N358H and H342Q) had the lowest impact on k_cat/K_m specificity constants, whereas C360A Ldt_Mt5 was the least active variant we tested. Interestingly, the K_{m(nitrocefin)} for the N358H variant was 2-fold lower than that of wild type, whereas the k_cat values were relatively comparable.

TABLE 4.

Summary of kinetic parameters for nitrocefin hydrolysis by Ldt_Mt5 and Ldt_Mt5 variants at pH 10

The Ldt_Mt5 variants are listed in order of decreasing catalytic efficiency.

Enzyme	Km	kcat	kcat/Km
	μm	min−1	min−1·m−1
LdtMt5 N358H	50 ± 3	0.61 ± 0.03	12.3 ± 0.4 × 103
LdtMt5	97 ± 19	0.79 ± 0.09	8.7 ± 1.9 × 103
LdtMt5 H342Q	151 ± 11	0.63 ± 0.01	4.2 ± 0.2 × 103
LdtMt5 H342A	153 ± 8	0.49 ± 0.01	2.9 ± 0.1 × 103
LdtMt5 T357V	262 ± 72	0.55 ± 0.08	2.4 ± 0.6 × 103
LdtMt5 N358A	125 ± 25	0.32 ± 0.02	2.4 ± 0.3 × 103
LdtMt5 N362A	175 ± 16	0.40 ± 0.01	2.2 ± 0.1 × 103
LdtMt5 C360A	211 ± 40	0.33 ± 0.02	1.1 ± 0.2 × 103

FIGURE 6.

Ldt_Mt5 active site variants catalyze nitrocefin hydrolysis at pH 10. Michaelis-Menten curves for Ldt_Mt5 and Ldt_Mt5 variants are shown. Error bars represent S.E.

Two residues at the ends of loop L_C that interact with the PG stem in the outer cavity of Ldt_Mt2 (10), His³⁵² and Trp³⁴⁰, are substituted with Asn³⁵⁸ and Met³⁴⁶, respectively, in Ldt_Mt5. Asn³⁵⁸ replaces this conserved motif histidine (His³⁵²) in Ldt_Mt2 that participates in recognition of the donor PG stem (10) and in Ldt_Mt5 that participates in recognition of the meropenem adduct (Fig. 3B). Trp³⁴⁰ in Ldt_Mt2 is proposed to provide tetrapeptide substrate specificity (10). In an attempt to make the Ldt_Mt5 active site more Ldt_Mt2-like, we generated the Ldt_Mt5 N358H/M346W double variant. Contrary to our expectations, mass spectrometry data indicate that the double variant was not acylated by the carbapenems tested (Table 3), and the double variant behaved like wild-type Ldt_Mt5 when probed for its ability to catalyze nitrocefin hydrolysis in a pH rate profile analysis (data not shown).

Loss of ldt_Mt5 Modestly Enhances Susceptibility to Doripenem and Faropenem

Recent studies have reported that, in addition to inhibiting dd-transpeptidase and carboxypeptidase activities, carbapenems and penems bind to and inhibit ld-transpeptidases (10, 24, 25, 29–31). We hypothesized that loss of Ldt_Mt5 may alter sensitivity to carbapenems as the mutant lacking this protein would have one less target for carbapenems to inhibit. Minimum inhibitory concentration studies were performed to evaluate whether or not loss of ldt_Mt5 affected the susceptibility of M. tuberculosis to carbapenems (Table 5). The ldt_Mt5::Tn strain reproducibly had modestly enhanced susceptibility to doripenem and faropenem (a penem) compared with wild type, but neither strain was susceptible to ertapenem or meropenem under the conditions that were tested. Both strains displayed similar susceptibilities to tebipenem pivoxil.

TABLE 5.

Susceptibility of M. tuberculosis strains to isoniazid and carbapenems (and faropenem)

Values are reported as μg/ml.

Strain	Isoniazid	Ertapenem	Meropenem	Doripenem	Faropenem	Tebipenem pivoxil
Wild type	0.1	20	12.5	2.5	5	10
ldtMt5::Tn	0.1	20	12.5	1.2	2.5	10

Mutations in the ldt_Mt5 Locus Could Not be Detected in Meropenem-resistant Mutants

We tested the hypothesis that Ldt_Mt5 is a target of meropenem and that meropenem-resistant M. tuberculosis strains would harbor a mutation in the gene encoding this enzyme. Toward this end, we generated genetically resistant strains by isolating mutants grown in the presence of 400 μg/ml meropenem. Nine independent strains were isolated, their genomic DNA was purified, and the ldt_Mt5 loci (which included ∼100 bp in both 5′- and 3′-UTRs) were PCR-amplified and sequenced. In addition, we sequenced the locus of the parent M. tuberculosis H37Rv that was used to generate the mutants. The DNA sequences of the ldt_Mt5 loci in all nine meropenem-resistant strains were identical to the parent M. tuberculosis H37Rv and to the sequence of the reference M. tuberculosis H37Rv genome (data not shown) (32).

Loss of ldt_Mt5 Enhances Sensitivity to Crystal Violet and Osmotic Shock

To determine the effects of loss of functional Ldt_Mt5, we probed the cell wall integrity of wild-type and ldt_Mt5::Tn strains using crystal violet and osmotic shock. In comparison with wild type, ldt_Mt5::Tn M. tuberculosis displayed a minor growth defect when grown in complete medium (Fig. 7A). When complete medium was supplemented with crystal violet, ldt_Mt5::Tn M. tuberculosis behaved similarly to cells lacking the dominant ld-transpeptidase Ldt_Mt2 (ldt_Mt2::Tn) as both strains were more susceptible to killing by the dye (Fig. 7B). Furthermore, ldt_Mt5::Tn cells or ldt_Mt2::Tn cells were 2–3 times less viable than wild-type cells when subjected to osmotic challenge (Fig. 7C). These findings suggest that loss of ldt_Mt5 alters cell wall permeability and sensitivity to crystal violet and compromises cell wall integrity. We also examined the cell morphology of M. tuberculosis lacking ldt_Mt5 by electron microscopy. Interestingly, no observable changes in cell size and morphology between wild-type and ldt_Mt5::Tn strains were observed (Fig. 8). The gene encoding Ldt_Mt5 is in an operon downstream of murB, another PG biosynthetic enzyme. We attempted to complement our ldt_Mt5::Tn strain with a wild-type copy of ldt_Mt5 under the control of its native promoter. We designed and tested eight different complemented strains, but none were able to restore growth phenotypes (data not shown).

FIGURE 7.

Loss of Ldt_Mt5 sensitizes M. tuberculosis to crystal violet and osmotic stress. Wild-type (closed circles), ldt_Mt5::Tn (open circles), or ldt_Mt2::Tn (triangles) M. tuberculosis were grown in 7H9 complete medium (A) or 7H9 medium supplemented with crystal violet (B). C, strains lacking Ldt_Mt2 or Ldt_Mt5 are less tolerant to osmotic shock. Error bars represent S.E.

FIGURE 8.

Transmission (A and C) and scanning (B and D) electron microscopy reveals no significant changes in M. tuberculosis cell morphology upon loss of a functional copy of Ldt_Mt5. A and B, wild-type M. tuberculosis; C and D, ldt_Mt5::Tn M. tuberculosis. Scale bars for transmission EM images (A and C) and scanning EM images (B and C) represent 100 and 200 nm, respectively.

Discussion

Recently, 3→3 cross-links have been identified in the PG of a variety of bacterial species (5, 33–36), and ld-transpeptidases have been identified as the enzymes that catalyze synthesis of this type of transpeptide bond (6–8, 12, 33, 37, 38). Ldt_Mt5 is a paralog of Ldt_Mt2 from M. tuberculosis and has been reported to catalyze formation of 3→3 transpeptide linkages in PG substrates derived from Mycobacterium abscessus (30).

There are significant structural differences within the CD of Ldt_Mt5 and consequently the active site despite overall architectural similarity to Ldt_Mt2. The β-hairpin flap that covers the active site is the smallest among paralogs and exhibits high mobility (high B factors in the apo structure and partial disorder observed in the meropenem-bound structure; Fig. 2D). This hairpin and loop L_C display the largest structural differences among M. tuberculosis ld-transpeptidases (Fig. 2A), and the distinctive structural changes observed upon meropenem adduct formation suggest that their mobility and flexibility could play a role in the catalytic mechanism. The outer cavity that is present in other characterized ld-transpeptidases is absent in apo-Ldt_Mt5; however, such an outer cavity is restored when meropenem acylates Ldt_Mt5. Upon acylation by meropenem, residues from the conserved motif, including His³⁴², Asn³⁵⁸, Cys³⁶⁰, and Asn³⁶², shift to positions resembling those occupied by equivalent residues in Ldt_Mt2 (Figs. 3, B and C, and 4A) and Ldt_Mt1, lining a cavity that could accommodate a PG stem (Fig. 3).

We observed catalytic residues Cys³⁶⁰ and His³⁴² in a nonproductive contact in our apo crystal form, and His³⁴² is not optimally poised for catalysis (Fig. 4A); however, the nonproductive contact appears to be released upon adduct formation (Fig. 4B). We did not observe acylated Ldt_Mt5 by mass spectrometry, likely the result of the presence of this nonproductive contact. However, we clearly observed a meropenem adduct on Ldt_Mt5 in our crystal form. Thus, if given enough time, meropenem will acylate Ldt_Mt5 over the extended incubation period that is required for co-crystallization. Alternatively, a component of the crystallization buffer may promote acylation of Ldt_Mt5 by meropenem.

The pK_a of a cysteine side chain is 8.3. Nitrocefin is a poor substrate for Ldt_Mt5, but we observed Ldt_Mt5-catalyzed nitrocefin hydrolysis at pH >9 (Fig. 5). It is feasible that the nonproductive contact of the catalytic residues may be released at pH ≥9 by weakening of the hydrogen bonds holding the residues in this conformation (Fig. 4A). None of the active site variants we designed fully abolished this activity, including C360A Ldt_Mt5; however,C360A Ldt_Mt5 was the least active variant. Furthermore, the N358H substitution affected nitrocefin recognition (K_m^N358H < K_m^WT). It has been demonstrated previously that substitutions to any of the catalytic residues of serine proteases significantly reduce the rate of peptide bond cleavage but do not completely abolish it (39), indicating that the remaining catalytic site environment after residue substitutions can still promote turnover albeit slowly. It is feasible that, under basic conditions, Cys³⁶⁰ is deprotonated and can hydrolyze nitrocefin and that excess hydroxide in the Ldt_Mt5 active site will still promote turnover of this unnatural substrate even in the absence of the catalytic cysteine. Alternatively, different Ldt_Mt5 residues may mediate nitrocefin hydrolysis.

In addition to catalyzing 3→3 transpeptidation in PG, ld-transpeptidases incorporate non-canonical d-amino acids into PG during stationary growth phase and catalyze attachment of Braun lipoprotein in some Gram-negative bacteria (12, 40). Unlike Ldt_Mt2, Ldt_Mt5 has a 33-residue ex-CTSD (residues 417–451). The ex-CTSD (Fig. 1) is disordered and contains proline-rich stretches (Fig. 2A). Proline-rich regions have been observed in other mycobacterial PG biosynthetic enzymes, including putative dd-transpeptidases PonA1, PonA2, and PonA3 and Ldt_Mt4, another paralog of Ldt_Mt2 (41, 42). Although these proline-rich regions are seemingly common among these M. tuberculosis cell wall biosynthetic enzymes, their role in M. tuberculosis physiology is still largely unknown. Interestingly, proline-rich sequence stretches frequently mediate protein-protein interactions (43). The proline-rich ex-CTSD of Ldt_Mt5 is in close proximity to the catalytic site. Thus, it is plausible that the Ldt_Mt5 ex-CTSD participates in the recognition of protein substrates and/or binding partners, and these interactions may drive the conformational changes required to release His³⁴² and Cys³⁶⁰ from their nonproductive contact. Likewise, it is reasonable to speculate that the active site of Ldt_Mt5 may have evolved to accommodate large substrates like proteins and play a role in anchoring them to the PG reminiscent of the role ld-transpeptidases serve in Gram-negative species in anchoring Braun lipoprotein (12, 40). Taken together, the major structural differences and divergent catalytic site suggest that Ldt_Mt5 and Ldt_Mt2 evolved to serve different functions in M. tuberculosis (Fig. 9).

FIGURE 9.

Loss of Ldt_Mt2 or Ldt_Mt5 compromises M. tuberculosis cell wall integrity. The M. tuberculosis PG is 3→3 and 4→3 cross-linked by ld- and dd-transpeptidases (not shown), respectively. Both Ldt_Mt2 (green) and Ldt_Mt5 (blue) have two BIg domains, whereas Ldt_Mt1 (red) has one, which likely differentially positions the ld-transpeptidases within the periplasm. Although loss of Ldt_Mt1 alone results in no discernible phenotype, loss of Ldt_Mt2 results in compromised cell wall integrity as a result of loss of 3→3 cross-links in PG. Ldt_Mt5 has a structurally distinct active site relative to both Ldt_Mt1 and Ldt_Mt2, and when Ldt_Mt5 is lost, M. tuberculosis is sensitized to chemical probes and osmotic stress similarly to when Ldt_Mt2 is lost. Although Ldt_Mt5 can catalyze 3→3 cross-link formation in vitro, its physiological function remains unclear.

It has been demonstrated that YbiS, an E. coli ld-transpeptidase, is a substrate of the thioreductase DsbG (44). In E. coli, DsbG reduces the catalytic cysteine of YbiS that is prone to sulfenylation in the periplasm. We have previously reported a crystal structure of Ldt_Mt2 that shows Cys³⁵⁴ oxidized to the sulfenic acid (10), suggesting that M. tuberculosis ld-transpeptidases are also susceptible to oxidation. Although we did not observe any sulfur adducts in our apo-Ldt_Mt5 structure, it is conceivable that Ldt_Mt5 requires binding of a protein partner to maintain the correct oxidation state of its catalytic cysteine in vivo.

Although all β-lactam antibiotics target dd-transpeptidases involved in 4→3 cross-link formation in PG maturation, only the carbapenem class of β-lactams (and faropenem, a penem) inhibit ld-transpeptidases. Furthermore, the genome of M. tuberculosis encodes for BlaC, an extended spectrum class A β-lactamase (45, 46). For these historical reasons, β-lactams are seldom considered for treatment of M. tuberculosis infection. However, carbapenems have been recently identified as poor substrates for BlaC (47). We have previously demonstrated that M. tuberculosis lacking Ldt_Mt2 is more susceptible to killing by β-lactams (8, 14). Sanders et al. (42) have reported that LdtC (homologous to Ldt_Mt5 in M. tuberculosis on the basis of sequence) is the primary ld-transpeptidase in Mycobacterium smegmatis. Strains lacking ldtC are hypersusceptible to imipenem, and ldt_Mt5 from M tuberculosis fully complements this phenotype in an ldtC mutant, suggesting that these enzymes are equivalent (42). We observed a modest enhancement in susceptibility of the ldt_Mt5::Tn strain to select carbapenems (Table 5) presumably due to synthetic lethality as these β-lactams may inactivate other targets. Although our meropenem adduct crystal form supported very slow acylation of Ldt_Mt5 over many days, we cannot rule out the possibility that Ldt_Mt5 was more rapidly inactivated by this class of β-lactams in vivo, particularly in the event that Ldt_Mt5 requires a protein-protein interaction for productive catalysis. To date, studies examining acylation of Ldt_Mt5 by carbapenems, including the data presented here, have been in vitro, and interestingly, Ldt_Mt5 is the only Ldt_Mt2 paralog that is not inactivated by carbapenems. The increased susceptibility of ldt_Mt5::Tn strains to osmotic shock and crystal violet coupled with the observed modest enhancement in susceptibility to carbapenems and our meropenem-Ldt_Mt5 crystal form suggest that Ldt_Mt5 is worth pursuing as a drug target.

Author Contributions

L. A. B. B. and G. L. conceived the study, and L. A. B. B., G. L., and M. A. B. designed the study. L. A. B. B. and G. L. designed and performed the biochemical and genetic experiments and analyzed the results. L. A. B. B. prepared all proteins and together with A. G. and M. A. B. performed the crystallization experiments and preliminary crystal quality analysis; J. J. collected the crystallographic data at synchrotron facilities. A. G. and M. A. B. carried out the crystallographic and structural analyses. Y. P. and M. A. B. performed the ITC experiments. L. A. B. B. and G. L. prepared samples for mass spectrometry, and E. P. L. and C. A. T. performed and analyzed the mass spectrometry data. L. A. B. B., G. L., and M. A. B. wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.