Inhibition of D - glycero -β- D - manno -heptose 1-phosphate adenylyltransferase from Burkholderia pseudomallei by epigallocatechin gallate and myricetin

Flavonoids play beneficial roles in various human diseases. In this study, a flavonoid library was employed to probe inhibitors of D - glycero -β- D - manno -heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei ( Bp HldC) and two flavonoids, epigallocatechin gallate (EGCG) and myricetin, have been discovered. Bp HldC is one of the essential enzymes in the ADP‐ L ‐ glycero ‐β‐ D ‐ manno ‐heptose biosynthesis pathway constructing lipopolysaccharide of B. pseudomallei . Enzyme kinetics study showed that two flavonoids work through different mechanisms to block the catalytic activity of Bp HldC. Among them, a docking study of EGCG was performed and the binding mode could explain its competitive inhibitory mode for both ATP and βG1P. Analyses with EGCG homologues could reveal the important functional moieties, too. This study is the first example of uncovering the inhibitory activity of flavonoids against the ADP‐ L ‐ glycero ‐β‐ D ‐ manno ‐ heptose biosynthesis pathway and especially targeting HldC. Since there are no therapeutic agents and vaccines available against melioidosis, EGCG and myricetin can be used as templates to develop antibiotics over B. pseudomallei. and 18.32 µM with myricetin. The values indicate a potent inhibitory activity of the two flavonoids against Bp HldC.


Introduction
Flavonoids are polyphenols that perform various biological activities such as antiinflammation, anti-oxidation, anti-cancer and cardiovascular protective action [1]. They are synthesized from phenylalanine and their scaffolds can be categorized into more than seven subclasses [2]. Many kinds of catalytic enzymes such as hydrolases, oxidoreductases, phosphatases, DNA synthases and RNA polymerases have been known to be inhibited by various flavonoids. They also attract the interests of people due to the increasing evidence of the versatile health benefits as dietary foods [3]. Flavonoids interact well with proteins due to their dual properties of hydrophilicity and hydrophobicity. Therefore, many X-ray crystal structures of flavonoids complexed with enzymes such as ecto-5'-nucleotidase (PDB ID: 4H2B), dihydroflavonol 4-reductase (3C1T) and UDP-glucose flavonoid 3-O glycosyl have been reported. Because of the binding characteristics, flavonoids and their derivatives have been developed as new drug candidates targeting various kinds of illnesses [4].
In drug discovery, flavonoids drew the attention to be utilized as lead compounds to develop new bactericidal drugs. The antibacterial activity of flavonoids may be occurred by three different mechanisms [5]; inhibition of nucleic acid synthesis, inhibition of energy metabolism and cytoplasmic membrane damage. Combined with available antibiotics, flavonoids work synergistically to cope with bacterial infections [6] Burkholderia pseudomallei is a pathogenic bacteria with relatively high mortality causing melioidosis that affects people, especially in the tropics. [7][8][9]. Death rates are comparable to measles, higher than leptospiration and dengue fever [10]. From pneumonia and sepsis to skin abscesses [11], various symptoms of melioidosis make their clinical diagnosis difficult.
Nevertheless, no vaccines are commercially available to prevent melioidosis infection, despite intensive research efforts [12][13][14]. As Gram-negative bacteria, Burkholderia spp. construct a condensed network of surface-exposed polysaccharides including capsular Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200677/900588/bcj-2020-0677.pdf by guest on 24 December 2020 polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS). They are known as the main virulence factors and their biosynthesis pathways play important roles in the pathogenesis and immunomodulation of melioidosis [15][16][17]. The LPS consists of lipid A, core and O-antigen domains in most Gram-negative bacteria [18]. The core oligosaccharide is divided into an outer part and an inner part. The inner part is composed of L-glycero-Dmanno-heptose (or heptose) and 3-deoxy-D-manno-octulosonic acid. Heptose is a greatly conserved component of the LPS core among some genera of bacteria. The precursors of heptose were synthesized through the ADP-L-glycero-β-D-manno-heptose biosynthesis pathway [19]. This pathway is well conserved in Gram-negative bacteria species [20]. Dglycero-β-D-manno-heptose-1-phosphate adenylyltransferase (HldC) involved in this pathway is one component of HldE from Escherichia coli, a bifunctional protein including both kinase (HldA) and HldC domains [21]. The deletion mutant of the hldE gene exhibited increased sensitivity to some antibiotics like erythromycin, telithromycin, novobicin, rifampicin, etc [22]. It implies that the inhibition of the ADP-L-glycero-β-D-manno-heptose biosynthesis pathway is a potential target to develop antibiotic adjuvants. Therefore, inhibitors targeting HldC from B. pseudomallei (BpHldC) can be good therapeutic adjuvants to shorten the duration of medication to cure melioidosis combined with known antibiotics [23].
The X-ray crystal structure of BpHldC revealed that it forms a homotetrameric structure with a flexible C-terminal helix presumed to be crucial for its enzymatic function. A MES buffer molecule occupies a predicted catalytic site in two subunits only indicating that BpHldC may act in an allosteric mode. Highly conserved residues, His40 and Lys69, interact with the MES molecule. In this study, a flavonoid library ( Figure S1) (Table S1) has been applied to search flavonoids blocking the catalytic activity of BpHldC. Furthermore, their inhibitory modes were analyzed based on the X-ray crystal structure of BpHldC. Since there Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200677/900588/bcj-2020-0677.pdf by guest on 24 December 2020 is no antagonist against HldC has been reported, the flavonoids found in this study are the first examples targeting this enzyme.

Preparation of the protein
The BphldC gene (NCBI Reference Sequence: WP_004189202.1) coding for BpHldC protein was amplified with a pair of primers. The PCR product was ligated into the amplified expression vector pB 2 via the ligation-independent cloning (LIC) method. The transformant was acquired by the use of Escherichia coli (E. coli) DH5a, amplified and sequenced for identifying. Transformation into E. coli BL21 (DE3), large-scale cultivation for protein overexpression and purification of BpHldC were performed with the method previously reported [24]. The expressed proteins contained non-cleavage N-terminal His 6 -tags followed by five glycines (MHHHHHH GGGGG). The protein was purified by Ni 2+ -affinity chromatography. The ion-exchange chromatography using a 5 ml Hi-Trap Q column followed for the secondary purification. The purity of the protein was at least 95% as considered by SDS/PAGE ( Figure S2). The pooled proteins were concentrated to the proper concentration for the enzyme assay.

Enzyme kinetics of the protein
The malachite green assay method [25] was used to get the steady-state kinetics of the enzyme. For the kinetic studies, components of the reaction mixture (40 μl) were the same as for the chemical screening. BpHldC was incubated with ATP at 0.0078 -1.5 mM in the presence of 1 mM βG1P and with βG1P at 0.0078 -2.5 mM in the presence of 0.5 mM ATP.
The reaction was incubated at RT for two hours. The steady-state curve was fitted using For blanks, an enzyme-free reaction mixture was made and also incubated with the same range of concentrations of flavonoids. The reaction was initiated by adding saturating two substrates and stood for two hours. After the reaction, 160 μl of the malachite reagent was treated and the absorbance was measured at 620 nm using the microplate spectrophotometer (Spectramax 190, Molecular Devices Corporation, Sunnyvale, CA, USA). Wavelengths of maximum absorption (λ max ) of EGCG was 274 nm [26] (All catechin derivatives, gallic acid were reported that they have λ max at 210 nm and from 275-280 nm [27]). λ max of myricetin was 328 nm and 369nm [28]. The difference in absorbance with and without enzyme in the reaction mixture was managed into the percentage reactivity (%Reactivity). The IC 50 curves of three flavonoids against BpHldC were fitted with nonlinear regression analysis using GraphPad software. from the spectrophotometer were used for fitting steady-state kinetic graphs and secondary plots using the GraphPad Prism program.

Ligand preparation, target preparation, and induced-fit docking
All the docking and scoring calculations were performed using the Schrödinger software suite (Maestro, version 11.8.012). The SDF file of EGCG was got from the PubChem database.
The file was imported into Maestro and prepared for docking using Ligand Preparation. The atomic coordinates of the crystal structure of BpHldC (PDB ID: 5X9Q) were saved from the Protein Data Bank and prepared by removing all solvent and adding hydrogens and minimal minimization using Protein Preparation Wizard. Ionizer was used to generate an ionized state of all compounds at the target pH 7.0 ± 2.0. The input for an induced-fit docking is the prepared low-energy ligand forms. The induced-fit docking protocol [29] was worked on the graphical user interface, Maestro 11.8.012 linked with the Schrödinger software. Receptor sampling and refinement were conducted for residues within 5.0 Å of each ligand for each ligand-protein complex. With Prime [30], energy minimizing with a side-chain sampling, prediction module, and the backbone of BpHldC, were carried out. A total of induced-fit receptor conformations with EGCG were generated and were scored using a combination of Prime and Glide Score scoring functions [31].

Chemical screening with a malachite green assay method
A malachite green test that can easily detect free phosphate was performed to find inhibitory compounds for BpHldC. The adenylyltransferase catalyzes the transfer of adenylyl groups to heptose and produced byproduct, pyrophosphates. Pyrophosphatase turns a pyrophosphate into two phosphates and the malachite green agent detects phosphates. An in-house  Figure S3).

Enzyme kinetics of the protein
Enzyme kinetic assays of BpHldC were performed with various concentrations of ATP at a fixed concentration of 1 mM βG1P and the kinetic parameters were determined. Enzyme assays with various concentrations of βG1P at a fixed concentration of 0.5 mM ATP were also carried out. Both ATP and βG1P versus velocity plots of BpHldC were well fitted by the allosteric sigmoidal model (Figure 1). The steady-state kinetic data of BpHldC was obtained through a nonlinear regression analysis by using GraphPad Prism 8.4.3 and organized in Table 1.

IC 50 values of flavonoids
To measure the dose-dependent inhibitory activities of EGCG and myricetin on BpHldC, a malachite green assay method was used at the saturated substrate concentrations of 0.5 mM ATP and 1 mM βG1P. The data were plotted as log inhibitor concentration versus percentage reactivity based on absorbance obtained from the spectrophotometer (Figure 2) (Figure S4).  inhibition type of EGCG was presented in competitive inhibition for ATP and βG1P. The inhibition type of myricetin revealed an uncompetitive mechanism for ATP and a mixed-type inhibition for βG1P. All parameters were calculated using GraphPad Prism program.

Docking
To deduce the binding modes of EGCG and GCG with BpHldC at the atomic level, an indepth theoretical investigation with an induced-fit docking study using the Schrödinger program was carried out (Figure 6). The crystal structures of BpHldC deposited in the Protein Data Bank was retrieved and docked with EGCG and GCG to predict its binding mode. Top-ranked structures with the highest Glide g-scores from the induced-fit docking results were surveyed. The top model has a Glide g-score of -9.53 and -9.19 with respect to EGCG and GCG, respectively and docked reasonably in the active site pocket. The predicted complex structure and 2D schematic representation are illustrated in Figure 6(c) and 6(d).

Discussion
Melioidosis causing 90,000 deaths across the tropics is a pathogenic disease triggered by the intracellular Gram-negative bacterium B. pseudomallei [10]. In poorly resourced regions, the overall mortality rate increases by up to 50% [10] [33]. However, if patients are properly administered with antimicrobials, the rate decreases to 10% [34]. The symptoms become severe if patients have underlying comorbidities such as diabetes [35]. Until now no vaccine is available. Therefore, the development of anti-melioidosis agents is required. In our previous study, 3-deoxy-D-manno-oct-2-ulosonic acid cytidylyltransferases (KdsBs) from two pathogenic microbes, B. pseudomallei and Pseudomonas aeruginosa, have been studied and proven to be directly inhibited by Rose Bengal. Since KdsB is one of the key enzymes in the CMP-3-deoxy-D-manno-oct-2-ulosonic acid biosynthesis pathway of B. pseudomallei, the scaffold of Rose Bengal can be used as a template to develop anti-melioidosis agents.
In this study, we have studied BpHldC involved in the ADP-L-glycero-β-D-manno-heptose pathways. Since there is no antibiotic agent currently available against these biosynthesis pathways, the enzymes playing here are emerging targets to develop multi-drug resistant microbes. To assay with BpHldC, a surrogate of D-glycero-β-D-manno-heptose-1-phosphate, βG1P was employed as a substrate. An in-house flavonoid library (Supplementary Table 1) was built and used to probe inhibitory compounds. Enzyme kinetics of BpHldC (Figure 1) indicated that the substrate was enough to detect their catalytic activity and could be applied The inhibitory enzyme kinetics of the two flavonoids were tried to survey its inhibitory mechanism. According to the steady-state inhibitory enzyme kinetic analysis (Figure 3), EGCG tends to act as a competitive inhibitor with respect to βG1P and ATP. The secondary plot (Figure 4 and 5 [32]. In both cases, the ratio of K i´ over K i was much larger than 1 confirming the competitive inhibitory mode of action of EGCG [36]. The K i´ and K i of myricetin were 4.99 µM and 3.75 µM for βG1P and the ratio of K i´ over K i was larger than 1 implying the mixed model. The K i´ and K i of myricetin were 1.44 µM and 5.89 µM for ATP and K i´ over K i was smaller than 1. Since the ratio is around 0.25, its mode of action may be not competitive. Therefore, two flavonoids act differently on their inhibition over BpHldC.
BpHldC is a tetrameric enzyme with a large C-terminal helix domain connected to a hinge loop [19]. For the proper catalytic action of BpHldC, its C-terminal domain should experience a conformational change. In addition, it requires two substrates. The uncompetitive inhibitory mode of myricetin over ATP and the mixed-mode over βG1P could be related to the complicated characteristics of BpHldC. However, EGCG showed the competitive inhibitory mode for both ATP and βG1P. In order to elucidate the inhibitory mode of EGCG at the molecular level, induced-fit molecular docking trials have been performed. In the previous X-ray crystal structure study [19], the nucleotide-binding site was Third, two residues in the C-terminal domain, Arg147 and Lys154, interact with the 4-oxide ion and 3-hydroxyl group of the trihydroxybenzoate moiety, respectively. The former forms an ionic bond and the latter a hydrogen bond. It is worthwhile to note that the flap domain and the C-terminal domain participate in the interaction with ATP. Therefore, the docking study of EGCG with BpHldC revealed that EGCG occupies some parts of the substrate and nucleotide-binding sites together. Since its binding has been turned out to be tight according to conditions of the tight-binding inhibition [32], the docking result is well-matched with the kinetic study. The predicted occupation of EGCG in the catalytic cavity explains its competitive mode of inhibitory action for both substrate and nucleotide.
Since some flavonoids are displaying the structural similarity with EGCG, two flavonoids, GCG and CG, were analyzed further to investigate their structure and function relationship.
GCG is the C-2 epimeric isomer of EGCG, Interestingly, its IC 50 value, 18.44 µM, is comparable with that of EGCG. Therefore, its docking mode was searched and analyzed compared with that of EGCG. At first, the best Glide g-score of GCG was -9.19. Intriguingly, Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200677/900588/bcj-2020-0677.pdf by guest on 24 December 2020 the gallocatechin moiety is inverted at the active site pocket. As a result, the 5-and 7hydroxyl groups of the chromene moiety form hydrogen bonds with Ser63 and Val64, respectively. Lys69 also binds with the trihydroxyphenyl ring through πcation interaction.
The three residues, Ser63, Val64 and Lys69 are on the flap domain of BpHldC. The 3-and 5hydroxyl groups of the trihydroxyphenyl moiety make hydrogen bonds with Tyr120 and Asn30, respectively. The carboxyl oxygen and the 5-hydroxyl group of the trihydroxybenzoate moiety also build hydrogen bonds with Asn30 and Glu127, respectively.
Accordingly, GCG may interact tightly in the active site pocket of BpHldC and thus displays inhibitory activity. The importance of the gallate moiety of GCG has been also proven with Gallocatechin. It does not have gallate attached and its inhibitory activity was severely reduced.
On the contrary, CG did not show inhibitory activity against BpHldC. In the chemical structure, it lacks only one hydroxyl (5-hydroxyl) group at the phenyl ring moiety compared with GCG. It implies that one of the key interactions is the hydrogen bond formed between Asn30 and the 5-hydroxyl group of the trihydroxyphenyl moiety of GCG. Actually, the IC 50 value of CG is 487.5 µM and is 26-fold higher than that of GCG. In summary, the 5-hydroxyl group of the phenyl ring moiety and the gallate moiety are two key factors of EGCG homologues to interact with BpHldC.
EGCG exhibits antibacterial activity through inhibition of efflux pumps in A. baumannii [37]. Acinetobacter species have been alerted due to their implicated in hospital-acquired and healthcare-associated infections. Ventilator-associated pneumonia is the frequent lifethreatening hospital-acquired infection caused by P. aeruginosa and A. baumannii [38].
ECGC together with GCG show antibacterial activity through inhibiting FabG/FabI reductases from E. coli [39]. Myricetin displayed antibacterial activity against several antibiotic-resistant pathogens including multidrug-resistant Burkholderia cepacia [40]. At the Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200677/900588/bcj-2020-0677.pdf by guest on 24 December 2020 molecular level, myricetin has been found to suppress E. coli DnaB helicase with an IC 50 value of 11.3 µM [41]. The compound displayed poor activity against Klebsiella pneumonia (MIC 50 128 mg/mL), but strong synergy was observed at a concentration of 32 µg/mL together with amoxicillin/clavulanate, ampicillin/sulbactam and cefoxitin [42]. Therefore, this study finds other potential antibiotic mechanisms of myricetin and EGCG together with its epimer GCG through targeting BpHldC. Since these flavonoids are the first known inhibitors against BpHldC, they can be good templates to develop anti-melioidosis agents.
Among the five enzymes in the ADP-l-glycero-β-d-manno-heptose biosynthesis pathway, heptose-7-phosphate analogues targeting GmhA [43] and triazine derivatives targeting the kinase domain of HldE from E. coli [22] have been reported. The triazine derivatives actually attenuated the Gram-negative bacterial virulence. Therefore, including the flavonoids found in this study, various scaffolds are being used to develop antibiotics targeting this pathway. A further study of these antibiotic candidates could provide new antibiotics working independently or synergistically.

Conflict of interest statement
The authors declare that there is no conflict of interest.  with respect to βG1P for BpHldC inhibitory activity.