Undecaprenyl Phosphate Phosphatase Activity of Undecaprenol

Subscriber access provided by GRIFFITH UNIVERSITY

Article

The undecaprenyl phosphate phosphatase activity of undecaprenol kinase regulates the lipid pool in Gram positive bacteria Lin-Ya Huang, Shih-Chi Wang, Ting-Jen R. Cheng, and Chi-Huey Wong Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00603 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 9, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

The undecaprenyl phosphate phosphatase activity of undecaprenol kinase regulates the lipid pool in Gram positive bacteria Lin-Ya Huang‡∇, Shih-Chi Wang#‡, Ting-Jen R. Cheng*, and Chi-Huey Wong#*

Genomics Research Center, Academia Sinica, Taipei 115, Taiwan

#Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan

‡ These authors contributed equally

∇ Current Address: CHO Pharma, Nangang district, Taipei 115, Taiwan

Corresponding Author * Genomics Research Center, Academia Sinica 128 Academia Road, Section 2, Nankang,

Taipei

115

(Taiwan).

E-mail:

[email protected].

Phone:

886-2-2789-9929; [email protected].

0

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 43

ABBREVIATIONS UK, undecaprenol kinase; UpP, Undecaprenyl phosphate phosphatase; DgkA, diacylglycerol kinase; C55OH, undecaprenol; C55P, undecaprenyl phosphate; C55PP, undecaprenyl pyrophosphate

1


Page 3 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

ABSTRACT

Bacteria cell walls contain many repeating glycan structures, such as peptidoglycans, lipopolysaccharides, teichoic acids and capsular polysaccharides. Their synthesis starts in the cytosol, and they are constructed from a glycan lipid carrier, undecaprenyl phosphate (C55P), which is essential for cell growth and survival. The lipid derivative undecaprenol (C55OH) is predominant in many Gram-positive bacteria but has not been detected in Gram-negative bacteria; its origin and role have thus remained unknown. Recently, a homologue of diacylglycerol kinase (DgkA) in E. coli was demonstrated to be an undecaprenol kinase (UK) in the Gram-positive bacterium Streptococcus mutans (S. mutans). In this study, we found that S. mutans UK was not only an undecaprenol kinase but also a Mg-ADP-dependent undecaprenyl phosphate phosphatase (UpP), catalyzing the hydrolysis of C55P to C55OH and a free inorganic phosphate. Furthermore, the naturally undetectable C55OH was observed in E. coli cells expressing S. mutans dgkA, supporting the phosphatase activity of UK/UpP in vivo. These two activities were indispensable to each other and utilized identical essential residues binding to their substrates, suggesting that both activities share the same active site and might involve a direct phosphoryl transfer mechanism. This study revealed a unique membrane enzyme displaying bifunctional activities determined by substrate binding and C55OH production. The reciprocal conversion of C55P and the undecaprenol pool efficiently regulate cell wall synthesis, especially in Gram-positive bacteria.

KEYWORDS. Undecaprenol kinase, Undecaprenyl phosphate phosphatase, polyprenyl phosphate metabolism 2


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 43

INTRODUCTION Polyprenyl phosphates are essential components ubiquitously distributed among all kinds of organisms. The biosynthesis of glycoproteins in eukaryotes and the assembly of cell wall components in bacteria require the participation of polyprenyl phosphates, which function as universal membrane-bound saccharide carriers in glycan biosynthesis.1 The bacterial cell wall provides structural integrity protecting cells from osmotic and environmental pressure. The backbone of cell wall layers such as peptidoglycans, LPS and teichoic acids is composed of many repeating glycan structures. The synthesis of cell wall involves the use of undecaprenyl phosphate (C55P) as carrier for loading glycan units to assemble mature cell walls. It serves as a limiting factor that controls the production of the bacterial cell wall.2 The production of undecaprenyl phosphate occurs through the dephosphorylation of undecaprenyl pyrophosphate (C55PP), which is generated from de novo synthesis or after the ﬁnal glycan transfer (Figure 1). In the de novo synthesis, farnesyl pyrophosphate (C15PP) is condensed with multiple isopentenyl pyrophosphates (IPPs) by a cytosolic undecaprenyl pyrophosphate synthase (UppS) to become C55PP.3 After the dephosphorylation of C55PP by undecaprenyl pyrophosphate phosphatase (UppP),4,5 C55P is loaded with glycan units and subsequently transferred to the cell wall for polymerization after translocation across the hydrophobic membrane. Under the polymerization process, the byproduct C55PP is released and subjected to dephosphorylation to produce C55P for recycling.6

3


Page 5 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 1. The metabolism pathway of C55P in Gram-positive bacteria. In bacteria, C15PP is condensed with isopentenyl pyrophosphates (IPP) by undecaprenyl pyrophosphate synthase (UppS) to form C55PP, which is dephosphorylated to C55P by undecaprenyl pyrophosphate phosphatase (UppP). C55P is then loaded with glycans for the polymerization of cell wall components. C55PP can also be replenished during bacterial cell wall synthesis. In Gram-positive bacteria, an enzyme, undecaprenol kinase (UK), can convert C55OH to C55P by using ATP, but the origin of C55OH remained unknown. One possibility is the dephosphorylation from C55P (dashed line).

Another undecaprenyl derivative, undecaprenol (C55OH), does not participate in the de novo biosynthesis pathway; however, it has long been reported to be a major form in the undecaprenoid pool in the membrane of many Gram-positive bacteria, such as Staphylococcus aureus (S. aureus), Enterococcus faecalis, Listeria monocytogenes and Lactobacillus plantarum. Intriguingly, in contrast to Gram-positive bacteria, Gram-negative bacteria have no detectable C55OH.7-12 How and why this lipid is produced in Gram-positive bacteria remain unknown. In the 1970s, the phosphorylation of C55OH and the dephosphorylation of C55P were detected in the membrane fractions of S. aureus.13.14 The genes involved in the activities were not successfully identified 4


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 43

until 2003 when a homologue of E. coli diacylglycerol kinase (DgkA) was demonstrated to be an undecaprenol kinase (UK) in Streptococcus mutans.15 Later, homologues of UK from other Gram-positive bacteria were also shown to exhibit undecaprenol kinase activity.16,17 The Gram-negative bacterium E. coli does not have detectable C55OH and exhibits extremely low UK activity, suggesting that the phosphorylation pathway from C55OH to C55P exists only in Gram-positive bacteria.11,15,17 In contrast to the finding of C55OH phosphorylation, the exact enzyme responsible for the C55P dephosphorylation activity has remained a mystery for half a century after the detection of C55OH. In this report, we analyzed the activity of UK from S. mutans and found that this enzyme was not only a kinase but also a Mg-ADP-dependent phosphatase.

5


Page 7 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

MATERIALS AND METHODS Materials ATP,

ADP,

adenosine

5’-(β,γ-imido)triphosphate

(AMPPNP),

β,γ-methyleneadenosine 5’-triphosphate (AMPPCP), adenosine 5’-(γ-thio)triphosphate (ATPγS), AMP, pyruvate kinase (PK), octyl glucoside (OG), cadiolipin and lauryldimethylamine oxide (LDAO) were purchased from Sigma. Phosphoenol pyruvate (PEP) was from Alfa Aesar. Undecaprenol (C55OH), undecaprenyl phosphate (C55P) and undecaprenyl pyrophosphate (C55PP) were from Larodan Fine Chemicals. All organic

solvents

and

silica

TLC

plates

were

from

Merck.

N-dodecyl-β-D-maltopyranoside (DDM) was from Avanti.

Protein Expression and Purification The gene dgkA from Streptococcus mutans was synthesized with a His-tag, followed by an enterokinase cutting site on its N-terminal, and cloned into vector pET-28a (Novagen) for expression.17 Mutants were constructed by site-directed mutagenesis of individual sites in dgkA and confirmed by DNA sequencing at Mission Biotech (Taiwan). E. coli BL-21(DE3) cells harboring the expression plasmids were grown at 37 °C in LB medium containing 100 µg/mL ampicillin. After being induced with 1 mM IPTG for 4 h, cells were harvested by centrifugation and suspended in buffer A (20 mM Tris pH 8.0, 300 mM NaCl and 5% glycerol) containing 1% DDM. After disruption by a Microfluidizer (Microfluidics), the lysate was clarified by centrifugation at 12,000 × g for 30 min. The supernatant was purified by nickel chelation chromatography (GE), and the desired proteins were eluted by buffer A containing 0.1% DDM with imidazole at concentrations ranging from 100-300 mM. The purified proteins were concentrated using an Amicon Ultrafiltration Unit (Millipore), and their 6


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 43

purity was checked by SDS-PAGE.

Activities Measured by Thin Layer Chromatography (TLC) For qualitative TLC analysis, a solution (10 µL) containing 50 mM Tris pH 8.0, 10 mM MgCl2, 2 µM UK/UpP, 4 mM undecaprenol (17.4 mol %), 20 mM LDAO and 10 mM ATP was used to monitor the phosphorylation activity. At indicated incubation times at 37 ºC, a small aliquot of sample (0.5 µL) was spotted on a silica TLC plate and developed with a solution of CHCl3: MeOH: H2O: NH3 (7N) = 88: 48: 10: 1 (v/v). The lipids were visualized by anisaldehyde staining and heating before the appearance of green DDM spots. To monitor the dephosphorylation of C55P, a solution (10 µL) containing 50 mM Tris pH 8.0, 10 mM MgCl2, 4 µM UK/UpP, 4 mM C55P, 20 mM LDAO and 2 mM ADP was incubated at 37ºC. At the indicated time points, a small aliquot of sample (0.5 µL) was spotted on a silica TLC plate and developed with a solution of EA: Hex = 1: 7 (v/v) to separate lipids or a solution of isobutyric acid: NH4OH (1 N) = 5: 2 (v/v) for nucleotide separation. The critical micelle concentration (CMC) of LDAO is approximately 1 mM, thus the mole fraction of C55OH or C55P in LDAO micelles was calculated based on the formula of [C55OH or C55P] / ([C55OH or C55P] + [LDAO] - 1 mM).18

Activities Measured by High-performance Liquid Chromatography (HPLC) HPLC was performed to quantify the amount of undecaprenol and undecaprenyl phosphate. Unless stated otherwise, the standard assay containing 50 mM Tris pH 8.0, 10 mM MgCl2, 2 mM LDAO, 200 µM C55OH (16.6 mol %) (for kinase activity or C55P for phosphatase activity), 200 nM enzyme and 200 µM ATP (for kinase activity or ADP for phosphatase activity) in a 20 µL solution was incubated at 37 ºC. The reactions were 7


Page 9 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

initiated through the addition of the nucleotide and MgCl2 and terminated with 25 µL of n-butanol containing 20 mM EDTA. After phase separation, the butanol layer was collected and analyzed by HPLC equipped with a C4 column (5 µm, 4.6 × 150 mm, AR-300, COSMOSIL) using a linear gradient of 25% aqueous 30 mM NH4HCO3 in 75% MeOH to 100% MeOH for 40 min and then 100% MeOH for further 20 min at room temperature at a flow rate of 0.6 mL/min. C55OH and C55P were monitored at λ = 210 nm, and the integrated areas of the peaks corresponding to C55P or C55OH were used to calculate the percentage conversion. To monitor the phosphatase reaction of UK/UpP, the reactions were performed at various concentrations of substrates (7.8 to 1000 µM ADP; 7.8 to 1000 µM C55P) with 200 nM enzyme while the C55P or ADP was kept at 2 mM. The produced C55OH was monitored using HPLC analysis at 0, 1, 2, 5, 10, 20, and 30 min and the initial velocity was used to obtain the kinetic parameters (SigmaPlot).

Determination of Free Inorganic Phosphate Free inorganic phosphate (Pi) was detected using the malachite green method19 in clear 384-well plates. At the indicated time points for both kinase and phosphatase activities, 2-µL reaction samples were mixed with 50 µL of malachite green mixture solution A: solution B (3:1 v/v), where A is 0.045% malachite green and 0.05% Tween 20, and B is 4.2% ammonium molybdate in 4 N HCl, and the absorbance at 660 nm was measured. The concentration of phosphates was calculated based on a standard curve derived from potassium phosphate.

Determination of ATP Concentration 8


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 43

The amount of ATP was determined using a CellTiter-Glo Luminescent Assay kit (Promega). Generally, reaction samples of 1 µL were mixed with 50 µL of assay reagents. The generated luminescence was monitored by using SpectraMax M5. The amount of ATP in the reaction was calculated using a standard curve derived from various concentrations of ATP. To monitor the kinase reaction of UK/UpP, the reactions were performed at various concentrations of substrates (15.6 to 2000 µM ATP; 15.6 to 4000 µM C55OH) with 100 nM enzyme while C55OH or ATP was kept at a saturated concentration of 6 mM or 2 mM, respectively. The initial velocity parameters were used to obtain the apparent kinetic parameters (SigmaPlot).

Membrane Lipid Extraction Transformed E. coli (DH5α) or Staphylococcus aureus (BCRC11863) bacteria were grown in 100 mL of LB with shaking at 37 ºC. Bacteria were washed with 0.85% NaCl and collected by centrifugation after OD reached 2.0. Bacterial membrane lipids were extracted following a previous report with minor modification.11 Briefly, bacteria were suspended in 0.5 mL of 60% KOH and 1 mL of MeOH. After the mixture was boiled for 1 h, the samples were cooled on ice and separated into two fractions, and 200 µL of acetic acid, 300 µL of PBS and 700 µL of chloroform were added. After centrifugation, the chloroform layer was collected, washed with H2O, and evaporated under vacuum. The lipids were then suspended in n-butanol and analyzed using the HPLC procedure described above.

31

P-Nuclear Magnetic Resonance Analysis of Protein Activity Experiments were carried out on a Bruker 600 spectrometer equipped with a probe

in 31P resonance mode at the frequency of 202.4 MHz. A solution (250 µL) containing 9


Page 11 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

50 mM Tris pH 8, 130 mM LDAO, 20 mM C55OH (13.4 mol %), 40 mM ATP and 50% D2O was measured as a control before the addition of enzyme. The reaction was initiated by mixing with 50 µL of 17 µM UK/UpP and 10 mM MgCl2 to a final volume of 300 µL in an Eppendorf and then transferred into a D2O Shigemi NMR microtube. After centrifugation to remove bubbles, the microtube was loaded into the spectrometer, and the field was locked by D2O. The

31

P-NMR spectra were recorded at 310 K, and

there was a delay of approximately 15-20 min for recording after the addition of enzyme. Spectra were processed within TopSpin 2.1, and the Pi turnover was calculated by the integral area of the individual peak relative to the sum of the integral area of the

αP-ATP and αP-ADP peaks.

10


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 43

RESULTS

The dephosphorylation of undecaprenyl phosphate by UK In our previous attempt to synthesize lipid II by using UK as the enzyme for the phosphorylation of C55OH to produce C55P, we were puzzled by the fact that C55P can only exist for a short period of time and disappears after a longer incubation.20 During the synthesis, C55OH was phosphorylated to C55P quickly; however, the product C55P was subsequently transformed back to C55OH (Figure 2A). TLC analysis excluded the possibility of a reversible kinase reaction since ATP was not regenerated from ADP but was exhausted upon the dephosphorylation of C55P. This phenomenon was also observed in the presence of mixed micelles containing phospholipids, octyl glucoside and cardiolipin (Figure S1A) or 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) containing decyl maltoside (DM) (Figure S1B). It was initially assumed that C55P might be unstable and become C55OH in solution. However, purified C55P itself can be stored stably in a buffer solution for a long period of time, indicating that dephosphorylation is not an intrinsic property of C55P but instead some unknown factors cause the removal of the phosphate group from C55P. Thus, various combinations of components in the kinase reaction, such as enzyme, MgCl2 and nucleotides, were tested (Figure 2B). It was surprising to find that only with the addition of both the enzyme and nucleotide, either ATP or ADP, the transformation of C55P to C55OH was observed. It was also noted that magnesium ion was required for the dephosphorylation activity since the reaction was completely abolished after elimination of Mg2+ through the addition of EDTA. The weak C55OH signal in the reaction without the addition of Mg2+ was possibly due to the presence of a small quantity of divalent cations in the enzyme or lipids.

11


Page 13 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 2. Mg2+-ADP dependent phosphatase activity of UK/UpP (A) Time-dependent reaction of UK/UpP with undecaprenol. The enzyme UK (2 µM) was incubated with 4 mM C55OH (17.4 mol %) and 10 mM ATP. At the indicated time points, aliquots of samples were analyzed by silica TLC to detect lipids (upper) or nucleotides (below). (B) The required factors for C55P phosphatase activity of UK. C55P (4 mM, 17.4 mol %) was incubated with various combinations of 4 µM enzyme, 10 mM MgCl2, 10 mM ADP or ATP at 37°C for 18 hr and analyzed by TLC. (C) ADP dependent phosphatase activity. UK/UpP (2 µM) was incubated at 37°C with 4 mM (17.4 mol %) C55P in the presence of 10 mM ADP (left), 10 mM ATP (middle) or 10 mM ATP with 5U pyruvate kinase and 40 mM PEP (right). At the indicated time points, aliquots were analyzed by silica TLC to detect lipids (upper) and nucleotides (below). The standards are C55OH (a), C55P (b), ATP (c), and ADP (d). (D) The requirement of β-phosphate of ADP for the phosphatase activity. UK was incubated with 200 µM (16.6 mol %) C55P in the presence of 200 µM ATP, ADP or AMP at 37°C. After incubation for various times, the reaction products were analyzed by HPLC and

12


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 43

the amount of C55P and C55OH was determined.

The substrate specificity of the dephosphorylation activities of UK In addition to C55P, other

substrates

were

also

investigated

for

the

dephosphorylation activities of UK. C55PP was first suspected as a candidate, as it is a precursor of C55P in the cell wall synthesis pathway (Figure 1) and has only one additional phosphate over C55P. However, neither undecaprenyl pyrophosphate phosphatase nor undecaprenyl pyrophosphate pyrophosphatase activity was detected in the presence of ADP (Figure S2A and B). The possibility that C55OH can be produced from C55PP by UK in the lipid metabolism pathway was also excluded. Further, we found that UK cannot act on p-nitrophenyl phosphate (PNPP), a substrate widely used to measure common phosphatase activity (Figure S2C), indicating that the phosphatase activity of UK is specific for specific lipid phosphates such as geranylgeranyl phosphate and undecaprenyl phosphate.20 The possibility of a contaminating phosphatase was also excluded since the purity of the purified recombinant enzyme was confirmed by SDS-PAGE (Figure S3A) as well as by size-exclusion chromatography (Figure S3B). These results demonstrated that UK was also an undecaprenyl phosphate phosphatase (UpP). Hereafter the enzyme was referred to as UK/UpP.

ADP dependence of C55P dephosphorylation Regarding the specificity of UK/UpP toward nucleotides, it was found that the kinase substrate ATP, in addition to ADP, could promote the C55P dephosphorylation activity of UK/UpP (Figure 2B). To test whether ATP was a direct activator or was converted to ADP, which then activated the dephosphorylation activity of UK/UpP, we mixed the enzyme and C55P with each of these two nucleotides, and we monitored the 13


Page 15 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

formation of ADP and C55OH on TLC at different time intervals. As shown in Figure 2C, C55P was quickly converted to C55OH in the presence of ADP, whereas the conversion was much slower in the presence of ATP. To monitor the status of ATP/ADP further, it was revealed that in the presence of ATP, ATP was transformed to ADP first, and the dephosphorylation of C55P occurred only when ATP was completely converted to ADP after 10 min of incubation (Figure 2C). To further confirm that C55P dephosphorylation was caused directly by ADP but not ATP, pyruvate kinase (PK) and phosphoenolpyruvate (PEP) were included to immediately convert ADP back to ATP. In this reaction, C55OH cannot be detected, demonstrating that the phosphatase activity was ADP-dependent, but not ATP-dependent. We further quantified the production of C55OH by HPLC. In accordance with the TLC result, the addition of ADP caused earlier appearance of C55OH comparing with the ATP group (Figure 2D). In contrast to ADP and ATP, adenosine monophosphate (AMP) did not affect the dephosphorylation of C55P. Although ADP is not a substrate, it is an activator of the enzyme, and the activities were measured and used to calculate kinetic parameters in order to make a general comparison between the kinase and phosphatase activities. The apparent Km of ATP was first measured at a saturated concentration of C55OH and then the apparent Km of C55OH was measured at a saturated concentration of ATP (Figure 3A). The phosphatase activities were measured by a similar method (Figure 3B). The Kd value for ADP in the phosphatase reaction was determined to be 24.8 ± 5.9 µM, which was lower than the apparent Km for ATP in the kinase reaction (218.0 ± 44.6 µM) (Figure 3). The catalytic mechanism of the kinase activity was further confirmed to be consistent with the random bi-substrate model described by Copeland21 (Figure S4). The kinase reaction was much faster than the phosphatase reaction such that the apparent kcat value for the kinase activity of UK was 3.4 ± 0.2 s-1; whereas, 14


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 43

whereas the apparent kcat value for the phosphatase activity of UpP was determined as to be 0.5 ± 0.1 s-1. This result revealed that the kinase activity of UK/UpP is very efficient, and only a low concentration of ADP was enough to activate the phosphatase reaction.

Figure 3. Kinase and phosphatase activity catalyzed by UK/UpP (A) To measure the kinase reaction of UK/UpP, UK/UpP (100 nM) in 50 mM Tris, pH 8, and 10 mM MgCl2 was incubated with 15–2000 µM ATP and 6 mM C55OH (13.3%) (top) or 15-4000 µM C55OH and 2 mM ATP (bottom) in a mixed micelle (C55OH : LDAO = 1:7) for an incubation at 37°C. (B) To measure the phosphatase reaction, UK/UpP (100 nM) in 50 mM Tris, pH 8, and 10 mM MgCl2 was incubated with 7–1000 µM ADP and 2 mM C55P (top) or 7–1000 µM C55P and 2 mM ADP (bottom) in a mixed micelle (C55P : LDAO = 1:7) at 37°C. The mole fraction of undecaprenoids in LDAO micelles was calculated based on the formula of [C55OH or C55P] / ([C55OH or C55P] + [LDAO] - 1 mM) and the critical micelle concentration (CMC) of 15


Page 17 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

LDAO was approximately 1 mM. The measurement of ATP as well as undecaprenoids were performed by using the methods described in Materials and Methods. The rates were presented as average + standard deviation from three independent experiments. Km,app: the apparent Km value;

kcat,app: the apparent kcat value.

Substrate discrimination between kinase and phosphatase Several ATP analogues were used to investigate the phosphoryl transfer activity of UK/UpP (Figure S5). Adenosine 5’-(β,γ-imido)triphosphate (AMPPNP) and β,γmethyleneadenosine 5’-triphosphate (AMPPCP) are non-hydrolysable analogues. These two analogues have an imide or methylene group connecting the two terminal phosphates. 5’-(γ-Thio)triphosphate (ATPγS) is a slowly hydrolysable ATP analogue that has a thiophosphate group replacing the γ-phosphate group of ATP.22 Similar to other protein kinases, the kinase reaction of UK/UpP used ATPγS as substrate, but not AMPPNP or AMPPCP, demonstrating that the phosphoryl transfer reaction occurred at the γ-phosphate of ATP. The rate of C55OH phosphorylation in the presence of ATPγS was similar to that in the presence of ATP in the first 15 min. However, when the C55P dephosphorylation was started, the dephosphorylation activity was not observed in the reaction containing ATPγS, indicating that the thiophosphoryl group was not accepted by the phosphatase activity of UK/UpP. Consistently, it is widely reported that most protein kinases accept the non-physiological substrate ATPγS, but thiophosphorylated proteins tend to be poor substrates for protein phosphatases.23,24

The release of inorganic phosphate To confirm that the phosphoryl group of C55P was released into the solution during the phosphatase reaction of UK/UpP, we measured the concentration of Pi with 16


Biochemistry

malachite green. Indeed, the concentration of free phosphates increased up to 200 µM when UK/UpP was incubated with 200 µM C55P in the presence of ADP (Figure 4A). With ATP replacing ADP, the reaction containing 200 µM C55P and 100 µM ATP would result in the release of Pi at 300 µM, indicating that an extra 100 µM Pi was generated from ATP. The earlier production of Pi in the ATP group suggested an ATPase activity in the presence of C55P. It is worth noting that no Pi release was observed when the enzyme was incubated with ATP alone or C55P alone, implying that the phosphoryl transfer reaction of UK/UpP required the coexistence of lipids and nucleotides. A 400

C55P+ATP C55P+ADP

Released Pi (µ µ M)

300

ATP ADP C55P

200

blank 100

0 0

B

5

10

30 Time (min)

60

120

360

1.2 1.0 Pi

Pi turnove r (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 43

C55 P

0.8

αADP 0.6

βADP γATP

0.4

α ATP 0.2

βATP

0.0 0

100

200

300

Time (min)

Figure 4. The release of inorganic phosphates by UK/UpP (A) The amount of released free phosphate (Pi) during C55P dephosphorylation. UK/UpP (200 nM) was incubated with 200 µM (16.6 mol %) C55P in the presence of 100 µM ADP or ATP. At the indicated time points, the released Pi was detected by malachite green as described in Materials and Methods. The signal gradually increased in the presence of both C55P and nucleotides, and the addition of ATP generated more Pi. (B) Monitoring of various phosphates measured by 17


Page 19 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

time-resolved 31P NMR. Before measurement, the 17 µM enzyme was pipetted into a premixed 20 mM (13.4 mol %) C55OH and 40 mM ATP and then the solution was transferred to the NMR tube as described in the Materials and Methods. The 31P NMR signals were recorded at 310K as described in Materials and Methods.

Because the activities of UK/UpP involve two phases, hydrophobic lipids and hydrophilic nucleotides as well as inorganic phosphates, different methods should be used to detect individual substrates. To further explore the dynamics of the phosphoryl transfer reaction,

31

P-NMR was used to simultaneously monitor the movement of

phosphates (Figure 4B and S6). Because there is no other phospholipid present in the reaction, all

31

P signals comes from the transfer of γ-phosphate of ATP. The result

showed a gradual decrease of ATP signal, corresponding to an augmentation of ADP and Pi signals. The signal of C55P was increased at the beginning and subsequently declined. The preparation of reaction mixture and the operation of NMR took approximately 15‒20 min, so the NMR spectra were recorded at the end period of the kinase reaction, when the amount of C55P had almost reached the peak and started to decay. Based on the changes of C55P, the reaction can be divided into three stages: before reaching the peak of C55P (0‒20 min), after the peak of C55P but before exhausting the supply of ATP (20 min‒2 h), and after the exhaustion of ATP. The dominant reaction of the first stage was the kinase activity, when the γ-phosphate of ATP was efficiently transferred to C55OH. We were curious whether the ATPase activity might be accompanied with the kinase activity. However, at this stage, most of the γ-phosphate from ATP was transferred to C55OH, but not released as free phosphates. This indicated that the C55OH phosphorylation occurred prior to C55P, and the ATPase activity was not dominant in the presence of ATP and C55OH. In the second stage, the supply of ATP gradually decreased 18


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 43

with increased amount of ADP. In addition, the detected amount of C55P slowly decreased. The result indicated that both the kinase and phosphatase activities of UK/UpP were measured. In the third stage, ATP was exhausted and therefore the phosphatase reaction become dominant. As a result, an increase in Pi with a decrease in C55P was observed.

The in vivo activity of UK/UpP Members of the bacterial diacylglycerol kinase and undecaprenol kinase family are encoded by the dgkA gene. Since C55OH has not yet been reported in Gram-negative bacteria, we used E. coli as a model strain to monitor whether the S. mutans dgkA gene can contribute to the production of C55OH in vivo. In the HPLC analysis results shown in Figure 5, C55OH was detected in the Gram-positive bacteria S. aureus but not in the Gram-negative bacteria E. coli, similar to a reported study.11 However, when S. mutans dgkA was expressed in E. coli, an extra peak emerged at an elution time identical to the C55OH standard (Figure 5C). This peak was further isolated and demonstrated to be C55OH by TLC and mass spectrometry analysis (Figure S7). This result demonstrated that dgkA from S. mutans was indeed responsible for the C55P dephosphorylation activity in vivo and that the enzyme could function in different species from Gram-positive S. mutans to Gram-negative bacteria E. coli.

19


Page 21 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 5. Analysis of C55OH produced in E. coli expressing S. mutans dgkA(UK/UpP). Total membrane lipids were extracted from S. aureus (A), E. coli containing a control vector (B), or E. coli containing an expression plasmid for S. mutans-dgkA (C), and then analyzed by HPLC as described in Materials and Methods. The membrane lipids obtained from E. coli containing a control vector (B) were also spiked with pure C55OH and the samples were analyzed in parallel (D). "*" marked the elution of C55OH in chromatographs.

Critical residues for the kinase and phosphatase activity of UK/UpP Further, we would like to know the relationship between the kinase and the phosphatase activities. Although the size of UK is small, only 137 amino acids, it is uncertain whether the catalytic residues of the phosphatase activity are the same as those for the kinase activity. Mutagenesis was performed to evaluate the contribution of specific residues. Based on alignments of homologues from Gram-positive and Gram-negative bacteria (Figure S8), we selected to mutate conserved and polar amino acids and then analyzed the catalytic activity of the mutants. The activity of UK/UpP 20


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 43

containing the mutation D90A or D106A is less than 10% of the activity of wild type, and UK/UpP containing the mutations H44E, E79A, N82A or E86A showed little activity (Figure 6). Comparing the kinase and phosphatase activities of various mutants, D7A and E25A has slightly better phosphatase activity than the kinase activity, whereas T19A, S23A, H44A, S83A, K102A and K105A performed relatively better activity as kinase than as phosphatase. Despite some minor variations, in general, those with lower kinase activity also showed compromised phosphatase activity. Mutations that lead to the sole C55OH phosphorylation or C55P dephosphorylation activity were not observed, indicating that these two activities were indispensable to each other and could not be differentiated. Further, the conversion of H44 to alanine or to glutamic acid resulted in different activity profiles, suggesting that the electrostatic status around this residue might have an important influence on the enzyme activities.

Figure 6. The critical residues for kinase and phosphatase activities of UK/UpP 21


Page 23 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(A) The UK/UpP variants containing the mutation at certain amino acid (100 µM) were incubated with 200 µM (16.6 mol %) C55OH and 500 µM ATP for 10 min for the measurement of the UK activity or with 200 µM (16.6 mol %) C55P and 100 µM ADP for 60 min for the measurement of the UpP activity. The reaction products were analyzed by HPLC as described in Materials and Methods. The amount measured in the reaction using wild-type was defined as 100%. (B, C) The predicted structure of S. mutans UK/UpP was generated using homology modeling by SWISS-MODEL workspace based on the template of E. coli DgkA (PDB 4UXX). Shown is the view in the membrane plane (B) or the view from the top (C). The critical amino acids, E79, N82, E86, D90 and D106 were labeled and shown in stick. AMPPCP representing the nucleotide binding site was colored in green. The trimer UK/UpP was displayed with chains A, B and C colored in bronze, gray and cyan, respectively.

It was noted that residue T19 or S23 might act as acceptor for the phosphoryl group of C55P before the inorganic phosphate was released to the solution, because the enzyme showed a weak phosphatase activity without altering the kinase activity, and these two residues are located in the N-terminal amphiphilic surface helix and possibly are proximal to the active site based on the homology modeling. However, we also observed that the UK/UpP variants with mutation at T19 or S23 aggregated easily. Thus, to assess the possible existence of a phosphoenzyme intermediate and to avoid the bias from the in vitro assays, we evaluated the dephosphorylation activity of these mutants in vivo. If T19 or S23 was phosphorylated from C55P, mutation of these residues would abolish the dephosphorylation activity of UK/UpP. The in vivo activity was measured based on the analysis of the lipid extracts from E. coli DH5α harboring different UK/UpP mutants, including T19A and S23A, and then the presence of C55OH and C55P were analyzed by HPLC. Table 1 summarizes the production of C55OH by UK/UpP 22


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 43

mutants. Only UK/UpP mutants E79A, N82A, E86A, D106A lost their phosphatase activity, and E38A, D90A and K105A retained the phosphatase activity, as a low amount of C55OH can be detected. These results excluded the possibility that T19 and S23 were phosphorylated to form a phospho-enzyme intermediate. In addition, the UK/UpP variants containing the mutation(s) on the other potential phosphate-attacking nucleophilic amino acids, such as serine, threonine, tyrosine, histidine, aspartate, glutamate, lysine and arginine were still able to produce C55OH, suggesting that a covalent phospho-enzyme intermediate might not be part of the phosphatase mechanism. This result was consistent with the in vitro activity, demonstrating that E79, N82, E86 and D106 were the most important residues for the phosphatase activity.

Table 1. Production of C55OH in E. coli expressing UK/UpP mutants mutant vector wt D4A R6A D7A K9A S11A K13A K14A K16A R18A T19A

C55OH + + + + + + + + + + +

mutant T21A S22A S23A E25A T29A T33A K36A E37A E38A R39A K42A K43A

C55OH + + + + + + + + -/+ + + +

mutant H44A S47A K59A S61A E64A S71A T77A E79A N82A S83A E86A D90A

C55OH + + + + + + + + -/+

mutant S93A D94A Y95A H96A S98A K102A K105A D106A S116A T122A K130A H137A

C55OH + + + + + + -/+ + + + +

“+” : with C55OH signal “-”: extremely weak or undetectable C55OH signal “-/+”: weak C55OH signal

23


Page 25 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

DISCUSSION In our previous studies where S. mutans UK(dgkA) was used to prepare C55P for the enzymatic synthesis of lipid II, the transformation of C55P back to C55OH was observed after C55OH was phosphorylated.20 This phenomenon prompted us to further explore the origin of C55P dephosphorylation. Our results demonstrated that S. mutans UK, referred to as UK/UpP in this study, is not only an undecaprenol kinase, but also a phosphatase that catalyzes the dephosphorylation of C55P in a Mg-ADP-dependent manner.

The biological function of UK/UpP C55P, which serves as a carrier lipid to transfer a variety of phosphate-linked glycans from the cytosol to the periplasmic space, plays a vital role in cell wall biosynthesis. It can be produced from the dephosphorylation of C55PP, which is synthesized de novo from condensation of short lipids or regenerated from cell wall synthesis byproducts (Figure 1). Although C55OH does not participate in the de novo biosynthesis pathway, it is the major form of undecaprenoids found in many Gram-positive bacteria.1,7-10,12 The identification of an undecaprenol kinase with undecaprenyl phosphate phosphatase activity not only links C55OH to the center of the undecaprenyl lipid metabolism pathway but also suggests the origin of C55OH from C55P. As the center of this metabolism pathway, the blockage of C55P production can inhibit bacterial growth. In fact, binding to C55PP and thus blocking the formation of C55P is the inhibition mechanism of the antibiotic bacitracin.25 Mutation of UK/UpP increased bacitracin susceptibility in Streptococcus mutans,15 which is a major pathogen for dental cavity formation, indicating the important role of the kinase activity in the replenishment of C55P from C55OH. The enzyme UK/UpP might mediate the acquisition of phosphates under limiting 24


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 43

growth. The phosphatase activity produces free phosphates, which could be a source for sustaining growth during starvation. In light of this fact, the dgkA gene in many Gram-positive bacteria, including S. mutans, is part of a phosphate regulon.26 This regulon contains PhoH, which is a putative ATPase and was identified as a phosphate starvation-inducible gene.27 The release of inorganic phosphate from C55P is meaningful, as it will replenish the limiting phosphate reservoir under phosphate starvation. In addition, UK/UpP was shown to be required for acid-resistance in S. mutans,28,29 which is known to use glycolytic products to acidify the microenvironment and kill competitors. The ability of S. mutans to survive under acid conditions might be accomplished by the release of phosphates from C55P. Moreover, UK/UpP is also required for the biosynthesis of certain lantibiotics,30 and biofilm formation by this oral pathogen;31 thus, this gene is considered to be a virulence factor and an anti-cariogenic target.28,32 It is also essential for the sporulation of Bacillus subtilis.33 The conditional requirements for this gene in bacteria emphasize the regulation function of UK/UpP in membrane turnover and the adaption of bacteria to environmental changes. The dominance of kinase or phosphatase reaction of UK/UpP depends on the availability of substrates. In the case of lipids, it is reported that undecaprenol represents a major component of undecaprenoid lipids in the biological membrane of Gram-positive bacteria.7-10,12 The binding affinities of UK/UpP toward C55OH and C55P were similar, but the efficiency of the kinase activity was higher than that of the phosphatase activity (0.03 s-1µM-1 vs. 0.003 s-1µM-1). Thus, when the supply of ATP is sufficient, the kinase activity is the dominant function. With regard to the nucleotides, the activity could also be influenced by the ATP-to-ADP ratio, which might reflect the growth stage of the bacteria. It has been reported that the concentration of ATP increases significantly during cells' outgrowth from the stationary phase, and is relatively 25


Page 27 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

consistent in the exponential phase regardless of different doubling rates, and decreases in the stationary phase.34,35 At the beginning of cells' outgrowth, C55P is in high demand for cell wall synthesis, and thus, a fast conversion of C55P from C55OH reservoirs would benefit bacterial growth and proliferation. In contrast, when bacteria are resting, the excess C55P could be dephosphorylated back to its lipid reservoir and the cell wall synthesis activity is reduced. The coexistence of the kinase and phosphatase activity on the same enzyme seems to be an effective way of linking energy sources and cell wall synthesis. In addition to UK/UpP reported herein, several proteins have been reported to catalyze both forward and reverse reactions in the same domain. For example, histidine kinase can phosphorylate and dephosphorylate their downstream response regulators in response to a variety of different stimuli.36,37 The kinase reaction involved a covalent linkage of phosphate to the histidine residue of the enzyme, whereas UK/UpP performs a direct phosphoryltransfer mechanism. Another example is isocitrate dehydrogenase kinase/phosphatase (AceK), which controls the metabolic switch between the Krebs cycle and the glyoxylate bypass by phosphorylation or dephosphorylation of isocitrate dehydrogenase.38 AceK consists of a canonical eukaryotic kinase scaﬀold, and its phosphatase activity has an absolute requirement of ATP. AceK is allosterically regulated by metabolites, which induce the inactivation or activation of the kinase or phosphatase activity.39 In contrast to the observation that the kinase and phosphatase activities of UK/UpP are indispensible to each other, one single mutation on AceK that retains its kinase activity but abolishes the phosphatase activity has been reported.40,41 It is worth to mention that the dgkA homolog of the gram-positive bacteria Clostridium difficile encodes a conventional DGK/UK domain in the N-terminal fused with a C-terminal domain homologous to members of the type 2 phosphatidic acid 26


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 43

phosphatase. This C-terminal domain appears to function as an undecaprenyl pyrophosphate phosphatase.42 This protein was expected to produce C55P both by phosphorylating C55OH and by dephosphorylating the terminal phosphate of C55PP. Another possibility of C55OH formation is through the sequential cleavage of the two phosphates from C55PP.

The proposed catalytic mechanism for UK/UpP The structure of DgkA from E. coli has provided insights into the mechanism of UK/UpP. This homologous protein exhibits very low undecaprenol kinase activity and is responsible for the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid, which is used in a periplasmic membrane-derived oligosaccharide cycle in response to osmotic stimulation.43 It has served as a model protein to study membrane protein structure, folding, assembly and activities for more than half a century. Its crystal structure was solved and shown a very unique homo-trimer with individual monomers having three transmembrane domains and an N-terminal amphiphilic helix.44,45 DgkA contains three active sites where the individual active site cavity is surrounded by three transmembrane domains of one monomer and a vertical amphiphilic helix of an adjacent monomer. Despite catalyzing different lipid substrates from DgkA, it was found that S. mutans UK/UpP also forms a homo-trimer with a molecular weight of approximately 55 kDa (Figure S3), and these two have 37% sequence identity. Using homology modeling, the structure of UK/UpP was predicted and shown in Figures 5B and 5C. The important amino acids, E79, N82, E86, D90 and D106 located in TM2 and TM3 are embedded in the active sites of UK/UpP and are conserved among different species. The most conserved region among most Gram (+) and Gram (-) bacteria is between TM2 and TM3, and it was predicted to be the site for 27


Page 29 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

nucleotide binding. The C-terminus of TM1 and TM3 and the N-terminal helix, which might be involved in the interaction with lipids, are more diverse resulting in a different preference for lipid substrates between UK/UpP and DgkA (Figure S8). It is reported that E. coli's DgkA confers basal ATPase activity, especially in the absence of diacylglycerol lipid substrates. With the addition of diacylglycerol substrates, the basal ATPase was greatly diminished. In our results, the hydrolysis of ATP only occurred in the presence of C55P. In other words, ATP was not hydrolyzed alone. The difference might be caused by the usage of different micelle systems. In our earlier studies, it was found that the substrate specificity of the kinase activities of both DgkA and UK/UpP were low.20 In addition, certain detergents, such as Triton X-100, can be phosphorylated by UK/UpP.20,46 To avoid the interferences from detergents, LDAO and DDM detergents were used to dissolve lipids and UK/UpP in our studies. It is well established that DGK has high activity only in phospholipid-containig mixed micellles, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/dodecylphosphocholine (DPC) micelles as a model membrane.47-49 The addition of phospholipids, such as cardiolipin, to the membranes can also promote DgkA's catalytic activities.18,50-52 In terms of UK/UpP, the phospholipid-containing mixed micelles promote both the kinase activity and the phosphatase activity (Figure S2). It is possible that phospholipids induce DgkA or UK/UpP to an active state and promote the phosphoryl transfer reaction from the γ-phosphate of ATP. The phosphoryl transfer mechanism could be a direct transfer or involve an enzyme-phosphate intermediate. The recent co-crystal structure of DgkA from E. coli with AMPPCP and monoacylglycerol showed that the γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid,45 and biochemical studies also emphasized a direct in-line phosphoryl transfer mechanism with a random 28


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 43

equilibrium kinetic pattern.18,53 The structure and biochemical analysis of E. coli DgkA provided insights into the mechanism of UK/UpP. Because of the similarity between DgkA and UK/UpP in sequence and trimer conformation, the kinase activity of UK/UpP was expected to have a direct phosphoryl transfer mechanism (Figure 6). Under this mechanism, the critical amino acids required for both the kinase and phosphatase activities may involve E86 and N82 to stabilize the terminal phosphate of ATP through divalent cations, and D106 to coordinate with the ribose of nucleotides. E79, which served as a general base, initiated the kinase catalysis by abstracting a proton from the proximal hydroxyl of C55OH to attack the γ-phosphate of ATP to form C55P and ADP. After the release of products, the kinase reaction was complete. We proposed that phosphatase catalytic mechanism might involve water and β-phosphate of ADP. In this mechanism, the water molecule in the active site might be activated by the β-phosphate of ADP, which was stabilized by divalent cations, to hydrolyze the phosphate from C55P, which might be stabilized by E79 and N82. The sequence alignment showed that the most diverse region of UK/UpP is located on the N-terminal part. The structure of the N-terminus of DgkA is an amphiphilic surface helix. This helix is vertical to the three parallel transmembrane helixes that form the active-site cavity and is across the interface where lipid and nucleotide substrates interact. This region was relatively dynamic,44,45,54 so many positive charges of residues in the region of UK/UpP might therefore influence the entry and the departure of phosphate-bearing substrates and water. It will be interesting to further study its structure to reveal the interaction of phospholipids with this small enzyme.

29


Page 31 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 6. The proposed catalytic mechanism of UK/UpP. (A) The reaction of UK/UpP. Circles represented lipid pools (upper) and nucleotide pools (below) in the reaction. Gray arrows indicated the phosphoryl transfer direction. Based on the concentration of substrates, the reaction can be roughly separated to three stages, and the dominant activity was labeled. The γ-phosphate from ATP was labeled in red. (B) The catalytic E79 residue may abstract a proton from the primary hydroxyl of lipid substrate C55OH to facilitate its attack on the γ-phosphorus of ATP, which may coordinate with magnesium ions, and stabilized by E86 and N82. The ADP produced coordinates with Mg2+ activated H2O, leading to the hydrolysis of the phosphate of C55P to produce C55OH and Pi. The transversion between kinase and phosphatase is a sum of different activities, which was still not clear.

CONCLUSION In summary, an ADP-dependent undecaprenyl phosphate phosphatase activity was found in S. mutans' undecaprenol kinase with the same active site. The activities of this enzyme required both lipids and nucleotides and were regulated by the presence of ATP 30


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 43

or ADP in the environment. In addition to the known de novo and recycling pathways, this bifunctional enzyme provides an alternative source for C55P production and storage and enables an efficient control of bacterial cell wall biosynthesis through regulating the undecaprenoid pool.

ASSOCIATED

CONTENT

Supporting information The Supporting Information is available free of charge on the ACS website. The supporting information contains figures showing the reaction of UK/UpP in phospholipid containing micelles (Fig. S1), detection of pyrophosphatase and general protein phosphatase activities of UK/UpP (Fig. S2), the purity and oligomeric status of recombinant UK/UpP (Fig. S3), the kinetics characterization of the kinase and phosphatase reaction of UK/UpP (Fig. S4), the substrate specificities of ATP analogues for UK/UpP (Fig. S5), the phosphoryl transfer reaction of UK/UpP (Fig. S6), detection of C55OH in the membrane extracts from E. coli expressing S. mutans dgkA by TLC and mass spectrometry (Fig. S7), alignment of the amino acid sequences of the UK/UpP and DGK enzymes (Fig. S8), and the production of C55OH in E. coli expressing S. mutans-dgkA mutants (Fig. S9)

AUTHOR INFORMATION 31


Page 33 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Funding This project is supported by Academia Sinica. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Dr. Ming-Daw Tsai and Dr. Tsung-Lin Li for their valuable suggestions on experiments. We also thank Dr. Wei-Chieh Cheng for C55P and Ms. Ya-Chih Chang for assistance in HPLC analysis.

32


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 43

REFERENCES 1.

Hartley, M. D., and Imperiali, B. (2012) At the membrane frontier: A prospectus on the

remarkable

evolutionary

conservation

of

polyprenols

and

polyprenyl-phosphates. Arch. Biochem. Biophys. 517, 83. 2.

van Heijenoort, J. (1998) Assembly of the monomer unit of bacterial peptidoglycan. Cell Mol. Life Sci. 54, 300.

3.

Liang, P. H., Ko, T. P., and Wang, A. H. (2002) Structure, mechanism and function of prenyltransferases. Eur. J. Biochem. 269, 3339.

4.

El Ghachi, M., Bouhss, A., Blanot, D., and Mengin-Lecreulx, D. (2004) The bacA gene of Escherichia coli encodes an undecaprenyl pyrophosphate phosphatase activity. J. Biol. Chem. 279, 30106.

5.

El Ghachi, M., Derbise, A., Bouhss, A., and Mengin-Lecreulx, D. (2005) Identification of multiple genes encoding membrane proteins with undecaprenyl pyrophosphate phosphatase (UppP) activity in Escherichia coli. J. Biol. Chem. 280, 18689.

6.

Manat, G., Roure, S., Auger, R., Bouhss, A., Barreteau, H., Mengin-Lecreulx, D., and Touze, T. (2014) Deciphering the metabolism of undecaprenyl-phosphate: the bacterial cell-wall unit carrier at the membrane frontier. Microb. Drug Resist. 20, 199. 33


Page 35 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

7.

Thorne, K. J., and Kodicek, E. (1966) The structure of bactoprenol, a lipid formed by lactobacilli from mevalonic acid. Biochem. J. 99, 123.

8.

Gough, D. P., Kirby, A. L., Richards, J. B., and Hemming, F. W. (1970) The characterization of undecaprenol of Lactobacillus plantarum. Biochem. J. 118, 167.

9.

Umbreit, J. N., Stone, K. J., and Strominger, J. L. (1972) Isolation of polyisoprenyl alcohols from Streptococcus faecalis. J. Bacteriol. 112, 1302.

10. Vilim, A., Woods, M. C., and Carroll, K. K. (1973) Polyprenols of Listeria monocytogenes. Can. J. Biochem. 51, 939. 11. Barreteau, H., Magnet, S., El Ghachi, M., Touze, T., Arthur, M., Mengin-Lecreulx, D., and Blanot, D. (2009) Quantitative high-performance liquid chromatography analysis of the pool levels of undecaprenyl phosphate and its derivatives in bacterial membranes. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877, 213. 12. Higashi, Y., Strominger, J. L., and Sweeley, C. C. (1970) Biosynthesis of the peptidoglycan of bacterial cell walls. XXI. Isolation of free C55-isoprenoid alcohol and of lipid intermediates in peptidoglycan synthesis from Staphylococcus aureus. J. Biol. Chem. 245, 3697. 13. Willoughby, E., Highasi, Y., and Strominger, J. L. (1972) Enzymatic dephosphorylation of C55 -isoprenylphosphate. J. Biol. Chem. 247, 5113.

34


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 43

14. Sandermann, H., Jr., and Strominger, J. L. (1972) Purification and properties of C 55 -isoprenoid alcohol phosphokinase from Staphylococcus aureus. J. Biol. Chem. 247, 5123. 15. Lis, M., and Kuramitsu, H. K. (2003) The stress-responsive dgk gene from Streptococcus mutans encodes a putative undecaprenol kinase activity. Infect. Immun. 71, 1938. 16. Hartley, M. D., Larkin, A., and Imperiali, B. (2008) Chemoenzymatic synthesis of polyprenyl phosphates. Bioorg. Med. Chem. 16, 5149. 17. Jerga, A., Lu, Y. J., Schujman, G. E., de Mendoza, D., and Rock, C. O. (2007) Identification of a soluble diacylglycerol kinase required for lipoteichoic acid production in Bacillus subtilis. J. Biol. Chem. 282, 21738. 18. Walsh, J. P., and Bell, R. M. (1986) sn-1,2-Diacylglycerol kinase of Escherichia coli. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence. J. Biol. Chem. 261, 6239. 19. Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., and Candia, O. A. (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100, 95.

35


Page 37 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

20. Huang, L. Y., Huang, S. H., Chang, Y. C., Cheng, W. C., Cheng, T. J., and Wong, C. H. (2014) Enzymatic synthesis of lipid II and analogues. Angew. Chem. Int. Ed. Engl. 53, 8060. 21. Copeland, R. A. (2004) Enzyme reactions with multiple substrates. Enzymes: a practical introduction to structure, mechanism and data analysis (Second ed.). John Wiley & Sons, p350-366. 22. Bagshaw, C. (2001) ATP analogues at a glance. J. Cell Sci. 114, 459. 23. Eckstein, F. (1985) Nucleoside phosphorothioates. Annu. Rev. Biochem. 54, 367. 24. Cassel, D., and Glaser, L. (1982) Resistance to phosphatase of thiophosphorylated epidermal growth factor receptor in A431 membranes. Proc. Natl. Acad. Sci. U. S. A. 79, 2231. 25. Stone, K. J., and Strominger, J. L. (1971) Mechanism of action of bacitracin: complexation with metal ion and C55 -isoprenyl pyrophosphate. Proc. Natl. Acad. Sci. U. S. A. 68, 3223. 26. Kazakov, A. E., Vassieva, O., Gelfand, M. S., Osterman, A., and Overbeek, R. (2003) Bioinformatics classification and functional analysis of PhoH homologs. In Silico Biol. 3, 3.

36


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 43

27. Kim, S. K., Makino, K., Amemura, M., Shinagawa, H., and Nakata, A. (1993) Molecular analysis of the phoH gene, belonging to the phosphate regulon in Escherichia coli. J. Bacteriol. 175, 1316. 28. Shibata, Y., van der Ploeg, J. R., Kozuki, T., Shirai, Y., Saito, N., Kawada-Matsuo, M., Takeshita, T., and Yamashita, Y. (2009) Kinase activity of the dgk gene product is involved in the virulence of Streptococcus mutans. Microbiology 155, 557. 29. Yamashita, Y., Takehara, T., and Kuramitsu, H. K. (1993) Molecular characterization of a Streptococcus mutans mutant altered in environmental stress responses. J. Bacteriol. 175, 6220. 30. Chen, P., Novak, J., Qi, F., and Caufield, P. W. (1998) Diacylglycerol kinase is involved in regulation of expression of the lantibiotic mutacin II of Streptococcus mutans. J. Bacteriol. 180, 167. 31. Yoshida, A., and Kuramitsu, H. K. (2002) Multiple Streptococcus mutans Genes Are Involved in Biofilm Formation. Appl. Environ. Microbiol. 68, 6283. 32. Shibata, Y., Kawada-Matsuo, M., Shirai, Y., Saito, N., Li, D., and Yamashita, Y. (2011) Streptococcus mutans diacylglycerol kinase homologue: a potential target for anti-caries chemotherapy. J. Med. Microbiol. 60, 625.

37


Page 39 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

33. Amiteye, S., Kobayashi, K., Imamura, D., Hosoya, S., Ogasawara, N., and Sato, T. (2003) Bacillus subtilis diacylglycerol kinase (DgkA) enhances efficient sporulation. J. Bacteriol. 185, 5306. 34. Murray, H. D., Schneider, D. A., and Gourse, R. L. (2003) Control of rRNA expression by small molecules is dynamic and nonredundant. Mol. Cell 12, 125. 35. Schneider, D. A., and Gourse, R. L. (2004) Relationship between growth rate and ATP concentration in Escherichia coli: a bioassay for available cellular ATP. J. Biol. Chem. 279, 8262. 36. Baker, M. D., Wolanin, P. M., and Stock, J. B. (2006) Signal transduction in bacterial chemotaxis. Bioessays 28, 9. 37. Igo, M. M., Ninfa, A. J., Stock, J. B., and Silhavy, T. J. (1989) Phosphorylation and dephosphorylation of a bacterial transcriptional activator by a transmembrane receptor. Genes Dev. 3, 1725. 38. LaPorte, D. C., and Koshland, D. E., Jr. (1982) A protein with kinase and phosphatase activities involved in regulation of tricarboxylic acid cycle. Nature 300, 458. 39. Zheng, J., Yates, S. P., and Jia, Z. (2012) Structural and mechanistic insights into the bifunctional enzyme isocitrate dehydrogenase kinase/phosphatase AceK. Philos. Trans. R. Soc. Lond. B Biol. Sci. 367, 2656. 38


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 43

40. Zheng, J., and Jia, Z. (2010) Structure of the bifunctional isocitrate dehydrogenase kinase/phosphatase. Nature 465, 961. 41. Wang, S., Shen, Q., Chen, G., Zheng, J., Tan, H., and Jia, Z. (2014) The phosphatase mechanism of bifunctional kinase/phosphatase AceK. Chem. Commun. (Camb) 50, 14117. 42. Bernard, R., El Ghachi, M., Mengin-Lecreulx, D., Chippaux, M., and Denizot, F. (2005) BcrC from Bacillus subtilis acts as an undecaprenyl pyrophosphate phosphatase in bacitracin resistance. J. Biol. Chem. 280, 28852. 43. Van Horn, W. D., and Sanders, C. R. (2012) Prokaryotic diacylglycerol kinase and undecaprenol kinase. Annu. Rev. Biophys. 41, 81. 44. Li, D., Lyons, J. A., Pye, V. E., Vogeley, L., Aragao, D., Kenyon, C. P., Shah, S. T., Doherty, C., Aherne, M., and Caffrey, M. (2013) Crystal structure of the integral membrane diacylglycerol kinase. Nature 497, 521. 45. Li, D., Stansfeld, P. J., Sansom, M. S., Keogh, A., Vogeley, L., Howe, N., Lyons, J. A., Aragao, D., Fromme, P., Fromme, R., Basu, S., Grotjohann, I., Kupitz, C., Rendek, K., Weierstall, U., Zatsepin, N. A., Cherezov, V., Liu, W., Bandaru, S., English, N. J., Gati, C., Barty, A., Yefanov, O., Chapman, H. N., Diederichs, K., Messerschmidt, M., Boutet, S., Williams, G. J., Marvin Seibert, M., and Caffrey, M.

39


Page 41 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(2015) Ternary structure reveals mechanism of a membrane diacylglycerol kinase. Nat. Commun. 6, 10140. 46. Walsh, J. P., Fahrner, L., and Bell, R. M. (1990) sn-1,2-diacylglycerol kinase of Escherichia coli. Diacylglycerol analogues define specificity and mechanism. J. Biol. Chem. 265, 4374. 47. Ullrich, S. J., Hellmich, U. A., Ullrich, S., and Glaubitz, C. (2011) Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR. Nat. Chem. Biol. 7, 263. 48. Czerski, L., and Sanders, C. R. (2000) Functionality of a membrane protein in bicelles. Anal. Biochem. 284, 327. 49. Koehler, J., Sulistijo, E. S., Sakakura, M., Kim, H. J., Ellis, C. D., and Sanders, C. R. (2010) Lysophospholipid micelles sustain the stability and catalytic activity of diacylglycerol kinase in the absence of lipids. Biochemistry 49, 7089. 50. Bohnenberger, E., and Sandermann, H., Jr. (1983) Lipid dependence of diacylglycerol kinase from Escherichia coli. Eur. J. Biochem. 132, 645. 51. Walsh, J. P., and Bell, R. M. (1986) sn-1,2-Diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence. J. Biol. Chem. 261, 15062.

40


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 43

52. Russ, E., Kaiser, U., and Sandermann, H., Jr. (1988) Lipid-dependent membrane enzymes. Purification to homogeneity and further characterization of diacylglycerol kinase from Escherichia coli. Eur. J. Biochem. 171, 335. 53. Badola, P., and Sanders, C. R. (1997) Escherichia coli diacylglycerol kinase is an evolutionarily optimized membrane enzyme and catalyzes direct phosphoryl transfer. J. Biol. Chem. 272, 24176. 54. Van Horn, W. D., Kim, H. J., Ellis, C. D., Hadziselimovic, A., Sulistijo, E. S., Karra, M. D., Tian, C., Sonnichsen, F. D., and Sanders, C. R. (2009) Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase. Science 324, 1726.

41


Page 43 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

GRAPHIC FOR THE TABLE OF CONTENTS

42


Undecaprenyl Phosphate Phosphatase Activity of Undecaprenol

Recommend Documents