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Crystal structure of α-acetolactate decarboxylase from Bacillus subtilis subspecies spizizenii
Korean J. Microbiol 2019;55(1):9-16
Published online March 31, 2019
© 2019 The Microbiological Society of Korea.

Jiyoung Eom1,†, Han Byeol Oh1,†, and Sung-il Yoon1,2,*

1Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon 24341, Republic of Korea
2Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 24341, Republic of Korea
Correspondence to: E-mail: sungil@kangwon.ac.kr;
Tel.: +82-33-250-8385; Fax: +82-33-259-5643
Received January 7, 2019; Accepted January 29, 2019.
Abstract

Acetoin is generated by numerous microorganisms using α- acetolactate decarboxylase (ALDC) to prevent overacidification of cells and their environment and to store remaining energy. Because acetoin has been used as a safe flavor enhancer in food products, industries have been interested in biotechnological production of acetoin using ALDC. ALDC is a metal-dependent enzyme that produces acetoin from α-acetolactate through decarboxylation reaction. Here, we report the crystal structure of ALDC from Bacillus subtilis subspecies spizizenii (bssALDC) at 1.7 Å resolution. bssALDC folds into a two-domain α/β structure where two β-sheets form a central core. bssALDC assembles into a dimer through central hydrophobic interactions and peripheral hydrophilic interactions. bssALDC coordinates a zinc ion using three histidine residues and three water molecules. Based on comparative analyses of ALDC structures and sequences, we propose that the active site of bssALDC includes the zinc ion and its neighboring bssALDC residues.

Keywords : Bacillus subtilis, acetoin, α-acetolactate decarboxylase, structure, zinc
Body

Acetoin (3-hydroxy-2-butanone) is an extracellular metabolite produced by diverse bacterial species, including Bacillus subtilis, Klebsiella pneumoniae, and Enterobacter cloacae, when they grow on glucose or other fermentable carbon sources (Levine, 1916; Xiao and Xu, 2007). Acetoin is generated by two catalytic steps from pyruvate. Two pyruvate molecules are first condensed into α-acetolactate through α-acetohydroxy acid synthase or α- acetolactate synthase, and then the resultant α-acetolactate is decarboxylated into acetoin by α-acetolactate decarboxylase (ALDC) (Halpern and Umbarger, 1959; Loken and Stormer, 1970). By converting acidic pyruvate into neutral acetoin, bacteria prevent overacidification of the cytoplasm and the extracellular environment (Tsau et al., 1992). Furthermore, acetoin can be reused as an energy source when glucose is exhausted (Grundy et al., 1993). Because acetoin is interconverted with 2,3-butanediol through NAD/NADH-dependent 2,3-butanediol dehydrogenase, acetoin has been regarded as a regulator of the cellular NAD/NADH ratio (Xiao and Xu, 2007).

Because acetoin is a safe, nontoxic substance with a pleasant yogurt creamy odor and a fatty butter taste, acetoin has been used to enhance the flavor of food products and the fragrance of cosmetics (Xiao and Lu, 2014). Moreover, acetoin is a precursor or an intermediate for the synthesis of a wide range of valuable chemicals, such as alkyl pyrazines, diacetyl, and acetylbutanediol (Xiao et al., 2009). Therefore, food and cosmetic industries have been interested in the development of acetoin production methods. Acetoin is commercially produced through traditional chemical processes. Recently, biotechnological production of acetoin using natural or engineered microorganisms has been reported (Bae et al., 2016; Shen et al., 2016; Bursac et al., 2017; Forster et al., 2017). Additionally, a cell-free acetoin biosynthesis method that employs thermostable acetolactate synthase and ALDC was developed (Jia et al., 2017).

ALDC is a metalloenzyme that catalyzes the decarboxylation of α-acetolactate into acetoin. Despite the significance of ALDC in bacterial survival and biotechnological applications, only three ALDC proteins from Brevibacillus brevis (bbALDC), B. subtilis strain 168 (bs168ALDC), and Klebsiella pneumoniae (kpALDC) have been structurally defined to date (Marlow et al., 2013; Ji et al., 2018; Wu et al., 2019). All the ALDC structures have been shown to accommodate a zinc ion using histidine residues, and thus zinc ion was proposed to play a key role in acetolactate decarboxylation. To further characterize the ADLC structure, we produced ALDC from B. subtilis subspecies spizizenii (bssALDC) using an Escherichia coli expression system and determined the crystal structure of bssALDC in space group (P21212), which differs from that of bs168ALDC (P31) and thus provides structural information in different crystal contacts. Our comparative structural analysis provides structural insights into the dimeric assembly and active site of ALDC.

Materials and Methods

Construction of protein expression vector

The DNA fragment corresponding to the bssALDC-encoding region was PCR amplified with DNA primers, containing either BamHI or SalI restriction enzyme recognition site, using the genomic DNA of B. subtilis subspecies spizizenii as a DNA template. The PCR product was digested by the BamHI and SalI restriction enzymes and ligated into a modified pET49b vector (pET49bm) that was designed to express bssALDC protein in N-terminal fusion with a hexa-histidine tag and a thrombin cleavage site (Park et al., 2017). The resulting bssALDC-pET49bm plasmid was transformed into E. coli DH5α strain. The nucleotide sequence of the bssALDC-encoding region in the bssALDC-pET49bm plasmid was verified by DNA sequencing.

Protein expression and purification

For bssALDC expression, the bssALDC-pET49bm plasmid was transformed into E. coli BL21 (DE3) strain. The resultant transformant cells were grown in LB medium at 37°C. When the optical density at 600 nm reached 0.6, isopropyl-β-D-1- thiogalactopyranoside was added into the culture to a final concentration of 1 mM to induce the overexpression of bssALDC protein. The cells were further grown for 3 h at 37°C. The culture was centrifuged, and the cell pellet was resuspended and sonicated in 50 mM Tris, pH 8.0, 200 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride.

The cell lysate was cleared by centrifugation and incubated with Ni-NTA resin in 50 mM Tris, pH 8.0, 200 mM NaCl, and 10 mM imidazole. The resulting resin was packed in an Econo- column and washed using 50 mM Tris, pH 8.0, 200 mM NaCl, and 10 mM imidazole. bssALDC protein was eluted from the resin using 50 mM Tris, pH 8.0, 200 mM NaCl, and 250 mM imidazole. The initially purified bssALDC protein was dialyzed against 20 mM Hepes, pH 7.4, and 150 mM NaCl and then digested using thrombin at 18°C for 3 h to remove the N- terminal hexa-histidine tag. The tag-free bssALDC protein was further purified by anion exchange chromatography using a Mono Q 10/100 column with a 0–0.5 M NaCl gradient. The purity and integrity of bssALDC protein were analyzed by SDS-PAGE in each purification step.

Protein crystallization

For crystallization, the purified bssALDC protein was concentrated to 21.9 mg/ml using a centrifugal filter. bssALDC was crystallized at 18°C by a sitting-drop vapor-diffusion method in a solution containing 18% PEG 4000, 0.08 M ammonium sulfate, 0.1 M sodium acetate, pH 4.5, and 12% glycerol.

X-ray diffraction of crystals

X-ray diffraction of a bssALDC crystal was carried out at Pohang Accelerator Laboratory beamline 7A. For X-ray diffraction, a bssALDC crystal was transferred to a solution containing 21% PEG 4000, 0.08 M ammonium sulfate, 0.1 M sodium acetate, pH 4.5, and 25% glycerol and flash-cooled at -173°C under a cryo-stream. X-ray diffraction data were collected using a detector distance of 200 mm with a rotation angle of 0.5° and 0.5 second exposure time for each frame. The diffraction data were indexed, integrated, merged, and scaled using the HKL2000 program (Table 1) (Otwinowski and Minor, 1997).

Crystallographic statistics of the bssALDC structure

bssALDC
Data collection
 Space group P21212
 Cell parameters a = 87.28 Å
b = 129.64 Å


c = 45.72 Å
 Wavelength (Å) 1.00004
 Resolution (Å) 30.00-1.70
 Highest resolution (Å) 1.73-1.70
 No. unique reflections 58,087
 Rmerge (%)a 7.0 (38.9)b
 I/sigma (I) 44.1 (7.2)b
 Completeness (%) 99.8 (100.0)b
 Redundancy 7.0 (6.9)b
Refinement
 Resolution (Å) 30.00-1.70
 No. of reflections (work) 54,766
 No. of reflections (test) 2,922
 Rwork (%)c 17.1
 Rfree (%)d 19.1
 No. atoms 3,988
  Protein 3,652
  Ligands (zinc) 2
  Water 334
 Average B-value (Å2) 21.6
 RMSD bonds (Å) 0.017
 RMSD angles (°) 1.58
 Ramachandrane (favored) 97.6%

         (outliers)
0.0%

Rmerge = ΣhklΣi | Ii(hkl) - <I(hkl)> | / ΣhklΣi Ii(hkl)

Numbers in parenthesis were calculated from data of the highest resolution shell.

Rwork = Σ| |Fobs|-|Fcalc| | / Σ|Fobs|, where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively.

Rfree = as for Rwork, but for 5% of the total reflections chosen at random and omitted from refinement.

Calculated using MolProbity (http://molprobity.biochem.duke.edu).



Structure determination

The crystal structure of bssALDC was determined by molecular replacement with the Phaser program using the structure of bbALDC (PDB ID 4BT4) as a search model. The final model of bssALDC was obtained through iterative cycles of rebuilding and refinement. Model building and refinement were performed using the Coot and Refmac5 programs, respectively (Emsley and Cowtan, 2004; Murshudov et al., 1997).

Gel-filtration chromatography

The oligomeric state of bssALDC was analyzed by gel- filtration chromatography. 100 μg of bssALDC protein in 300 μl of 20 mM Hepes, pH 7.4, and 150 mM NaCl was loaded onto a Superdex 200 10/300 column, and a mobile phase containing 20 mM Hepes, pH 7.4, and 150 mM NaCl was continuously flowed through the column. Protein elution was monitored by measuring absorbance at 280 nm.

Accession number

The atomic coordinates and the structure factors for bssALDC (PDB ID 6J3D) have been deposited in the Protein Data Bank (www.rcsb.org).

Results and Discussion

Overall structure of bssALDC

bssALDC protein was recombinantly overexpressed in E. coli cells and purified to homogeneity by Ni-NTA affinity chromatography and anion exchange chromatography (Fig. 1). bssALDC protein was crystallized in space group P21212 in a PEG 4000 solution at pH 4.5. The bssALDC crystal diffracted X-ray to 1.7 Å resolution. The bssALDC structure was determined by molecular replacement and refined to 1.7 Å resolution with an Rfree value of 19.1% (Table 1). The asymmetric unit of the bssALDC crystal contains two bssALDC molecules (chains A and B). Because the two bssALDC chains exhibit essentially identical structures with a root-mean-square-deviation (RMSD) value of 0.25 Å for 232 Cα atoms, the structure of chain A will be described as the prototype of the bssALDC structure unless otherwise specified.

Fig. 1.

SDS-PAGE analysis of purified bssALDC protein.



bssALDC folds into a zinc-containing α/β structure that consists of two domains, namely, N and C (Figs. 2 and 3). In the middle of the bssALDC structure, each domain presents a wide β-sheet. The β-sheet of domain N (N-β-sheet) is composed of seven β-strands in an order of the β15, β1, β8, β3, β4, β5, and β6 strands, and one side of the N-β-sheet is covered by an α-helix (α2) and two short β-strand (β2 and β7). The β-sheet of domain C (C-β-sheet) is constituted by five β-strands in an order of the β9, β14, β11, β12, and β13 strands. The β14, β11, and β12 strands in the middle of the C-β-sheet are elongated, making a slanted circle, and occludes one side of the C-β-sheet along with the β10 strand and the α3 helix. The two domains are fastened by hydrophobic interactions between N-β-sheet and C-β-sheet in the middle of the bssALDC structure and further joined by four α-helices (α1, α4, α5, and α6).

Fig. 2.

Amino acid sequence alignment of bssALDC and its orthologs (bs168ALDC, bbALDC, and kpALDC). bssALDC residues near a zinc ion are colored in red. The bssALDC sequence that differs from the bs168ALDC sequence is colored in blue. The dimerization interface residues of bssALDC are highlighted in bold. The secondary structure elements of bssALDC are shown above the sequence of bssALDC.


Fig. 3.

Overall structure of bssALDC monomer. The monomeric structure of bssALDC is shown in rainbow ribbons (N-terminus, blue; C-terminus, red). A zinc ion and its coordinating histidine residues are shown as a black sphere and magenta sticks, respectively.



bssALDC dimerization

Two bssALDC chains in the asymmetric unit form a dimer with a buried surface area of ∼1040 Å2 on each chain (Fig. 4A). The dimeric organization of bssALDC is also observed in its orthologs, bs168ALDC, bbALDC, and kpALDC, suggesting that ALDC forms a homodimer irrespective of bacterial species (Fig. 4B) (Marlow et al., 2013; Ji et al., 2018; Wu et al., 2019). To be consistent with the dimeric assembly in the crystal, bssALDC also adopts a dimer in solution. In gel-filtration chromatography, bssALDC was eluted in a single peak between 44 kDa and 158 kDa protein standards with an apparent molecular size of 61 kDa, indicating that bssALDC forms a dimer in solution (calculated molecular weight of bssALDC, 29.3 kDa) (Fig. 4C).

Fig. 4.

bssALDC dimer and its dimerization interface. (A) The dimeric structure of bssALDC. The chains A and B of a bssALDC dimer are shown as green and cyan ribbons, respectively. Zinc ions and their coordinating histidine residues are shown as black spheres and magenta sticks, respectively. (B) Structural overlays of bssALDC (magenta), bs168ALDC (PDB ID 5XNE; yellow), and bbALDC (PDB ID 4BT2; light blue) dimers. (C) Gel-filtration chromatography analysis of bssALDC using a Superdex 200 10/300 column. The elution of each gel-filtration standard is indicated by a vertical line.



bssALDC dimerizes primarily using residues from the α3-β11 loop, the β12-β13 loop, the β15 strand, and the α5 helix (Fig. 4A). The dimerization interface residues of bssALDC are highly conserved in the orthologous sequences of ALDC (Fig. 5A). The central region of the dimerization interface of bssALDC is highly hydrophobic and is surrounded by hydrophilic residues (Fig. 5B). The extensive hydrophobic surface of the dimerization interface suggests that the monomeric form of bssALDC is unstable in solution and that the structural stability of bssALDC is achieved when bssALDC forms a dimer.

Fig. 5.

Dimerization interface of bssALDC and its sequence conservation. (A) High sequence conservation of the dimerization interface residues of bssALDC. One chain of the bssALDC dimer is shown as surfaces, and its sequence conservation is color coded (high sequence conservation, magenta; low sequence conservation, white). The other chain is represented by cyan lines. Sequence conservation was calculated using the ConSurf server (Ashkenazy et al., 2016). (B) Dimerization interface of bssALDC. The hydrophobic (T, P, L, F, Y, I, M, and A) and hydrophilic (Q, S, N, Q, and R) residues of one bssALDC chain in the dimerization interface are shown as yellow and magenta surfaces, respectively. The dimerization interface residues of the other bssALDC chain are represented by sticks (carbon, cyan; nitrogen, blue; oxygen, red; sulfur, orange).



Active site of bssALDC

The bssALDC structure presents a zinc ion between two domains in the center (Figs. 3, 6A and B). The zinc ion is coordinated by three histidine residues (H191, H193, and H204) from the β13 and β14 strands and is additionally liganded by three water molecules (Fig. 6B). Zinc coordination by histidine residues is a conserved structural feature of ALDC orthologs because zinc ion is liganded commonly by three histidine residues (H191, H193, and H204 in bs168ALDC; H194, H196, and H207 in bbALDC; H198, H200, and H211 in kpALDC) in the bs168ALDC, bbALDC, and kpALDC structures. Notably, the three histidine residues are absolutely conserved in the sequences of ALDC orthologs (Fig. 6C). However, zinc coordination by three water molecules is limited to B. subtilis ALDC. In the bssALDC and bs168ALDC structures, zinc ion is indirectly liganded by the W1, W2, and W3 water molecules, which are secured by the E62, R142, and E251 residues through hydrogen bonds (Fig. 6B and D). However, in the kpALDC structure, kpALDC E69 that corresponds to bssALDC E62 is closely located to zinc ion and directly coordinates zinc ion by displacing the W1 and W2 molecules of the bssALDC structure (Wu et al., 2019). Although W3 is observed in the kpALDC structure, kpALDC E258 that is equivalent to the W3-stabilizing E251 residue of bssALDC is disordered. Moreover, in the bbALDC structure, an oxygen atom of Glu253 displaces the W3 molecule of the bssALDC structure and directly coordinates zinc ion unlike the indirect water-mediated coordination of bssALDC (Marlow et al., 2013). To be consistent, the bssALDC E251 residue is found in only 33% of ALDC orthologs and is missing in 64% of ALDC orthologs although the bssALDC E62 and R142 residues exhibit 100% and 99% sequence identities (Fig. 6C).

Fig. 6.

Zinc binding of bssALDC. (A) High sequence conservation of the zinc-binding residues of bssALDC. The bssALDC structure is shown as surfaces, and its sequence conservation is color coded (high sequence conservation, magenta; low sequence conservation, white). A zinc ion and its neighboring bssALDC residues and water molecules are included in a box. Sequence conservation was calculated using the ConSurf server (Ashkenazy et al., 2016). (B) Zinc ion (gray sphere) and its neighboring bssALDC residues (sticks) and water molecules (red spheres) in the bssALDC structure. (C) Sequence conservation of zinc-surrounding bssALDC residues. The sequence identity was determined using 149 ALDC ortholog sequences in reference of the bssALDC sequence. (D) Zinc ion (gray sphere) and its neighboring bs168ALDC residues (sticks) and water molecules (red spheres) in the bs168ALDC structure (PDB ID 5XNE).



The zinc-neighboring site was previously proposed as an active site of ALDC based on structural analysis of bbALDC in complex with transition state analogues or substrate mimics (Marlow et al., 2013). To be consistent, the ALDC residues that constitute the proposed active site are highly conserved in ADLC orthologs (Fig. 6A). Interestingly, in the structure of bbALDC in complex with a substrate mimic, ethane-1,2-diol, the oxygen atoms of ethane-1,2-diol are located in replace of the W1 and W2 molecules of the bssALDC structure, suggesting that substrate would directly interact with zinc ion and then be modified to product (Marlow et al., 2013). However, in the ethane-1,2-diol-bound structure of kpALDC, ethane-1,2-diol binds ∼5Å away from zinc ion (Wu et al., 2019). In both unbound and ethane-1,2-diol-bound structures of kpALDC, the two terminal oxygen atoms of kpALDC E69 that are positionally equivalent to the W1 and W2 molecules of the bssALDC structure do not change their conformations and coordinate zinc ion. These observations suggest that the substrate mimic does not directly interact with zinc ion and does not structurally modify kpALDC. Therefore, to reveal the exact catalytic mechanism of ALDC, further structural studies on ALDC in complex with substrate or product are required.

Similar structural features of bssALDC and bs168ALDC

bssALDC differs from bs168ALDC at four residues, including L87R, E112D, I120M, K124R changes (Fig. 2). Moreover, bssALDC and bs168ALDC were crystallized in different space groups (P21212 and P31, respectively), indicating that bssALDC and bs168ALDC make different crystal contacts (Table 1). Despite these differences, the two structures exhibit highly similar structures, with an RMSD value of 0.35 Å for 233 Cα atoms, and present essentially identical active site, suggesting that bssALDC and bs168ALDC adopt similar structures even in solution irrespective of environment and drive catalytic reaction using an identical enzymatic mechanism (Figs. 4B, 6B and D) (Ji et al., 2018). Taken together, our comparative structural study of bssALDC and bs168ALDC reveals the conserved structural features of B. subtilis ALDC.

적 요

다양한 미생물은 세포와 주변의 과산화를 방지하고 여분의 에너지를 보관하기 위해 α-acetolactate decarboxylase(ALDC)를 이용해 아세토인을 생성한다. 아세토인은 안전한 식품 향미 개선제이기 때문에 ALDC를 이용한 아세토인 생합성에 많은 산업체가 관심을 가지고 있다. ALDC는 α-acetolactate의 탈카르복실화 반응을 통해 아세토인을 생산하는 금속 의존 효소이다. 본 논문에서는 고초균 아종 spizizenii의 ALDC (bssALDC) 결정구조를 1.7 Å 해상도에서 보고한다. bssALDC는 두 개의 β-sheet가 중앙부를 형성하는 α/β 구조를 가진다. bssALDC는 중앙부의 소수성 상호작용과 주변부의 친수성 상호작용을 통해 이합체를 형성한다. bssALDC는 세 개의 histidine 잔기와 세 개의 물 분자를 이용해 아연 이온에 배위결합한다. 구조와 서열의 비교 분석에 기초하여 아연 이온과 이 주변부 bssALDC 잔기들이 bssALDC의 효소 활성부위임을 제안한다.

Acknowledgements

This research was supported by a grant from the National Research Foundation of Korea (2016R1D1A1B03930540 to SIY) and by 2016 Research Grant from Kangwon National University (520160456 to SIY). X-ray diffraction was carried out at the Pohang Accelerator Laboratory at beamline 7A. There is no conflict of interest to declare.

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