Structural Insight into the Substrate Gating Mechanism by <i>Staphylococcus aureus</i> Aldehyde Dehydrogenase

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Oct 2020Structural Insight into the Substrate Gating Mechanism by Staphylococcus aureus Aldehyde Dehydrogenase Xuan Tao†, Zhemin Zhang†, Xiao Zhang, Hongyan Li, Hongzhe Sun, Zong-Wan Mao and Wei Xia Xuan Tao† MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou, 510275 , Zhemin Zhang† MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou, 510275 , Xiao Zhang MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou, 510275 , Hongyan Li Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong. , Hongzhe Sun *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong. , Zong-Wan Mao MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou, 510275 and Wei Xia *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou, 510275 https://doi.org/10.31635/ccschem.020.202000219 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Staphylococcus aureus produces staphyloxanthin, a C30 carotenoid with golden color, as an antioxidant to promote bacterial resistance to reactive oxygen species. The biosynthesis pathway of staphyloxanthin involves a series of catalytic enzymes. Aldehyde dehydrogenase (AldH) is a dehydrogenase recently identified to convert 4,4'-diaponeurosporen-aldehyde into 4,4'-diaponeurosporenoic acid during staphyloxanthin biosynthesis. Here, we present the crystallographic structures of apo- and holo-forms of S. aureus AldH. The dimeric enzyme contains a unique C-terminal helix, which resembles a "gatekeeper" helix found in human membrane-bound fatty aldehyde dehydrogenase (FALDH). Particularly, the helix adopts "open" and "closed" conformations in apo- and holo-AldH, respectively, to control the access of the substrate tunnel. Mutagenesis in combination with in vitro and in vivo activity assays identifies several residues essential for S. aureus AldH substrate recognition and enzyme catalytic turnover. Our results provide insights into substrate recognition of S. aureus AldH toward polyunsaturated long-chain aldehydes at atomic resolution. Download figure Download PowerPoint Introduction Staphylococcus aureus is a widespread human pathogen that can cause a series of infectious disorders ranging from relatively benign to life-threatening diseases.1 Due to the abuse of antibiotics, the emergence of drug-resistant S. aureus, such as methicillin-resistant (MRSA) and vancomycin-resistant S. aureus strains (VRSA), have become serious global public health threats.2 Meanwhile, there has been a lack of productivity in the discovery of new antibiotics, creating an urgent need for the development of novel anti-infective therapies. Via neutralization of virulence factors to render pathogen bacteria susceptible to human immune system clearance, antivirulence therapy has become an alternative approach to combat bacterial infections.3,4 Such a strategy poses less selective pressure, which may reduce the emergence of drug resistance.5 More than 90% of S. aureus clinical isolates generate a golden pigment staphyloxanthin, a C30 carotenoid virulence factor.6 Previous studies have demonstrated that staphyloxanthin functions as an antioxidant to protect the bacteria from oxidative stress.7 Bacteria that lack the pigment can grow normally but are susceptible to human neutrophil killing.8 A recent study demonstrates that staphyloxanthin also condenses in the functional membrane micro-domain of S. aureus to maintain bacterial membrane integrity.9,10 Given the essential role of staphyloxanthin, blocking its biosynthetic pathway is a potential antivirulence strategy to combat S. aureus infection.11–13 The genes involved in staphyloxanthin biosynthesis were initially identified to be in a crtOPQMN operon, which can encode five enzymes to catalyze the stepwise conversion of two farnesyl diphosphate molecules into staphyloxanthin.14,15 Recently, the sixth pathway enzyme, S. aureus 4,4'-diaponeurosporen-aldehyde dehydrogenase (SaAldH), was identified to catalyze the oxidation reaction of 4,4'-diaponeurosporen-4-al into 4,4'-diaponeurosporenoic acid. When SaAldH is coexpressed with CrtOPQMN in an Escherichia coli host, staphloxanthin- and staphyloxanthin-like compounds can be produced (Figure 1).16 Figure 1 | Staphyloxanthin biosynthetic pathway in S. aureus and reconstructed biosynthetic pathway in E. coli. Structures of carotenoids produced in recombinant E. coli host, but not in S. aureus, are shown in the dashed box. Download figure Download PowerPoint SaAldH belongs to the superfamily of aldehyde dehydrogenases (ALDHs), which are homo-oligomeric enzymes catalyzing the NAD(P)+-dependent oxidation of aldehydes to carboxylic acids.17 ALDHs process a diverse array of substrates, ranging from small acetaldehyde to long fatty aldehyde.18–20 Therefore, ALDH family enzymes usually have substrate binding pockets with different sizes and shapes to accommodate their specific substrates. For example, sheep liver ALDH1 substrate tunnel is more open than its counterpart in ALDH2. Therefore, ALDH1 prefers larger, bulkier retinal as a substrate, whereas ALDH2 prefers acetaldehyde.18,21,22 Notably, the substrate of SaAldH, 4,4'-diaponeurosporen-4-al, is an extremely long polyunsaturated aldehyde (C30). Therefore, a key unresolved question in staphyloxanthin biosynthesis is how SaAldH recognizes 4,4'-diaponeurosporen-4-al and achieves substrate specificity. Herein, we report the crystal structures of apo- and holo-forms of SaAldH. Unexpectedly, the bacterial ALDH family protein contains a unique C-terminal α-helix, which resembles a membrane-bound human fatty aldehyde dehydrogenase (FALDH). Particularly, the C-helix adopts different conformations in apo- and holo-SaAldH to control the substrate tunnel access. In vitro enzymatic assay, H2O2 sensitivity, and macrophage killing assays reveal key residues essential for SaAldH catalytic turnover. Our studies provide the first structural basis for the substrate recognition and catalysis of SaAldH in the staphyloxanthin biosynthetic pathway. Experimental Methods Detailed experimental methods are available in the Supporting Information. Results and Discussion Apo-SaAldH forms a dimer with a unique C-terminal helix SaAldH cDNA was cloned into a pET47b vector and expressed as a His6-fusion protein in E. coli( Supporting Information Table S1). To obtain a stable protein sample, the dehydrogenase active site C244 of SaAldH was mutated to serine. The fusion protein was purified by a Ni affinity column and subsequently digested by PreScission protease to remove the His6-fusion. SaAldHC244S was eluted from Tricorn Superdex 200 column at 13.3 mL with a calculated molecular weight of 100 kDa, corresponding to a dimeric form (calculated molecular weight of monomeric SaAldH is 51.7 kDa). Purified apo-form SaAldHC244S was assayed for crystallization and yielded three-dimensional crystals for X-ray diffraction experiments (see Supporting Information Table S2). The crystals of apo-SaAldHC244S belong to the P22121 space group and diffract to a resolution of 1.8 Å. Initial phases were obtained by molecular replacement using human FALDH (PDBID: 4QGK) as a search model. The final structure model contains only one chain of SaAldH in one asymmetric unit and was refined to a Rwork of 21.3% and Rfree of 23.9%. Apo-SaAldH structure adopts the typical aldehyde dehydrogenase fold with a NAD(P)+-binding domain (residues 2–84 and 106–211), a catalytic domain (residues 212–424), and a bridging domain connecting the two subunits of the dimer (residues 85–105 and 425–446) (Figure 2a).23 Clear but discontinuous electron density is found at the NAD(P)+-binding domain, which is likely due to the low occupancy of the NAD(P)+ cofactor. In the SaAldH structure, the NAD(P)+-binding site consists of residues I111, F114, N115, E141, T143, V169, T173, I177, G188, V191, I195, V196, F234, and F338. Sequence alignments show that each residue is similar among the ALDH homologues, indicative of a highly conserved NAD(P)+-binding site in the ALDH superfamily ( Supporting Information Figure S1). Figure 2 | Structure of SaAldH. (a) SaAldH is a symmetrical homodimer with a large dimerization interface. One subunit is shown as a cartoon model, the catalytic domain (cyan residues 212–424), NAD(P)+-binding domain (wheat residues 2–84 and 106–211), bridging domain (green residues 85–105 and 425–446), and C-terminal helix (magenta residues 447–458) are shown. The other subunit is shown as a surface model in gray. The substrate tunnel is shown in yellow. (b) The C-terminal helix is stabilized by both polar and hydrophobic interactions. The T-shaped π–π stacking interactions among residues F456, F457, and Y92, hydrogen bonding between K449 and residues 440–445, and the salt bridge between K454 and E41 from adjacent subunits (E41*) are shown in the stick model. All residues visible in electron density are shown. 2Fo–Fc density at 1σ is shown as mesh. Download figure Download PowerPoint Opposite the NAD(P)+-binding site is the substrate binding tunnel, which is built up by residues A61, T62, I66, Y116, Q119, T243, S244, V245, M395, L397, A398, and F404 from one subunit and Y92, L93, H442, I453, F456, and F457 from the adjacent subunit of the dimer. Intriguingly, distinct from the highly conserved NAD(P)+-binding site, the residues built up from the substrate tunnel are highly variable in different ALDH homologues ( Supporting Information Figure S1), which is probably attributable to different substrate specificity. Unexpectedly, the C-terminal residues 449–459 form a short α-helix and are located near the substrate tunnel entrance, which resembles a "gatekeeper" helix identified in the human membrane-associated FALDH.20 The helix is stabilized by both polar and hydrophobic interactions, such as a hydrogen-bonded network between the K449 side chain and backbone atoms in a loop region consisting of residues 440–445 and a salt bridge between K454 and E41 from the adjacent subunit. Particularly, side chains of F456 and F457 point to the substrate tunnel and form T-shaped π–π stacking interactions with Y92 (Figure 2b). Previous studies have demonstrated that the FALDH C-helix induces a 93° kink in the direction of the substrate cavity.20 Intriguingly, although SaAldH C-helix also lies close to the access of the substrate tunnel, no such kink was observed for the SaAldH substrate cavity as calculated using the MOLE toolkit ( Supporting Information Figure S2).24 Structure superimposition of SaAldH and FALDH reveals that the two structures are quite similar with a backbone r.m.s.d value of 0.811 Å. However, the orientations of the C-helices are different. SaAldH C-helix forms an 18.2° angle with the corresponding C-helix in FALDH (Figure 3a). Moreover, side chains of W450, F454, and L438 in FALDH C-helix point to the entry and block the access to the cavity, which prevents the direct entry of the substrate. In contrast, the SaAldH C-helix does not block the entry, so the substrate can enter the tunnel (Figures 3b and 3c). Figure 3 | The C-terminal helices of human FALDH and SaALDH adopt different conformations. (a) Structure superimposition between human FALDH (orange) and apo-SaAldH (marine). The two C-helices adopt different orientations. (b and c) Cross-section through the active sites of FALDH and apo-SaAldH. The substrate tunnels are characterized using the MOLE toolkit for PyMOL. In FALDH, the C-helix (highlighted in dashed circle) creates a kink in the tunnel (shown in magenta). While the linear arrangement of the substrate tunnel of apo-SaAldH (shown in yellow) is not restricted by the C-helix. Download figure Download PowerPoint The substrate tunnel in holo-SaAldH adopts a "closed" conformation To understand, in detail, how the substrate is bound in SaAldH, we obtained the crystal structure of substrate-bound (holo) SaAldH. In a previous study, it was reported that reconstitution of crtM, crtN, and crtP genes in E. coli led to the accumulation of SaAldH substrate 4,4'-diaponeuros-poren-4-al.16 We envision that coexpression of CrtM, CrtN, CrtP, and an inactive SaAldHC244S mutant can result in a substrate-bound state of SaAldH. As expected, the purified SaAldHC244S mutant coexpressed with CrtM/N/P gave a reddish color. Mass spectrometric analysis of a lipid extract of the purified SaAldHC244S sample indicated the presence of 4,4'-diapoly-copen-4-al, a fully unsaturated analogue of 4,4'-diaponeuros-poren-4-al (Figure 1 and Supporting Information Figure S3). The substrate analogue is usually found in E. coli artificial synthetic systems as described previously.16 Holo-SaAldH crystals with reddish-brown color were grown by a vapor diffusion method, and mass spectrometric analysis confirmed the presence of 4,4'-diapolycopen-4-al substrate in the protein crystals ( Supporting Information Figure S3). The crystals belong to the space group I422 and are diffracted to a resolution of 2.6 Å. Analysis of the holo-SaAldH structure reveals only a triangular-shaped electron density peak at the catalytic site with an approximate distance of 2.7 Å away from the catalytic residue C244 (mutated to S244). Unexpectedly, no electron density was observed in the substrate tunnel, although mass spectra data confirmed the presence of substrate, which is probably attributed to the long and potentially flexible chain of the substrate. Intriguingly, a significant orientation change of the C-helix was observed in holo-SaAldH by superimposing the apo- and holo-SaAldH structures. Holo-SaAldH C-helix has a 10.6° swing-in toward the tunnel entrance compared to the apo-form (Figure 4a). This C-helix swing-in results in substantial movement of F456 and F457 side chains, which insert into the substrate tunnel. Notably, no electron density was observed for Y92 side chain in holo-SaAldH. All these data suggest the disruption of the T-shaped π–π stacking interactions among F456, F456, and Y92 found in apo-SaAldH. Furthermore, the residues V441 and H442 in a preceding loop also move toward the substrate tunnel in holo-SaAldH (Figure 4b). It is worth noting that the swing-in C-helix in holo-SaAldH blocks the entrance of the substrate tunnel (Figures 4c and 4d). Moreover, this conformational change also induces a 52.6° kink in the direction of the substrate tunnel ( Supporting Information Figure S2). This configuration suggests that this unique C-helix might function as an important "gate" for the substrate tunnel. In the apo-state, the C-helix adopts an "open" conformation and allows enzyme substrate entry. Once the specific substrate obtains access into the substrate tunnel, the C-helix undergoes a conformational change to block the tunnel entrance and lock the substrate, exhibiting a "closed" conformation. Figure 4 | The substrate tunnels in S. aureus apo- and holo-AldH adopt "open" and "closed" conformations, respectively. (a) Structural superimposition of S. aureus apo- (green) and holo-AldH (magenta). The "swing-in" movement of C-helix is shown in the cartoon model. (b) The side chains of residues Y92, V441, H442, F456, and F457 are shown as stick models in apo- (green) and holo-AldH (magenta). (c and d) The "open" and "closed" conformations of the S. aureus AldH substrate tunnel. The structures are shown in the surface model. The C-helices in apo- and holo-AldH are highlighted in cyan and magenta, respectively. The catalytic residue C244 (mutated to S244) is colored in red. Download figure Download PowerPoint A substrate-bound structure model of SaAldH The "open" to "closed" conformational change of SaAldH C-helix is likely attributed to substrate binding. To further elucidate the substrate binding pattern, the binding of 4,4'-diapolycopen-4-al to holo-SaAldH was modeled using the molecular docking program, Autodock4.25 The grid box was set to cover the substrate tunnel calculated by the MOLE toolkit. The calculated structure model with the lowest energy was selected to represent the substrate–SaAldH complex ( Supporting Information). As shown in Figure 5a, the 4,4'-diapolycopen-4-al molecule can fit well into the substrate tunnel. The aldehyde group is close to the catalytic residue C244 (mutated to S244) with a distance of 3.7 Å. The kink of the substrate tunnel induces a distortion of the substrate molecule 4,4'-diapolycopen-4-al. The substrate forms intensive hydrophobic interactions with the residues located in the substrate tunnel, including A61, I66, Y116, P90, A398, P400, I453, F456, and F457 from the adjacent subunit (Figure 5a). In particular, Y116 forms a π–σ interaction with the 2-methyl group, while the F456 at the C-helix forms a parallel-displaced π–π stacking with F457 and hydrophobic interactions with the two terminal methyl groups of the substrate. This structural model indicates that the C-helix is likely to participate in substrate recognition during enzymatic catalysis. Figure 5 | Structure model of S. aureus holo-AldH binding to the substrate. (a) Cross-section through the substrate tunnel of holo-AldH (cyan). The C-helix is highlighted in green. The 4,4'-diapolycopen-4-al substrate (yellow) and the residue side chains lining the substrate tunnel are shown in the stick model. The distance between the aldehyde group and the catalytic residue (S244) is 3.7 Å. (b) Analysis of the interactions between 4,4'-diapolycopen-4-al and AldH residues in the substrate tunnel. The residues involved in hydrophobic interactions are colored in green. The σ–π interaction between the substrate methyl group and Y116 side chain is colored in cyan. The interaction between the substrate aldehyde group and catalytic residue (S244) side chain is colored in purple. Download figure Download PowerPoint Critical residues for SaAldH enzymatic activity The structures of apo- and holo-forms of SaAldH demonstrate that the unique C-helix might exert an important role in controlling the access of the substrate tunnel and recognizing a specific substrate. Several residues were identified as conformation stabilizers of the C-helix. For example, the K449 residue is engaged in a hydrogen bond network with the backbone oxygen atoms of V441, L443, and P445 in apo-SaAldH. Mutagenesis of the corresponding residue in human FALDH (K447 in FALDH) reduced the enzyme catalytic capacity for long-chain fatty aldehyde.20 The F456, F457, and Y92 form T-shaped π–π stacking to stabilize the C-terminus of the helix in apo-SaAldH. Furthermore, the F456 and F457 are also engaged in substrate recognition in the holo-SaAldH structure model. We envision that conformational disturbance of the C-helix interferes with SaAldH enzymatic activity. To test our hypothesis, we mutated residues K449, Y92, F456, and F457 to alanine and compared their activities using either benzaldehyde or 4,4'-diapolycopen-4-al as substrates. When benzaldehyde was used as a substrate, the SaAldHK449E mutant showed similar enzymatic activity compared to that of wild-type (WT) SaAldH. The SaAldHY92A and the double-mutant SaAldHF456A/F457A retain approximately 80% activity, as compared to WT-SaAldH, indicating that these mutations do not significantly compromise SaAldH catalytic capacity for benzaldehyde (Figure 6a and Supporting Information Table S3). Remarkably, however, when the long-chain 4,4'-diapolycopen-4-al was used as a substrate, the mutants SaAldHK449E and SaAldHY92A have no detectable enzyme activities. Single mutations of F456A and F457A resulted in substantially lower enzyme activities with 48% and 66% WT-SaAldH activity being retained, respectively, while the double-mutant SaAldHF456A/F457A completely lost the catalytic capacity for the 4,4'-diapolycopen-4-al substrate (Figure 6b). Consistent with the enzymatic assay data in vitro, coexpression of these SaAldH mutants with CrtM, CrtN, and CrtP in E. coli also resulted in significant accumulation of 4,4'-diapolycopen-4-al, which could not be further metabolized ( Supporting Information Figure S4). This indicates that the residues stabilizing the C-helix conformation are not directly involved in enzyme catalysis but are essential for recognition and efficient metabolism of 4,4'-diapolycopen-4-al substrate. Figure 6 | The C-helix residues are important for S. aureus AldH activity. (a) Comparison of the catalytic capacity (kcat/Km) of wide-type (WT) S. aureus AldH and different mutants to metabolize benzaldehyde. The K449E and Y92A mutants have the same catalytic capacities as WT-AldH, while the F456A/F457A double-mutant only causes 15% decrease in catalytic efficiency. (b) Comparison of the dehydrogenase activity of WT-AldH and mutants toward 4,4'-diapolycopen-4-al. F456 and F457 single mutants lead to 50% and 40% decreases on catalytic efficiency, respectively. K449E, Y92A, and F456A/F457 mutants have no detectable activities. Resistance capability comparison of S. aureus Newman and different mutants against H2O2 (c) and macrophage phagocytosis (d) aldH gene knockout significantly attenuates the resistance capability of S. aureus against H2O2 and macrophage phagocytosis. Complementation of aldH Y92A and F456A mutant genes cannot restore the capability. Each experiment was performed in triplicates. The data are shown in mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n. s. not significant. Download figure Download PowerPoint It has been reported that staphyloxanthin functions as an antioxidant to protect S. aureus against exogenous reactive oxidative as well as the staphyloxanthin biosynthesis pathway can lead to of S. aureus to oxidative or Therefore, we further these SaAldH mutations S. aureus to oxidative and macrophage aldH mutant of S. aureus Newman aureus was using the recently reported and S. aureus mutant was with or mutated aldH genes using the as described (see Supporting When with H2O2 for the of S. aureus mutant was only of that for S. aureus, that SaAldH is essential for S. aureus to H2O2 oxidative (Figure Complementation of genes can substantially to In contrast, aldH genes with Y92A or F456A mutants were to the of the Consistent with H2O2 macrophage killing experiments also demonstrate that Y92A and F456A mutants significantly decrease S. aureus during macrophage indicating the essential role of staphyloxanthin to protect S. aureus from human (Figure The structures of S. aureus AldH reveal a novel C-terminal α-helix in a bacterial aldehyde which access to the substrate cavity and a role in recognizing a specific polyunsaturated aldehyde substrate. SaAldH is a bacterial aldehyde demonstrates that SaAldH has a with human membrane-bound In contrast, it only has and with two other ALDH homologues in S. aureus Newman strains and ( Supporting Information Figure S1). SaAldH contains a hydrophobic C-helix as identified in human FALDH, whereas no such C-terminal is found in two other S. aureus ALDH homologues, indicating that this C-helix is unique for staphyloxanthin biosynthesis. of SaAldH that conformation significantly the catalytic capacity of SaAldH toward 4,4'-diapolycopen-4-al, but not that the C-helix is important for long-chain aldehyde substrate our structural model of the holo-SaAldH complex with 4,4'-diapolycopen-4-al reveals intensive hydrophobic between the substrate and residues on C-helix. Moreover, our structural analysis demonstrates that the C-helix can adopt two distinct an "open" conformation in apo-SaAldH and a "closed" conformation in holo-SaAldH. we not the substrate electron density in the holo-SaAldH structure, the is that the long-chain aldehyde can enter the "open" substrate cavity of apo-SaAldH. the hydrophobic interactions between the substrate and C-helix cause the "swing-in" movement of the which the entry of the substrate tunnel and prevents the of the substrate enzymatic catalysis. Previous studies have demonstrated that the of the ALDH is with the Therefore, the of the from SaAldH enzymatic catalysis might cause a conformational change at the which to from the substrate tunnel. It is worth noting that of the lining the substrate tunnel are hydrophobic ( Supporting Information Figure S1). Therefore, the side chains of these may participate in substrate by a hydrophobic cavity to accommodate the 4,4'-diapolycopen-4-al substrate, which contains a hydrophobic Furthermore, analysis of the substrate tunnels indicates that the of SaAldH substrate tunnel is Å. Therefore, it could a substrate than human FALDH, which has a substrate tunnel of Å ( Supporting Information Figure S2). these factors are likely to to specific substrate recognition by SaAldH. Previous studies of 3 ALDHs suggest that the C-helix may be a distinct of membrane-associated ALDH FALDH has an domain, while human is to membrane C-terminal or In both the enzyme substrate tunnel and C-helices are toward the lipid to their toward fatty aldehyde resulted from oxidative SaAldH contains a similar it is SaAldH is a membrane-associated Intriguingly, the crystal structure of S. aureus AldH with K449, and on the residues and a surface potential ( Supporting Information Figure Previous studies have demonstrated that the final biosynthetic staphyloxanthin is in the micro-domain of the S. aureus membrane to maintain bacterial membrane Intriguingly, the two enzymes in the staphyloxanthin biosynthetic and were identified to be membrane-associated It is to that this surface might SaAldH to with the bacterial membrane so that the hydrophobic from SaAldH can be to the biosynthetic enzymes. 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