Crystal structure of betaine aldehyde dehydrogenase from Burkholderia pseudomallei.

Burkholderia pseudomallei infection causes melioidosis, which is often fatal if untreated. There is a need to develop new and more effective treatments for melioidosis. This study reports apo and cofactor-bound crystal structures of the potential drug target betaine aldehyde dehydrogenase (BADH) from B. pseudomallei. A structural comparison identified similarities to BADH from Pseudomonas aeruginosa which is inhibited by the drug disulfiram. This preliminary analysis could facilitate drug-repurposing studies for B. pseudomallei. open access.

Introduction

Burkholderia pseudomallei is a rod-shaped, motile, flagellated, soil-dwelling Gram-negative proteobacterium of the Burkholderiaceae family that thrives in tropical and sub­tropical regions (Gassiep et al., 2021 ▸). B. pseudomallei causes melioidosis, a deadly emerging opportunistic infection mainly of the immunocompromised (Hall et al., 2019 ▸; Poe et al., 1971 ▸; Veluthat et al., 2021 ▸). B. pseudomallei is transmitted through open wounds, contact with contaminated soil and water, ingestion or inhalation, and it is also a potential biological warfare agent (Goarant et al., 2021 ▸). Melioidosis is endemic in South Asia and Northern Australia, with ∼165 000 cases annually; however, the global distribution of B. pseudomallei is unknown, as the associated disease is underreported and misdiagnosed (Patil et al., 2016 ▸; Poe et al., 1971 ▸; Veluthat et al., 2021 ▸). Typically, about 12 cases of melioidosis are reported annually in mainland USA, and most patients had traveled internationally; however, B. pseudomallei occurs naturally in Puerto Rico and the US Virgin Islands (Hall et al., 2019 ▸). Melioidosis symptoms include localized pain and swelling, ulcer, cough, headache, anorexia, joint pain, brain infection, seizures, fever, pneumonia, low blood pressure and abscesses (Hall et al., 2019 ▸; Poe et al., 1971 ▸; Veluthat et al., 2021 ▸). Thus, melioidosis may be misdiagnosed as tuberculosis, pneumonia or other diseases (Veluthat et al., 2021 ▸). Symptoms may appear a few days or several years after exposure, and the mortality rate of untreated melioidosis is around 90% (Loveleena et al., 2004 ▸; Patil et al., 2016 ▸; Poe et al., 1971 ▸; Veluthat et al., 2021 ▸). Melioidosis is currently treated with two to eight weeks of intravenous antimicrobial therapy (ceftazidime or meropenem) followed by 3–6 months of oral antimicrobial therapy (amoxicillin/clavulanic acid or trimethoprim–sulfamethoxazole), but still results in ∼40% mortality (Fen et al., 2021 ▸). As a part of efforts to develop new therapeutics and diagnostics for melioidosis, the Seattle Structural Genomics Center for Infectious Disease (SSGCID) has determined the crystal structure of betaine aldehyde dehydrogenase (BADH) from B. pseudomallei (BpBADH). BADH catalyzes the irreversible oxidation of betaine aldehyde to the osmoprotectant betaine and is being investigated as a drug target for drug-resistant bacteria, notably Pseudomonas aeruginosa, because its inhibition blocks choline catabolism and leads to the accumulation of the highly toxic betaine aldehyde (González-Segura et al., 2009 ▸).

Materials and methods

Production of BpBADH

Cloning, expression and purification were conducted as part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID; Myler et al., 2009 ▸; Stacy et al., 2011 ▸) following standard protocols as described previously (Bryan et al., 2011 ▸; Choi et al., 2011 ▸; Serbzhinskiy et al., 2015 ▸). The full-length betaine aldehyde dehydrogenase gene from B. pseudomallei (BpBADH; UniProt Q3JLL8) encoding amino acids 1–489 was PCR-amplified from genomic DNA using the primers shown in Table 1 ▸.

Table 1

Macromolecule-production information

Source organism	Burkholderia pseudomallei 1710b
DNA source	Dr Samuel I. Miller, University of Washington, USA
Forward primer	5′-ATGTCCGTATACGGTCTGCAGC-3′
Reverse primer	5′-GAACACCGGTTGATAGCGGCC-3′
Expression vector	pMCSG26
Expression host	E. coli BL21(DE3)R3 Rosetta cells
Complete amino-acid sequence of the construct produced	MSVYGLQRLYIAGAHADATSGKTFDTFDPATGELLARVQQASADDVDRAVASAREGQREWAAMTAMQRSRILRRAVELLRERNDALAELEMRDTGKPIAETRAVDIVTGADVIEYYAGLATAIEGLQVPLRPESFVYTRREPLGVCAGIGAWNYPIQIACWKSAPALAAGNAMIFKPSEVTPLSALKLAEIYTEAGVPAGVFNVVQGDGSVGALLSAHPGIAKVSFTGGVETGKKVMSLAGASSLKEVTMELGGKSPLIVFDDADLDRAADIAVTANFFSAGQVCTNGTRVFVQQAVKDAFVERVLARVARIRVGKPSDSDTNFGPLASAAQLDKVLGYIDSGKAEGAKLLAGGARLVNDHFASGQYVAPTVFGDCRDDMRIVREEIFGPVMSILSFETEDEAIARANATDYGLAAGVVTENLSRAHRAIHRLEAGICWINTWGESPAEMPVGGYKQSGVGRENGITTLEHYTRIKSVQVELGRYQPVFGHHHHHH

The gene was cloned into the ligation-independent cloning (LIC; Aslanidis & de Jong, 1990 ▸) expression vector pMCSG26 (Eschenfeldt et al., 2010 ▸) encoding a noncleavable C-terminal 6×His fusion tag (ORF-GHHHHHH). Plasmid DNA was transformed into chemically competent Escherichia coli BL21(DE3)R3 Rosetta cells. The plasmid containing Q3JLL8 was expression-tested, and 2 l of culture was grown using auto-induction medium (Studier, 2005 ▸) in a LEX Bioreactor (Epiphyte Three Inc.) as described previously (Serbzhinskiy et al., 2015 ▸). The expression clone BupsA.00020.b.AE1.GE43326 is available at https://www.ssgcid.org/available-materials/expression-clones/.

BpBADH-His was purified using a two-step protocol consisting of an immobilized metal-affinity chromatography (IMAC) step and size-exclusion chromatography (SEC). All chromatography runs were performed on an ÄKTApurifier 10 (GE) using automated IMAC and SEC programs according to previously described procedures (Bryan et al., 2011 ▸). Thawed bacterial pellets were lysed by sonication in 200 ml lysis buffer {25 mM 4-(2-hydroxyethyl)-1-piperazineethane­sulfonic acid (HEPES) pH 7.0, 500 mM NaCl, 5% glycerol, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 30 mM imidazole, 10 mM MgCl2, 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 250 µg ml−1 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 0.025% sodium azide}. After sonication, the crude lysate was clarified with 20 µl (25 units µl−1) Benzonase and incubated while mixing at room temperature for 45 min. The lysate was clarified by centrifugation at 10 000 rev min−1 for 1 h using a Sorvall centrifuge (Thermo Scientific). For the IMAC step, the clarified supernatant was passed over an Ni–NTA HisTrap FF 5 ml column (GE Healthcare) which had been pre-equilibrated with loading buffer (25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 30 mM imidazole, 1 mM TCEP, 0.025% sodium azide). The column was washed with 20 column volumes (CV) of loading buffer and was eluted with loading buffer and 250 mM imidazole in a linear gradient over 7 CV. Peak fractions, as determined by UV absorbance at 280 nm, were pooled and concentrated using an Amicon concentrator to a volume of 5 ml for SEC. For SEC, a SEC column (Superdex 75, GE) was equilibrated with running buffer [25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 2 mM dithiothreitol (DTT), 0.025% sodium azide]. The eluted peak fractions were collected and analyzed for the presence of BpBADH by SDS–PAGE. The SEC peak fractions containing BpBADH eluted as a single large peak at a molecular mass of ∼77 kDa. A dimer of BpBADH is expected to have a molecular mass of ∼106 kDa, while a monomer has a molecular mass of ∼53 kDa. BpBADH may be a monomer in the absence of a cofactor, while it dimerizes in the presence of the cofactor or other ligands. Further biophysical analysis is required to determine whether BpBADH dimerizes in the presence of NAD in solution. Interestingly, the dimer has been reported in more than 175 reported BADH structures with ligands, cofactors and inhibitors in the PDB and is consistent with the observed crystal form of BpBADH with NAD (Fig. 1 ▸).

Figure 1

Structure of B. pseudomallei betaine aldehyde dehydrogenase (BpBADH). (a) Monomer of apo BpBADH (rainbow colored from blue at the N-terminus to red at the C-terminus. (b) Dimer of BpBADH with NAD (monomers are shown as aquamarine and cyan ribbons, with NAD shown as sticks).

The peak fractions were pooled and concentrated to 34.72 mg ml−1 using an Amicon concentrator (Millipore). The protein concentration was assessed using the OD280 and a molar extinction coefficient of 46 870 M −1 cm−1. Purified protein was aliquoted into 200 µl aliquots, flash-frozen in liquid nitrogen and stored at −80°C until use for crystallization. The purified protein (batch BupsA.00020.b.AE1.PS38619) is available at https://www.ssgcid.org/available-materials/ssgcid-proteins/.

Crystallization

Purified BpBADH-His was screened for crystallization in 96-well sitting-drop plates against the JCSG++ HTS (Jena Bioscience), MCSG1 (Molecular Dimensions) and Morpheus (Rigaku Reagents; Gorrec, 2009 ▸, 2015 ▸) crystal screens. The protein solution for the apo structure did not contain NAD, whereas 4 mM NAD was added to the protein solution for the NAD-bound complex (Table 2 ▸). Equal volumes of protein solution (0.4 µl) and precipitant solution were set up at 287 K against reservoir (80 µl) in sitting-drop vapor-diffusion format. The final crystallization precipitant was JCSG+ condition F7 for the apo form and Morpheus condition H11 for the NAD-bound form (see Table 2 ▸). After cryoprotectant exchange into crystallization solution supplemented with 20% ethylene glycol, the crystals were harvested and flash-cooled by plunging them into liquid nitrogen.

Table 2

Crystallization

Method	Sitting-drop vapor diffusion
Plate type	96-well Compact 300, Rigaku
Temperature (K)	287
Protein concentration (mg ml−1)	34.72
Buffer composition of protein solution
Apo crystals	25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 2 mM DTT, 0.025% azide
NAD-bound crystals	25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 2 mM DTT, 0.025% azide, 4 mM NAD
Composition of reservoir solution
Apo structure	JCSG+ condition F7: 0.8 M succinate pH 7.0
NAD-bound structure	Morpheus condition H11: 10% PEG 4000, 20% glycerol, 0.02 M sodium L-glutamate, 0.02 M DL-alanine, 0.02 M glycine, 0.02 M DL-lysine, 0.02 M DL-serine, 0.1 M bicine/Trizma pH 8.5
Volume and ratio of drop	0.4 µl protein plus 0.4 µl reservoir
Volume of reservoir (µl)	80
Cryoprotectant	20% ethylene glycol

Data collection and processing

Data were collected at 100 K on beamline 21-ID-F at the Advanced Photon Source (APS), Argonne National Laboratory (see Table 3 ▸). Data sets were reduced with XSCALE (Kabsch, 2010 ▸). Raw X-ray diffraction images are available from the Integrated Resource for Reproducibility in Macromolecular Crystallography at https://www.proteindiffraction.org/.

Table 3

Data collection and processing

Values in parentheses are for the outer shell.

PDB code	6wsa	6wsb
Ligand	—	NAD
Diffraction source	21-ID-F, APS	21-ID-F, APS
Wavelength (Å)	0.97872	0.97872
Temperature (K)	100	100
Detector	RayoniX MX300HE CCD	RayoniX MX300HE CCD
Crystal-to-detector distance (mm)	270	200
Rotation range per image (°)	1	1
Total rotation range (°)	60	150
Space group	P6222	P21212
a, b, c (Å)	107.86, 107.86, 233.53	99.27, 156.70, 76.23
α, β, γ (°)	90, 90, 120	90, 90, 90
Mosaicity (°)	0.143	0.103
Resolution range (Å)	49.51–2.05 (2.10–2.05)	43.09–1.55 (1.59–1.55)
Total No. of reflections	362438 (26952)	1054995 (75766)
No. of unique reflections	51154 (3702)	172302 (12622)
Completeness (%)	99.9 (99.9)	99.9 (100.0)
Multiplicity	7.09 (7.28)	6.12 (6.00)
〈I/σ(I)〉	12.86 (3.50)	16.47 (3.02)
R r.i.m.	0.101 (0.548)	0.083 (0.621)
Overall B factor from Wilson plot (Å2)	32.748	20.398

Structure solution and refinement

The structures were solved by molecular replacement with Phaser (McCoy et al., 2007 ▸) from the CCP4 suite of programs (Collaborative Computational Project, Number 4, 1994 ▸; Krissinel et al., 2004 ▸; Winn et al., 2011 ▸) using PDB entry 2wox (Díaz-Sánchez et al., 2011 ▸) as the search model. The structure was refined using iterative cycles of Phenix (Liebschner et al., 2019 ▸) followed by manual rebuilding of the structure using Coot (Emsley & Cowtan, 2004 ▸; Emsley et al., 2010 ▸). The quality of both structures was checked using MolProbity (Chen et al., 2010 ▸). All data-reduction and refinement statistics are shown in Table 4 ▸. The structures of apo BpBADH and BpBADH with NAD were refined to resolutions of 2.05 and 1.55 Å, respectively. Structural figures were analyzed and prepared using PyMOL (DeLano, 2002 ▸). Multiple sequence alignments were generated with Clustal Omega (Li et al., 2015 ▸). Coordinates and structure factors have been deposited in the Protein Data Bank (https://www.rcsb.org/; Berman et al., 2000 ▸) with accession numbers 6wsa and 6wsb for apo BpBADH and BpBADH in complex with NAD, respectively.

Table 4

Structure solution and refinement

Values in parentheses are for the outer shell.

PDB code	6wsa	6wsb
Ligand	Glycerol	NAD
Resolution range (Å)	49.51–2.05 (2.08–2.05)	43.09–1.55 (1.57–1.55)
Completeness (%)	96.1	96.4
σ Cutoff	F > 0.000σ(F)	F > 0.000σ(F)
No. of reflections, working set	49171 (1957)	166209 (4765)
No. of reflections, test set	2932 (109)	10050 (297)
Final R cryst	0.140 (0.2203)	0.144 (0.2064)
Final R free	0.173 (0.2521)	0.169 (0.2342)
Cruickshank DPI	0.183	0.070
No. of non-H atoms
Protein	3666	7322
Ion	1	—
Ligand	60	100
Solvent	501	1388
Total	4228	8810
R.m.s. deviations
Bond lengths (Å)	0.006	0.006
Angles (°)	0.775	0.85
Average B factors (Å2)
Protein	33.0	15.3
Ion	32.1	—
Ligand	58.9	31.7
Water	42.6	30.8
Ramachandran plot
Most favored (%)	97.1	97.1
Allowed (%)	2.9	2.9

Results and discussion

The two structures reported here are of apo BpBADH and its complex with the cofactor NAD (Fig. 1 ▸). The monomers are very similar and have an r.m.s.d. of ∼0.17 Å for main-chain atoms. The 489 amino acids in each monomer fold as 20.4% β-strand, 39.3% α-helix, 2.5% 310-helix and 37.8% loops, forming six β-α-β motifs that contain five β-sheets (four mixed and one antiparallel). The structure also contains 21 helices, 21 strands, four β-hairpins, four β-bulges and 25 helix–helix interactions. BpBADH has a prototypical BADH topology and shares considerable structure and sequence similarity with the ortholog from P. aeruginosa (PaBADH; Fig. 2 ▸). The 489-amino-acid sequence of PaBADH folds as 19.6% β-strand, 38.2% α-helix, 2.5% 310-helix and 39.7% loops (Fig. 2 ▸).

Figure 2

Structural and primary-sequence alignment of BpBADH and PaBADH. The secondary-structure elements shown are α-helices (α), 310-helices (η), β-­strands (β) and β-turns (TT). Identical residues are shown in white on a red background and conserved residues are shown in red.

The structural similarities and motifs associated with the BADHs from both organisms may accelerate drug-discovery efforts. PaBADH is known to be inhibited by disulfiram through the catalytic cysteine (Velasco-García et al., 2006 ▸); thus, we hypothesize that BpBADH will likewise be inhibited by disulfiram. Disulfiram binds irreversibly to Cys286 in PaBADH, which is in the highly conserved cofactor-binding cavity of PaBADH and BpBADH; the corresponding residue is Cys285 in BpBADH (Figs. 2 ▸ and 3 ▸). Disulfiram is an irreversible inhibitor that leads to a buildup of betaine aldehyde, which becomes toxic in bacterial cells. The toxicity in the bacterial cells stops bacterial growth (Velasco-García et al., 2006 ▸). Since disulfiram is FDA-approved for treating chronic alcoholism, preliminary studies suggest that it could be repurposed as a lead compound for melioidosis. Furthermore, due to the structural similarity between PaBADH and BpBADH, the lessons learned in drug discovery for the former could accelerate efforts in addressing melioidosis.

Figure 3

LIGPLOT analysis reveals that the cofactor-binding domains of BpBADH (PDB entry 6wsb) and PaBADH (PDB entry 4caz) are well conserved (circles indicate identical residues). Both structures show the conserved catalytic cysteine irreversibly inhibited by disulfiram.

Conclusion

The high-resolution structures of betaine aldehyde dehydro­genase from B. pseudomallei (BpBADH) and P. aeruginosa (PaBADH) reveal a conserved NAD-dependent topology and structural similarity. Since the key amino-acid residues in inhibitor-binding sites are conserved, the previous studies on PaBADH could facilitate the development of small-molecule inhibitors of BpBADH.

PDB reference: betaine aldehyde dehydrogenase, 6wsa

PDB reference: bound to cofactor, 6wsb