1 Introduction

DNA helicases are motor proteins essential for DNA replication, repair, and recombination [8, 15, 16, 18]. During DNA replication, the leading strand is directly synthesized by DNA polymerase. On the lagging strand, the primase interacts with the hexameric helicase to synthesize short RNA primers. These primers are used to generate the Okazaki fragments necessary for progression of the replication fork. The most widely studied replicative helicase is Escherichia coli DnaB helicase (EcDnaB) [1, 21]. EcDnaB is a multifunctional ATPase that catalyzes the unwinding of double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA) intermediates at the replication fork to provide ssDNA templates for DNA polymerases [21]. Recently, we resolved the three-dimensional structures of the Geobacillus kaustophilus DnaB-family protein both in the apo state and complexed it with ssDNA and the non-hydrolysable NTP ATPγs, and showed that ATP hydrolysis may drive the movement of the helicase toward the 3′ end of the lagging strand [13].

Klebsiella pneumoniae (Kp) is a ubiquitous opportunistic pathogen that colonizes at the mucosal surfaces in humans and causes severe diseases such as septicemia, pneumonia, urinary tract infections, and soft tissue infections [17]. Despite advances in treatment and prevention, K. pneumoniae still poses a major threat to public health worldwide. Although K. pneumoniae strains are generally susceptible to some antibiotics, such as cephalosporins, many clinical strains of K. pneumoniae are highly resistant to antibiotics. Currently, few therapies are effective against these extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae strains [7, 30]. Discovering virulence factors and identifying novel targets for drug development and new therapies against K. pneumonia are critical for public health [4, 27, 29]. Since DnaB is required for DNA replication, blocking the activity of DnaB would be detrimental to bacterial survival [22]. Because of the distinct differences between eukaryotic and prokaryotic DnaB-like helicases [21, 22], the K. pneumoniae DnaB helicase may be a promising target in developing antibiotics.

Flavonoids are the most common group of plant polyphenols, and are responsible for much of the flavor and color of fruits and vegetables [19]. To date, over 5,000 different flavonoids have been described; many of these compounds display structure-dependent biological and pharmacological activities [19, 23, 26]. The 6 major subclasses of flavonoids are: flavonols, flavones, flavanones, catechins (flavanols), anthocyanidins, and isoflavones [19]. Flavonols are polyphenol compounds with in vitro anti-oxidant and anti-radical activity [3, 26], as well as antibacterial activity [5]. A flavonol is composed of 2 aromatic rings linked by a heterocyclic pyran-4-one ring.

In this study, we have cloned, expressed, and purified KpDnaB, and found that the 4 flavonols (Fig. 1) myricetin (Myr), quercetin (Que), kaempferol (Kae), and galangin (Gal) can interact with KpDnaB and prevent dNTP binding. These flavonols may be potential leads in anti-Kp drug development.

Fig. 1
figure 1

Molecular structure of Gal, Kae, Que and Myr

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2 Materials and Methods

2.1 Materials

All restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs (Ipswich, MA, USA) unless explicitly stated otherwise. All custom oligonucleotide primers were obtained from Invitrogen Corporation (Carlsbad, CA, USA). All chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless explicitly stated otherwise.

2.2 Construction of the KpDnaB Expression Plasmid

KPN04439, the gene encoding the putative KpDnaB, was amplified by PCR using genomic DNA of K. pneumoniae subsp. pneumoniae MGH 78578 as the template. The forward (5′-GGGGAATTCACAGCACAATCCCAGGTATTGAAA-3′) and the reverse (5′-GGGAAGCTTCTCATCATCATACTGAGGACCGGC-3′) primers were designed to introduce unique EcoRI and HindIII restriction sites (underlined) into KpDnaB, permitting the insertion of the amplified gene into the pET21e vector. The pET21e vector was engineered from the pET21b vector (Novagen Inc., Madison, WI, USA), to avoid having the N-terminal T7 tag fused with the gene product [24]. Briefly, the region containing the NheI site of the pET21b vector (CATATGGCTAGC) was mutated to introduce a new EcoRI site (CATATGGAATTC) by using the primers E1 (5′-AAGGAGATATACATATGGAATTCATGACTGGTGGACAG-3′) and E1′ (5′-TGCTGTCCACCAGTCATGAATTCCATATGTATATCTCCT-3′). The resultant plasmid was digested with EcoRI to remove the DNA fragment spanning from the original NheI site to the EcoRI site, and then religated onto itself at the EcoRI site to generate the expression vector pET21e. The KpDnaB DNA fragment was then inserted into pET21e to produce the plasmid pET21e-KpDnaB for KpDnaB expression. Therefore, the expected gene product expressed by pET21e-KpDnaB will have 2 additional artificial residues, EF, introduced by the EcoRI site located at the N-terminus, and a C-terminal His tag (KLAAALEHHHHHH), useful for purifying the recombinant protein.

2.3 Protein Expression and Purification

Recombinant KpDnaB protein was expressed and purified using the same protocol as described previously for the protein PriB [10], with a minor modification. Briefly, E. coli BL21(DE3) cells were transformed with the wild-type pET21e-KpDnaB plasmid and grown to 0.9 OD600 at 37 °C in Luria–Bertani medium containing 250 μg/mL ampicillin with rapid shaking. Overexpression of the KpDnaB construct was induced by incubating with 1 mM isopropyl thiogalactoside (IPTG) for 3 h at 37 °C. The cells overexpressing the protein were chilled on ice, harvested by centrifugation, resuspended in Buffer A (20 mM Tris–HCl, 5 mM imidazole, 0.5 M NaCl; pH 7.9) and disrupted by sonication with ice cooling between pulses. The KpDnaB was purified from the soluble supernatant by Ni2+-affinity chromatography (HiTrap HP; GE Healthcare Bio-Sciences, Piscataway, NJ, USA). Protein purity remained greater than 95% as determined by Coomassie-stained SDS–PAGE.

2.4 Dissociation Constant of dNTPS and KpDnaB Determined by Fluorescence Spectrophotometer

An aliquot amount of nucleotides was added into the solution containing KpDnaB (1 μM), 50 mM HEPES at pH 7.0 with a final volume of 2 mL in a quartz cuvettes of 1 cm square cross-section. The decrease in intrinsic fluorescence of protein was measured at 330 nm upon excitation at 280 nm and 25 °C with a spectrofluorimeter (Hitachi F-2700; Hitachi High-Technologies, Tokyo, Japan). Each data point was duplicated, and the difference was within 10%. An aliquot amount of nucleotides was added with or without 10 μM flavonol to a predetermined concentration of KpDnaB to obtain at least 7 data points for each dissociation constant (K d). The K d was obtained by the equation: ΔF = ΔFmax−K d(ΔF/[nucleotide]) (Enzyme Kinetics module of Sigma-Plot; Systat Software, Chicago, IL, USA).

3 Results

The purpose of this study was to identify some naturally occurring compounds that can inhibit the activity of bacterial replicative helicase(s). The results presented here show that 4 flavonol compounds may be useful in developing anti-K. pneumoniae antibiotics.

3.1 Fluorescence Quenching of KpDnaB by dATP, dTTP, dCTP, or dGTP

The fluorescence emission spectra of KpDnaB quenched with dATP, dTTP, dCTP, and dGTP are shown in Fig. 2a–d. Fluorescence intensity of KpDnaB decreased remarkably with increasing concentrations of dNTP (0–10−3 M). At 10−3 M, dCTP (Fig. 2c) and dGTP (Fig. 2d) quenched the fluorescence intensity of KpDnaB almost completely, whereas dATP (Fig. 2a) only quenches approximately 80% of the KpDnaB fluorescence intensity. Adding dNTPs resulted in a significant blue shift (~10 nm) of the emission wavelength (λem) of KpDnaB, indicating an interaction between dNTP and KpDnaB and suggesting formation of a dNTP-KpDnaB complex. The dissociation constant (K d) values of KpDnaB bound to dATP, dTTP, dCTP, and dGTP determined from the titration curves (Fig. 3) were 159.8 ± 60, 106.0 ± 30, 60.3 ± 13, and 63.2 ± 16 μM, respectively, meaning that KpDnaB exhibited the strongest binding activity towards dCTP.

Fig. 2
figure 2

The fluorescence quenching of KpDnaB by a dATP, b dTTP, c dCTP or d dGTP. dNTP, from the top down, are as follows: 0, 10−9, 10−8, 10−7, 10−6, 10−5, 10−4, and 10−3 M. An aliquot amount of the nucleotide was added into the solution containing KpDnaB (1 μM), 50 mM HEPES at pH 7.0 and 25 °C. After the addition of the nucleotide, the reaction solution was equilibrated for 10 min until no fluorescence change could be observed

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Fig. 3
figure 3

The fluorescence quenching of KpDnaB plotted as relative fraction of (F0−F)/F0 against the concentrations of the dNTP

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3.2 Fluorescence Quenching of KpDnaB by Flavonols

The fluorescence emission spectra of KpDnaB quenched by Gal, Kae, Que, and Myr are shown in Fig. 4a–d. Due to the compounds’ solubility, 10 μM of each flavonol was used. KpDnaB fluorescence intensity decreased significantly with increasing concentrations of flavonols (0–10 μM). The maximum λem of these flavonols alone in response to excitation at 280 nm was approximately 520 nm (data not shown). In addition, these 4 flavonols resulted in a slight blue shift (~1 nm) of the maximum λem of KpDnaB. Thus, the quenching of KpDnaB fluorescence mainly depended on the formation of a complex between the flavonol and KpDnaB (Fig. 4). The titration curves shown in Fig. 5 suggest that KpDnaB binds most strongly to Myr, and, in the order of decreasing affinity, to the other flavonols as follows: Myr > Kae > Gal > Que.

Fig. 4
figure 4

The fluorescence quenching of KpDnaB by a Gal, b Kae, c Que or d Myr. Flavonol, from the top down, are as follows: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μM. An aliquot amount of the flavonol was added into the solution containing KpDnaB (1 μM), 50 mM HEPES at pH 7.0 and 25 °C. After the addition of the flavonol, the reaction solution was equilibrated for 10 min until no fluorescence change could be observed

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Fig. 5
figure 5

The fluorescence quenching of KpDnaB plotted as relative fraction of (F0−F)/F0 against the concentrations of the flavonol

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3.3 Fluorescence Quenching of KpDnaB by dATP, dTTP, dCTP and dGTP in the Presence of Myr, Kae, Gal, or Que

To investigate whether the flavonols can inhibit the binding of KpDnaB to dNTP, we analyzed KpDnaB fluorescence quenching by dATP in the presence of Myr, Kae, Gal, or Que (Fig. 6a). The interaction of KpDnaB with dATP was much more sensitive to Gal than to Que. Fluorescence quenching of KpDnaB was not obvious until the concentration of dATP increased to 10−4 M, indicating that the binding affinity between KpDnaB and dATP was decreased by the presence of Gal, Kae, Que, or Myr in the reaction solution.

Fig. 6
figure 6

The fluorescence quenching of KpDnaB in the presence of 10 μM flavonol plotted as relative fraction of (F0−F)/F0 against the concentrations of a dATP, b dTTP, c dCTP or d dGTP. The reaction solution (1 μM KpDnaB, 50 mM HEPES at pH 7.0) was pre-incubated with 10 μM flavonol for 10 min, and then an aliquot amount of the nucleotide was added to the KpDnaB solution. After the addition of the nucleotide, the reaction solution was equilibrated for further 10 min until no fluorescence change could be observed

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To test whether the flavonols inhibit the binding of KpDnaB to other dNTPs, we also analyzed KpDnaB fluorescence quenching by dTTP (Fig. 6b), dCTP (Fig. 6c), and dGTP (Fig. 6d) in the presence of Myr, Kae, Gal, or Que. However, the results of these experiments were quite different from those of the experiment with dATP. dTTP, dCTP, and dGTP quenched KpDnaB fluorescence by almost 50% even at 10−4 M of the flavonols, suggesting that Myr, Kae, Gal, and Que specifically disrupt the KpDnaB-dATP interaction (Fig. 6).

3.4 Inhibition of KpDnaB-dNTP Interaction by Gal was the Strongest

Figure 7 shows the effects of flavonol on the interaction between KpDnaB and dATP, dTTP, dCTP, or dGTP. KpDnaB-dATP binding was inhibited by flavonoids as follows, in the order of decreasing efficiency: Gal > Myr > Kae > Que (Fig. 7a). None of the 4 flavonols disrupted dGTP binding to KpDnaB (Fig. 7d). Among the flavonols, Gal inhibited KpDnaB-dNTP interaction most strongly, although Myr showed the strongest binding to KpDnaB (Fig. 5). Thus, the ability of flavonols to inhibit KpDnaB action in general may be dNTP-dependent.

Fig. 7
figure 7

The quantitative analysis of fluorescence quenching of KpDnaB plotted as relative fraction of (F0−F)/F0 in the presence of 10 μM flavonol and 10 μM of a dATP, b dTTP, c dCTP or d dGTP

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3.5 The Flavonoids Inhibited Growth of K. Pneumoniae

To test whether these flavonoids can inhibit growth of K. pneumoniae, K. pneumoniae cells were grown to 0.5 OD600 at 37 °C, and then added 10 μM of Gal, Kae, Que, or Myr into the medium. Figure 8 shows that these 4 flavonoids inhibited growth of K. pneumoniae; Gal had a greater inhibitory effect than the others.

Fig. 8
figure 8

Growth of K. pneumoniae cells in Luria–Bertani medium supplemented with 10 μM of the flavonoids (Myr, Que, Kae, or Gal)

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4 Discussion

DNA replication is one of the most basic biological functions and should be a prime target in antibiotic development. In fact, it is the target of the bactericidal fluoroquinolone class of antibiotics that interferes with DNA gyrase [9] and topoisomerase [2]. Since DNA helicases are important components of the cellular replication machinery in all organisms, inhibition of helicase activity would be detrimental to bacterial survival as well [1, 6, 20, 21, 28]. In this study, we used fluorescence quenching to analyze the interaction of KpDnaB with 4 flavonols, Gal, Kae, Que, and Myr (Fig. 1), which contain different numbers of hydroxyl substituent on the aromatic rings. Our results demonstrated that these flavonols were capable of inhibiting the interaction of KpDnaB with dNTPs. The extent of KpDnaB fluorescence quenching induced by dATP in the presence of a flavonol was much smaller than that of dATP alone, indicating inhibition of KpDnaB-dATP binding by the flavonol (Figs. 3, 6). In addition, the inhibition depended not only on the flavonol (with Gal displaying the strongest inhibition), but also on the dNTP used (the inhibition was most specific to dATP binding).

Binding and hydrolysis of NTP cofactors by the DnaB helicase before association with ssDNA are essential processes that induce and modulate a high affinity conformation of the enzyme that can bind to ssDNA [14, 21, 25]. Although the primary replicative helicase can hydrolyze all NTPs [20], our studies indicate that KpDnaB has a preference for dCTP over other nucleotides (Fig. 3). Thus, other compounds similar to dCTP may be useful in inhibiting KpDnaB.

Other studies have shown that Myr non-competitively inhibits E. coli DnaB helicase [6] and RSF1010 RepA helicase [28], with IC50 of approximately 10 and 50 μM, respectively. In this study, we found that 10 μM of Gal, Kae, Que, or Myr can inhibit dNTP binding to KpDnaB. Although it is well established that flavonoids have several hydroxyl groups and thus have marked potentials to bind (any) proteins, the strength of the inhibition in dNTP binding of KpDnaB was not correlated with the number of hydroxyl substituent on the aromatic rings of the flavonols (Fig. 7).

Although Myr binds to KpDnaB with the highest affinity among the flavonols (Fig. 5), it did not display the highest inhibition of dNTP-KpDnaB binding (Fig. 7). Based on these results, we propose that these flavonols may inhibit dNTP binding to KpDnaB in 2 possible ways. First, these 4 flavonols may not bind to the active site of KpDnaB, or only partially occupy the active site. Second, since DnaB helicase binding to dNTP causes a large conformational change [11, 12, 20], these flavonols may inhibit the conformational change itself, thereby causing varying degree of inhibition. The inhibition of KpDnaB by these flavonols appeared to be dNTP-dependent, and, thus, neither of these possibilities can be dismissed.

Our crystal structure of Geobacillus kaustophilus helicase in complex with ssDNA and the non-hydrolysable NTP analogue ATPγs [13] revealed that ATP hydrolysis may drive the movement of the helicase toward the 3′ end of the lagging strand. In addition, the dNTP-binding site of the helicase at loop I, part of the Walker B motif, is adjacent to the DNA interaction site. From these results, we speculate here that 1 flavonol molecule is enough to bind to the empty active sites of 6 KpDnaB subunits to shut down and lock the enzyme in the dNTP-unbound state. We have prepared a crystal of KpDnaB in complex with Gal to further investigate this hypothesis, and the resulting information may be useful in designing compounds that fit more precisely into helicase active sites.