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Kinetic Characterization of PA1225 from Pseudomonas aeruginosa PAO1 Reveals a New NADPH:Quinone Reductase Elias Flores, and Giovanni Gadda Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00090 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 1, 2018

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Biochemistry

PA1225, a novel FAD-containing NADPH:quinone Reductase

Kinetic Characterization of PA1225 from Pseudomonas aeruginosa PAO1 Reveals a New NADPH:Quinone Reductase Elias Flores1 and Giovanni Gadda1234* 1

Department of Chemistry, 2Department of Biology, 3Center for Diagnostics and Therapeutics, 4 Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30302, United States Running Title: PA1225, a novel FAD-containing NADPH:quinone Reductase

* Corresponding Author Prof. Giovanni Gadda, Department of Chemistry, Georgia State University, PO Box 3965, Atlanta, GA 30302-3965, U.S.A., Telephone: 404-413-5537; E-mail: [email protected].

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ABSTRACT The gene pa1225 of Pseudomonas aeruginosa strain PAO1 was cloned and the resulting enzyme (PA1225) was purified and revealed to be an NADPH:quinone reductase. By using kinetics, fluorescence, and mass spectrometric analyses, PA1225 was shown to utilize FAD to transfer a hydride ion from NADPH to quinones. The enzyme could also use NADH, but with an efficiency that was 40-fold lower than NADPH as suggested by the kcat/Km values at pH 6.0. Similar initial rates of reaction were determined with 1,4-benzoquinone or 2,6-dimethoxy-1,4benzoquinone in the range between 25 and 200 µM, suggesting a low Km value for the quinone oxidizing substrate. Lack of inhibition by NADP+ versus NADPH at saturating concentrations of 1,4-benzoquinone was consistent with a Ping-Pong Bi-Bi mechanism. The reductive halfreaction at pH 6.0 had Kd values of 0.07 mM with NADPH and 1.8 mM with NADH; the kred for flavin reduction was pH independent with values of ~10 s-1 with NADPH and ~5 s-1 with NADH. Thus, the enzyme specificity for the reducing substrate arises primarily from a tighter binding of NADPH versus NADH. At pH 6.0, the kcat value with NADPH and 1,4-benzoquinone was 10.1 s1

, consistent with the hydride transfer from NADPH to FAD being fully-rate limiting for the

overall turnover of the enzyme. The enzyme showed negligible NADPH oxidase and azoreductase activities. This study enables annotation of the pa1225 gene as NADPH:quinone reductase, elucidates the enzymatic function of PA1225 in P. aeruginosa PAO1, and establishes that PA1225 is not an azoreductase as previously proposed.

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INTRODUCTION The latest information (2013) from the Center for Disease Control and Prevention (CDC) reports that 2 million people become infected with drug resistant bacteria each year in the United States with at least 23,000 deaths each year resulting from infection. The National Nosocomial Infections Surveillance System reported Pseudomonas aeruginosa as the leading cause of nosocomial infections.1,

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P. aeruginosa is responsible for 10% of all hospital-acquired

infections2 for which the CDC reports an estimated 51,000 healthcare-associated infections occur in the United States each year and more than 13% of P. aeruginosa are multi-drug resistant. To add to the challenges of treating infection, recent studies from P. aeruginosa infection collected from patients with cystic fibrosis show subpopulations undergo high phenotypic diversification over time.3,

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Furthermore, a recent study of whole genome sequence analyses of 185 P.

aeruginosa isolates from hospitalized patients suggests that characteristic drug resistance genes vary depending on geographical location of exposure.5 Therefore new strategies for combatting Pseudomonas are needed that are less dependent on antibiotics. Whole genome sequencing has been applied towards this aim, but its utility is greatly hampered by incorrect or superficial annotation. Computational algorithms, although a valuable tool in the arsenal of biologists, mis-annotate a significant fraction of genes and require functional and biochemical validation. It is estimated that the functional annotation of prokaryotic genes based on biochemical evidence represents 2,000 genes out of the 5,570 ORFs identified in P. aeruginosa PAO1 remained annotated as “hypothetical proteins” in 2016.8, 10 One of the hypothetical proteins of P. aeruginosa, PA1225, is currently annotated as an NAD(P)H dehydrogenase with a flavodoxin-like fold. Based on ~20-30% amino acid sequence identity to human NAD(P)H:quinone reductase 1 (NQO1) and NRH:quinone oxidoreductase (NQO2), one could speculate that PA1225 possesses a similar activity.11,

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Our interest in

PA1225 stems from the gene encoding for PA1225 being upregulated 80-fold by the deletion of the LysR regulator PA4203.13 Such a deletion also upregulates by 10- and 16-fold the genes encoding for a hypothetical heme oxygenase and a Class I nitronate monooxygenase (NMO).13 NMO is a well-characterized enzyme that oxidizes the mitochondrial toxin propionate 3nitronate to malonate semialdehyde, which is proposed to be further converted enzymatically to acetate.14 No direct binding of LysR to the upstream intergenic region of the monocistronic pa1225 gene was seen in electrophoretic mobility shift assays13, suggesting that overexpression of PA1225 may be due to the expression of NMO. These results suggest that PA1225 may be involved in detoxification mechanisms. However, in the absence of a biochemical validation of the enzymatic activity the possible role of PA1225 in detoxification cannot be formulated. In this study, the pa1225 gene was cloned with a C-terminal polyhistidine tag, overexpressed in Escherichia coli Rosetta(DE3)pLysS and the resulting enzyme was purified using a nickelNTA column. Potential reducing and oxidizing substrates were investigated using steady-state kinetics. The oxidation of NADPH and NADH by PA1225 was studied using rapid kinetics as a function of pH to gain insights on the steps of flavin reduction and substrate binding. The results established the enzymatic mechanism of PA1225 and support the conclusion that PA1225 is an NADPH:quinone reductase without azoreductase activity.

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MATERIALS AND METHODS Materials. Genomic DNA of P. aeruginosa PAO1 was a kind gift from Dr. Jim Spain, Georgia Institute of Technology, Atlanta, GA. The enzymes NdeI, XhoI, EcoRI, calf intestinal alkaline phosphatase, and T4 DNA ligase were from New England Biolabs (Ipswich, MA). Phosphodiesterase I was purchased from Worthington (Lakewood, NJ). Pfu DNA polymerase, DNase, and RNase were purchased from Agilent (Santa Clara, CA). Oligonucleotides for PCR were obtained from Sigma Genosys (The Woodlands, TX). The QIAprep Spin Miniprep Kit and the QIAquick PCR Purification Kit were obtained from Qiagen (Valencia, CA). Escherichia coli DH5α was purchased from Life Technologies, Inc. E. coli Rosetta(DE3)pLysS and the expression vector pET20b(+) were from Novagen (Madison, WI). HiTrapTM chelating HP 5-mL affinity column and prepacked PD-10 desalting columns were purchased from GE Healthcare (Piscataway, NJ). IPTG was purchased from Promega (Madison, WI). Quinones were purchased from Sigma-Aldrich (St. Louis, MO). NADH and NADPH disodium salts were purchased from VWR (Radnor, PA). All other reagents were of the highest purity commercially available. Cloning. The pa1225 gene was amplified from the genomic DNA of P. aeruginosa strain PAO1 by PCR in the presence of 6% DMSO and ligated to expression vector pET20b(+) using standard cloning PCR procedures. The primers incorporated the sequences encoding for Nde1 and EcoR1 endonuclease sites. 1 U Pfu DNA polymerase was used. DNA bands, visualized by 1% agarose gel electrophoresis, correlated with the expected length of 627 nucleotides, confirming the amplification of pa1225 DNA. The amplicon and pET20b(+) vector were independently digested at 37 oC with 1 U NdeI and 1 U EcoRI for 2 h followed by purification using QIAprep PCR Purification Kit. The pET20b(+) plasmid was further dephosphorylated with 1 U calf intestine alkaline phosphatase for 1 h at 37 oC and the purified using QIAprep PCR 5


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Purification Kit. The pET20b(+) vector and pa1225 gene were ligated by incubation at 16 oC with 1 U T4 ligase. The pa1225/pET20b(+) recombinant DNA was transformed into chemically competent E. coli strains DH5α or Rosetta(DE3)pLysS using heat shock at 42 oC for 30 s. Colonies grown at 37 oC on Luria-Bertani agar plates containing 50 µg/mL ampicillin were screened for the presence of the desired insert by using Macrogen DNA sequencing services (Rockville, MD). The DNA sequencing confirmed the correct insertion of the gene in the plasmid vector without undesired mutations. To incorporate a poly-histidine tag at the C-terminus of the protein the recombinant plasmid pa1225/pET20b(+) was used as template for subcloning to remove the EcoRI restriction site and the stop codon and incorporate an XhoI restriction site plus a C-terminal his-tag. Standard cloning PCR procedures was used as described above. The DNA sequencing confirmed the correct insertion of the gene in the plasmid vector without undesired mutations. Enzyme Purification. E. coli expression strain Rosetta(DE3)pLysS transformed with pa1225/pET20b(+) was used to inoculate 1.5 L of Luria-Bertani broth containing 100 µg/mL ampicillin and 34 µg/mL chloramphenicol and incubated at 37 oC for 18 h with shaking at 200 rpm. When the cell culture reached an O.D.600 of 0.6, IPTG was added to a final concentration of 0.2 mM and the temperature was lowered to 18 oC. After ∼20 h, the cells were harvested by spinning down at 12,000 x g for 25 min and the resulting wet cell paste was resuspended with 50 mL lysis buffer containing 10 mM imidazole, 200 mM NaCl, 10% (v/v) glycerol, 5 mM MgCl2, 1 mM PMSF, 2 mg/mL lysozyme, 5 µg/mL DNase, and 5 µg/mL RNase, pH 8.0. The resuspended cells were subjected to 20 min sonication while on ice. After centrifugation at 12,500 x g for 25 min to obtain the cell-free extract, the sample was loaded onto a HiTrapTM chelating HP 5 mL column equilibrated with Buffer A (20 mM sodium phosphate, 200 mM

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NaCl, 10 mM imidazole, 10% glycerol (v/v), pH 8.0). The column was washed with 5 column volumes of 10% Buffer B (Buffer A + 500 mM imidazole) at a flow rate of 4 mL/min. PA1225 was eluted with 40% Buffer B, dialyzed against 20 mM Tris-Cl, 150 mM NaCl, 10% glycerol (v/v), and stored at -20 oC. Cofactor Identification. Fluorescence emission spectra were recorded in 0.11 M Tris-Cl, 0.11 M NaCl, and 15 MgCl2 at pH 8.9 using a Shimadzu model RF-5301 PC spectrofluorometer (Kyoto, Japan) with 1 cm path length quartz at 25 oC. The release of PA1225 cofactor was achieved by heating at 100 oC for 15 minutes followed by centrifugation at 20,000 x g for 20 min to remove denatured protein. The released cofactor was passed through a prepacked, disposable PD10 column for buffer exchange with 0.11 M Tris-HCl, 0.11 M NaCl, and 15 mM MgCl2 at pH 8.9, which are the conditions in which phosphodiesterase I works optimally. The concentration of free flavin was calculated by measuring the absorbance at 450 nm after the solution was brought to a final volume of 3 mL. PA1225 at 2 µM was excited at 460 nm (444 nm for free flavin), and the emission scan was determined from 460 to 650 nm. After 1 U phosphodiesterase I was added to the cofactor sample, the fluorescence emission and excitation were recorded for 40 min until no change in peak intensity was observed. A positive control using 2 µM FAD was used to ensure phosphodiesterase I was active. The extracted cofactor was independently analyzed by mass spectrometry. The cofactor was extracted by heat denaturation at 100 oC for 20 min, followed by centrifugation and desalting by passing through a PD10 column equilibrated with water. The extracted flavin (5 µM) was mixed with α-cyano-4-hydroxycinnamic acid and analyzed on a Bruker Daltonics ultrafleXtreme MALDI-TOF in negative ion mode at the Core Facility of Georgia State University.

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Spectroscopic Properties. UV-visible absorption spectra were recorded with an Agilent Technologies diode-array spectrophotometer model HP 8453 PC (Santa Clara, CA) equipped with a thermostated water bath in 20 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 8.0, at 25 o

C. The extinction coefficient of the enzyme-bound flavin was determined by heating PA1225 at

100 oC for 20 min, followed by centrifugation at 20,000 x g for 20 min to remove the denatured protein, and recording of the UV-visible absorption spectrum of the extracted flavin. The concentration of the extracted FAD was determined spectroscopically by using the extinction coefficient of free FAD Ԑ450 = 11,300 M-1 cm-1.15 The extinction coefficient of enzyme-bound flavin was experimentally calculated by taking the ratio of A450/ Ԑ450 value for the extracted FAD and multiplying by the A460 value of the enzyme-bound flavin, yielding Ԑ460 = 10,100 M-1cm-1. The concentration of NAD(P)H was determined spectroscopically by using Ԑ340 = 6,220 M-1cm-1 and that of methyl red by using Ԑ430 = 23,360 M-1cm-1.

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The total protein concentration was

quantitated using Bradford assay with bovine serum album as a standard.17 Enzymatic Activity. Initial rates of reaction were normalized based on the concentration of enzyme-bound flavin, and were determined in 20 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 6.0 and 25 oC. The buffer was exchanged by passage of the enzyme over a PD-10 column that had been equilibrated in buffer just prior to use. The final concentration of enzyme in the assays was 0.4 µM. When the concentration of NADH or NADPH was varied, a fixed saturating concentration of 0.2 mM 1,4-benzoquinone was used. The quinone reductase activity was tested at varying concentrations of 1,4-benzoquinone, tetramethyl-1,4-benzoquinone, 2,6dimethoxy-1,4-benzoquinone, Coenzyme Qo, or 1,4-naphthoquinone, and a fixed concentration of 0.2 mM NADPH or NADH. With the exception of 2,6-dimethoxy-1,4-benzoquinone, which was prepared in DMSO, all quinones were prepared in 200-proof ethanol; quinone

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Biochemistry


concentrations were determined spectroscopically. The initial rates were normalized by subtracting control reactions in the absence of enzyme and were carried out in triplicate. The oxidase activity of PA1225 was assayed at atmospheric oxygen, i.e., 253 µM, with 0.2 mM NADPH as reducing substrate. The azoreductase activity was assayed with methyl red at a concentration of 0.1 mM or 0.2 mM and a fixed concentration of 0.2 mM NADPH. Flavin-monitored Turnover and Reductive Half-Reaction. All stopped-flow studies were performed at 25 oC using a Hi-Tech Scientific SF-61 DX2 stopped-flow spectrophotometer, equipped with a photo-diode array or a photomultiplier detector, under aerobic conditions. Flavin-monitored turnover of the enzyme was followed by monitoring the changes in the absorbance at 460 nm over 4 min upon mixing aerobically PA1225 with NADPH in 20 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 6.0; concentrations of enzyme and substrates after mixing were: 11 µM PA1225, 40 µM NADPH, and 253 µM oxygen. The reductive half-reaction was carried out at varying concentrations of NADPH or NADH under pseudo first-order conditions where the concentration after mixing of the enzyme was ~10 µM and that of the reducing substrate was between 0.5 and 15.0 mM (NADH) or 0.5 and 8.0 mM (NADPH). The enzyme solution was equilibrated by passing through a PD 10 column against 20 mM sodium phosphate, pH 6.0, 20 mM Tris-HCl, pH 8.0 and 9.0, or 20 mM sodium pyrophosphate, pH 10.0, containing 150 mM NaCl and 10% glycerol. Equal volumes of enzyme and reducing substrate were mixed in the stopped-flow spectrophotometer following established procedures. In all cases, the range of substrate concentrations was chosen to ensure proper determination of the Kd value and each measurement was carried out in triplicate. Steady-State Kinetic Mechanism. The steady-state kinetic mechanism could not be determined by changing the concentrations of both NADPH and 1,4-benzoquinone because the

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initial rates of reaction were the same with 1,4-benzoquinone between 25 and 200 µM. Instead, the inhibition pattern of NADP+ versus NADPH was determined by varying NADP+ from 0 to 800 µM and NADPH from 25 to 100 µM at a fixed, saturating 1,4-benzoquinone concentration of 200 µM in 20 mM sodium phosphate, 150 mM NaCl, pH 6.0 and 25 oC. Data Analysis. Kinetic data were fit using KaleidaGraph (Synergy Software, Reading, PA) and Kinetic Studio software suite Enzfitter (Hi-Tech Scientific, Bradford on Avon, U.K.). The steady-state kinetic parameters at varying concentrations of NADPH and fixed concentration of quinone were determined by using the Michaelis-Menten equation for a single substrate. Stopped-flow traces of time-resolved flavin reduction were fit to Eq. 1, which describes a single-exponential process for flavin reduction where kobs represents the observed first-order rate constant for the reduction of the enzyme-bound flavin associated with the absorption changes at 460 nm, A represents the absorbance at 460 nm at any given time, B is the amplitude of the decrease in absorbance, t is the time, and C is the offset value at infinite time that accounts for the non-zero absorbance of the fully reduced enzyme bound flavin. The concentration dependence of the observed rate constants for flavin reduction was analyzed with Eq. 2, which describes a hyperbolic trend where kobs represents the observed firstorder rate constant for the reduction of the enzyme-bound flavin at any given substrate concentration (S), kred is the limiting first-order rate constant for flavin reduction at saturating substrate concentrations and Kd is the apparent equilibrium constant for the dissociation for the enzyme-substrate complex into free substrate and enzyme. The pH dependence of the Kd values with NADPH or NADH as a reducing substrate was fit using Eq. 3, which describes a curve with two plateau regions; Kd(L) and Kd(H) indicate lower and

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Biochemistry


higher limiting values at low and high pH, respectively, and Ka is the dissociation constant of the ionizable group. = + kobs =

(1)

kred S

(2)

Kd + S

logKd =log

() ()

(3)

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RESULTS Enzyme Purification. The pa1225 gene was initially cloned from the genomic DNA of Pseudomonas aeruginosa PAO1, and the gene product PA1225 was purified using DEAE anionexchange chromatography. During purification, the protein-bound cofactor was progressively released from the protein during exposure to high salt concentrations of ammonium sulfate and elution from the column with 1 M NaCl (data not shown). SDS-PAGE analysis of the partially purified enzyme also revealed several protein bands, indicating that the enzyme preparation would require further purification steps needed to isolate PA1225 for biochemical studies. For these reasons, an alternate strategy to produce and isolate the protein was considered. To shorten purification time and minimize salt exposure, which resulted in the loss of cofactor during purification, the pa1225 gene was sub-cloned from plasmid pET20b(+)/pa1225 to incorporate a poly-histidine tag at the C-terminus of the protein. The recombinant PA1225 protein with an engineered poly-histidine tag was overexpressed in Escherichia coli strain Rosetta(DE3)pLysS and was purified with a HiTrapTM chelating High-Performance affinity column as determined by SDS-PAGE (Figure S1). The purified enzyme was stable and active for several months when stored in 20 mM Tris-HCl, 150 mM NaCl, 10% (v/v) glycerol, pH 8.0, at 20 oC. Cofactor Identification. PA1225 is annotated in the Pfam consortium database19 as an NAD(P)H-dehydrogenase with a flavodoxin-like fold, suggesting the presence of either FMN or FAD as a cofactor. Consistent with the enzyme being a flavoprotein, the UV-visible absorption spectrum of PA1225 exhibited maxima at 380 nm and 460 nm at pH 6.0 (Figure 1A). The identity of the cofactor was established with fluorescence emission spectroscopy and MALDITOF spectrometry. The enzyme was heat-denatured by boiling at 100 oC for 20 min, and the

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flavin was isolated after removal of the denatured protein by centrifugation at 12,000 x g. The extracted cofactor emitted light at 525 nm upon excitation at 460 nm (Figure 1A inset, solid line). Upon addition of 1 U snake venom phosphodiesterase I, the fluorescence emission of the extracted flavin increased 4-fold (Figure 1A inset, dashed line), ruling out FMN as the cofactor.20 As shown in Figure 1B, a negative ion mode MALDI-TOF spectrometric analysis of the flavin extracted from PA1225 showed a peak at 784.1 m/z(-), which matched the expected monoisotopic mass of FAD. The extinction coefficient (Ԑ460nm) of the enzyme-bound FAD was calculated to be 10,100 M-1cm-1 from the extinction coefficient of FAD in solution after boiling the enzyme to extract the flavin and removing the denatured protein by centrifugation. A flavin/protein monomer stoichiometry of ~0.8 was determined.

Figure 1. Cofactor identification of PA1225. (A) UV-visible absorption spectrum of PA1225 in 20 mM Tris-HCl, 150 mM NaCl, 10% glycerol (v/v), pH 8.0 and 25 oC. Inset: fluorescence emission spectrum of the heat-extracted flavin with λex at 460 nm before (solid line) and after (dashed line) treatment with 1 U snake venom phosphodiesterase I in 110 mM Tris-HCl, 110 mM NaCl, 15 mM MgCl2, pH 8.9 and 25 oC. (B) MALDI-TOF(-) of the heat-extracted flavin.

Identification of the Reducing Substrate. NADPH was chosen as a reducing substrate based on an amino acid sequence alignment of PA1225 to NQO1 showing 21% identity, suggesting that the enzyme may oxidize NADPH or NADH. The initial rates of reaction of the

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enzyme were determined at varying concentrations of NADPH between 25 and 200 µM at a fixed concentration of 200 µM 1,4-benzoquinone at pH 6.0 and 25 oC. The initial rates of reaction showed a hyperbolic dependence on the concentration of NADPH (Figure 2A), allowing for the determination of the apparent steady-state kinetic parameters at a fixed concentration of 1,4-benzoquinone (Table 1). When NADH was used as a substrate, it was not possible to saturate the enzyme and obtain app

app

kcat and

app

Km values, as shown in Figure 2B. However, an accurate

(kcat/Km) value could be determined from the slope of the initial rate of reaction as a function

of the concentration of NADH (Table 1).

Figure 2. Concentration dependence of the initial rate of reaction of PA1225 with NADPH (A) and NADH (B). Measurements were conducted with 0.2 mM 1,4-benzoquinone in 20 mM sodium phosphate, 150 mM NaCl, pH 6.0 and 25 oC, in duplicate, with the average values being displayed. Standard errors are smaller than the symbol size.

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Table 1. Apparent steady-state kinetic parameters of PA1225 for NADPH or NADH at pH 6.0 a. reducing substrate

app

kcat, s-1

app

Km, µM

app

(kcat/Km), M-1s-1

NADPH 10.1 ± 0.3 82 ± 5 124,000 ± 5,000 NADH NDb NDb 3,000 ± 100 a Conditions: 20 mM sodium phosphate, 150 mM NaCl, pH 6.0 and 25 oC, with 200 µM 1,4-benzoquinone; [NADPH] from 25 to 200 µM, [NADH] from 50 to 200 µM; b ND, not determined because saturation could not be achieved.

Identification of the Oxidizing Substrate. We tested the hypothesis that PA1225 is a quinone reductase, suggested by the 21-30% sequence identity with NQO1 and NQO2.21-23 Initial rates of PA1225-mediated quinone reduction were determined at varying concentration of 1,4-benzoquinone and a fixed concentration of 200 µM NADPH at pH 6.0 and 25 oC. Between 25 and 200 µM 1,4-benzoquinone there was no difference in the initial rate of reaction (Table S1), consistent with the enzyme being saturated with the substrate in this concentration range (Table 2). The enzyme showed a similar behavior when 2,6-dimethoxy-1,4-benzoquinone was tested as an oxidizing substrate (Table S1, Table 2). With tetramethyl-1,4-benzoquinone, Coenzyme Q0, and 1,4-naphthoquinone, substrate inhibition was observed at concentrations >25 µM (data not shown), so these oxidizing substrates were not further investigated. PA1225 was previously proposed by others to have azoreductase activity.24 To test this hypothesis, 0.2 mM methyl red was used as an oxidizing substrate with 200 µM NADPH at pH 6.0 and 25 oC. No changes in the absorbance at 340 nm were seen, indicating that PA1225 did not react with methyl red and that the enzyme did not possess azoreductase activity (Table 2). Molecular oxygen was also tested as an oxidizing substrate for PA1225 because its FAD cofactor is known to reduce oxygen in solution and when bound to oxidases and monooxygenases.25 With 200 µM NADPH and 253 µM O2 at pH 6.0 and 25 oC, PA1225 displayed a negligible NADPH:oxidase activity (Table 2).

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Table 2. Apparent kcat values of PA1225 with various oxidizing substrates at pH 6.0 a. app

kcat, s-1

oxidizing substrate

1,4-benzoquinone b 5.9 ± 0.1 2,6-dimethoxy-1,4-benzoquinone b 5.5 ± 0.1 atmospheric oxygen c 0.4 ± 0.2 methyl red d ∼0 a o Conditions: 20 mM sodium phosphate, 150 mM NaCl, pH 6.0 and 25 C, with 200 µM NADPH as reducing substrate; b [quinone] from 25 to 200 µM; c atmospheric oxygen, i.e., 253 µM; d [methyl red] = 200 µM. All assays were carried out in duplicate.

Steady-State Kinetic Mechanism. As a first step in the mechanistic characterization of PA1225, the steady-state kinetic mechanism of the enzyme was elucidated with NADPH and 1,4-benzoquinone as substrates at pH 6.0 and 25 oC. The initial rates of reaction being the same between 25 and 200 µM 1,4-benzoquinone (Table S2) prevented the use of the traditional approach of varying the concentrations of both NADPH and 1,4-benzoquinone. Consequently, the inhibition pattern of the NADP+ product versus NADPH at a fixed concentration of 1,4benzoquinone was used to characterize the steady-state kinetic mechanism of PA1225. Since the enzyme was saturated with 1,4-benzoquinone in the range from 25 and 200 µM (Table S1), the latter was used in the experiment. The initial rates of reaction with the NADPH between 25 and 100

µM were the same irrespective of the absence or presence of NADP+ up to 800 µM (Table S2), consistent with NADP+ not being an inhibitor when the enzyme is saturated with 1,4benzoquinone.

Flavin-monitored Turnover. To learn whether the reductive half-reaction of the enzyme could be studied aerobically, PA1225 was mixed aerobically with NADPH in a stopped-flow spectrophotometer and the oxidation state of the enzyme-bound flavin was monitored at 460 nm over time. With 40 µM NADPH and 253 µM O2 at pH 6.0 and 25 oC, the absorbance at 460 nm

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Biochemistry


of the enzyme-bound flavin decreased within ~1 s, consistent with a rapid reduction of the enzyme with NADPH (Figure 3). After that, the trace at 460 nm remained unchanged for up to 4 min incubation (Figure 3), indicating negligible oxidation of the enzyme-bound flavin and allowing for the reductive half-reaction to be studied aerobically.

Figure 3. Optically-monitored reaction of the enzyme-bound flavin (11 µM after mixing) during turnover with atmospheric oxygen and NADPH for 4 min at pH 6.0 and 25 oC. For clarity, one experimental point out of every five is shown (vertical lines). The experiment was carried out by mixing the content of the syringes shown in the figure; both syringes contained also 20 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 6.0.

Reductive Half-Reaction at pH 6.0. To gain information on the hydride transfer reaction from the NADPH to the enzyme-bound flavin, the reduction of the FAD cofactor was followed by monitoring the decrease in absorbance at 460 nm at pH 6.0 and 25 oC in a stopped-flow spectrophotometer. The experiment was carried out aerobically because of the negligible activity of PA1225 with oxygen as an oxidizing substrate (see above). PA1225, at a final concentration of 10 µM, was fully reduced by NADPH in a monophasic pattern (Figure 4A). The stopped-flow traces were fit with Eq 1 to extract the observed rate constants for flavin reduction (kobs) at different concentrations of NADPH under pseudo-first order conditions. The kobs value increased hyperbolically with increasing NADPH concentration (Figure 4B, solid circles), defining a

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limiting rate constant for flavin reduction at saturating concentrations of NADPH, kred, and a dissociation constant, Kd, for the equilibrium of the free enzyme and NADPH with the enzymeNADPH complex that proceeds to catalysis (Table 3). The rate constant for flavin reduction was also investigated with NADH as a reducing substrate. As for the case of NADPH, the stopped-flow traces were monophasic at all the concentrations of NADH used (Figure S2), yielding rate constants for flavin reduction that increased hyperbolically with increasing NADH concentration. The kinetic parameters associated with the reductive half-reaction of PA1225 with NADH are summarized in Table 3. Table 3. Reductive half-reaction at pH 6.0 a. substrate

kred, s-1

Kd, mM

kred/Kd, M-1 s-1

10.4 ± 0.1 NADPH 0.070 ± 0.002 150,000 ± 6,000 4.7 ± 0.2 1.8 ± 0.5 NADH 2,600 ± 700 a Conditions: 20 mM sodium phosphate, 150 mM NaCl, 10% glycerol at 25 oC.

R2 0.993 0.998

Figure 4. Reductive half-reaction of PA1225 with NAD(P)H in 20 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 6.0, at 25 oC. (A) Stopped-flow traces at 460 nm with varying NADPH concentrations and fit of the data to Eq. 1. Note the log time scale. For clarity, one experimental point out of every five is shown (vertical lines). Instrument dead time is 2.2 ms. (B) Concentration dependence of the kobs values for flavin reduction with NADPH (solid circles) and NADH (hollow circles). Data with NADPH were fit to Eq. 2; data with NADH were fit to Eq. 3; each data point was done in triplicate. Standard errors are smaller than the symbol size.

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Effect of pH on the Reductive Half-reaction To gain insights into possible ionizations associated with substrate binding and flavin reduction, the effect of pH on the reductive half-reaction with NADPH or NADH was investigated in a stopped-flow spectrophotometer between pH 6.0 and 10.0. Qualitatively, the stopped-flow traces and concentration dependences of the kobs versus [NAD(P)H] were similar to those observed at pH 6.0 (data not shown). Data could not be collected at pH 7.0 due to enzyme instability probably arising from aggregation or unfolding of the enzyme at a pH close to its computed pI value of 6.9. In all cases, the stopped-flow traces were monophasic, yielded fully reduced enzyme, and demonstrated hyperbolic concentration dependence of the kobs value. The kred values were independent of the pH with both NADPH and NADH, with an average value of ~10 s-1 for NADPH and ~5 s-1 with NADH (Fig. 5A). The Kd values, instead, decreased with decreasing pH, defining pKa values of 7.0 ± 0.2 with NADPH and 7.2 ± 0.2 with NADH (Fig. 5B). The limiting Kd values with NADPH were 0.022 ± 0.002 mM at low pH and 2.3 ± 0.5 mM at high pH. With NADH, the limiting Kd values were 0.16 ± 0.01 mM at low pH and 10.5 ± 0.4 mM at high pH.

19


Biochemistry


1.5

, s-1

0.5

red

1.0

log k

A

0.0 6.0

7.0

8.0

9.0

10.0

9.0

10.0

pH 1.5 1.0

B

0.5 0.0

d

log K , mM

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

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-0.5 -1.0 -1.5 6.0

7.0

8.0

pH

Figure 5. Effect of pH on the reductive half-reaction of PA1225 with NAD(P)H as the reducing substrate. Panel A shows the pH profiles of kred, yielding average values of 10 ± 1 s-1 with NADPH (solid circles) and 5 ± 1 s-1 with NADH (empty circles). Panel B shows the pH profiles of the Kd values, in which the data were fit with Eq 3.

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Biochemistry

PA1225, a novel FAD-containing NADPH:quinone Reductase DISCUSSION

Advances in genome sequencing allow for new protein sequences to be rapidly identified. However, because experimental determination of protein activity and function is considerably more labor intensive much of the annotation in databases is based on sequence similarity to proteins whose function may also be inferred rather than determined. An effective approach is to biochemically validate the function of annotated protein sequences established through bioinformatics because amino acid variations among similar sequences can yield altered activity or binding specificity. In this regard, two proteins with high sequence identity may have evolved different function, thereby making enzyme annotation without biochemical validation inaccurate.26, 27 This is especially important for pathogenic organisms like P. aeruginosa because it can misdirect efforts to develop new drugs. Here, we have cloned and expressed the gene for PA1225 from P. aeruginosa PAO1, which previous studies suggested it may be involved in detoxification mechanisms associated with the mitochondrial toxin P3N.13 Our biochemical investigation of the purified PA1225 provides evidence that the enzyme, which based on bioinformatics was annotated as an NAD(P)H:dehydrogenase, is actually an FAD-containing NADPH:quinone reductase. The work thereby adds to our understanding of the biochemical arsenal of P. aeruginosa and provides clues to PA1225 relevance in P. aeruginosa PAO1. FAD is the cofactor of PA1225, as established with fluorescence emission and MALDI-TOF spectrometric analyses obtained with the flavin after heat-denaturation of the enzyme. In contrast, FMN is the cofactor for most of the prokaryotic NAD(P)H:quinone reductases characterized thus far28, with the exception of Helicobacter pylori29, 30 and P. aeruginosa MdaB that use FAD. In this respect, PA1225 resembles the quinone reductases of eukaryotic organisms, such as mammals, plants, and fungi, which have been shown to have FAD as a cofactor28.

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NADPH is the preferred reducing substrate of PA1225, as indicated by the steady-state kinetics and reductive half-reaction results with NADPH or NADH. The enzyme turns over in the presence of NADPH or NADH and 1,4-benzoquinone. The second-order rate constant for the capture of the nicotinamide substrate into enzyme-substrate complexes that proceed to catalysis, kcat/Km, demonstrated a 40-fold preference of the enzyme for NADPH as compared to NADH at pH 6.0 (Table 1). The preference for NADPH is independently supported by the 60-fold difference observed in the kred/Kd values determined upon mixing PA1225 and the nicotinamide substrate in a stopped-flow spectrophotometer at pH 6.0 (Table 3). The enzyme preference for NADPH originates mostly from binding of the substrate in the active site of PA1225 rather than catalytic effects, as demonstrated in the reductive half-reaction of the enzyme showing a 25-fold smaller Kd value for NADPH as compared to NADH at pH 6.0 (Table 3). In the absence of structural data that could inform on the interactions of the enzyme with the substrate, a rationale to explain these differences cannot be provided. However, given the similarity of the structure of NADPH and NADH one could postulate that an enzyme group that interacts with the 2’phosphate of NADPH is likely responsible for the substrate specificity of the enzyme. Since lower pH enhances binding of NADPH more than it does for NADH, it is possible that a group near the 2’ phosphate of NADPH may acquire a proton below pH 7.0. The kcat/Km value of 105 M-1s-1 determined for PA1225 with NADPH and 1,4-benzoquinone at pH 6.0 agrees well with the values in the range 104-106 M-1s-1 previously reported for other NAD(P)H:quinone reductases in P. aeruginosa, such as NQR (PA1024),31 WrbA (PA0949),31 and MdaB (PA2580).31 While MdaB and PA1225 share the same cofactor and the reducing substrate, i.e., FAD and NADPH,32 both NQR and WrbA contain FMN and use NADH and NADPH, respectively.32 Thus,

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Biochemistry


prokaryotic NAD(P)H:quinone reductases utilize different flavin cofactors and reducing substrates to reduce quinones. Quinones are good oxidizing substrates of PA1225 with Km values ≤2.5 µM, as demonstrated by steady-state kinetic analysis. When the enzyme turned over with 200 µM NADPH and 1,4benzoquinone in the range from 25 µM to 200 µM there was no difference in the initial rates of reaction (Table S1), consistent with PA1225 being saturated with the oxidizing substrate and limiting us to determination of a appkcat value of ~6 s-1 (Table 2). The appkcat value of ~6 s-1 agrees well with the value of ~7 s-1 calculated for the overall turnover of the enzyme saturated with the quinone and 70% saturated with NADPH, i.e., 1

!

&

= "#$ %& (, with the values for kcat = 10.1 s'

and Km = 82 µM determined by varying NADPH at a fixed saturating concentration of 1,4-

benzoquinone (Table 1). A low Km value that is at most 10-times lower than the lowest concentration of quinone tested was estimated from the data. A similar low Km value was seen also with 2,6-dimethoxy-1,4-benzoquinone as oxidizing substrate for the enzyme. Similar to PA1225, previous results showed that both NQO1 and NQO2 have Km values for the quinone oxidizing substrate in the 2.0-4.0 µM range.33 The hydride transfer from NADPH to the enzyme-bound flavin is pH-independent. Evidence for this conclusion comes from the stopped-flow kinetic results on the reductive half-reaction catalyzed by PA1225 as a function of pH. The rate constant for flavin reduction, kred, with NADPH had a value of ~10 s-1 between pH 6.0 and 10.0 (Table 3). The lack of pH effects on the hydride transfer reaction can be explained if aromatic stacking of the nicotinamide ring of the substrate and the isoalloxazine ring of the flavin are sufficient for the proper orientation of the substrate C4 atom and flavin N5 atom in the enzyme-substrate complex required for the reaction. When NADPH is substituted with NADH, a minimal different orientation of the substrate C4

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atom and flavin N5 atom in the enzyme-substrate complex may explain the 2-fold difference in the observed kred values of ~10 s-1 and ~5 s-1 with NADPH and NADH (Table 3). Aromatic stacking of the nicotinamide and the flavin was previously reported with NQO1, in which the aromatic ring of NADP+ stacks 3.4 Å apart onto the flavin and shares the same binding site with quinones.28, 34 The hydride transfer from the NADPH to the enzyme-bound flavin is fully rate-limiting for the overall turnover of the enzyme at pH 6.0. Evidence to support this conclusion comes from the steady-state and reductive half-reaction kinetics of PA1225 with NADPH. At pH 6.0, the kred and kcat values were 10.4 s-1 and 10.1 s-1, respectively, establishing hydride transfer as the ratelimiting step for enzyme turnover. PA1225 operates through a Ping-Pong Bi-Bi steady-state kinetic mechanism. Evidence supporting this conclusion comes for the results of product inhibition studies with NADP+ present during turnover of the enzyme with NADPH and 1,4-benzoquinone. The lack of enzyme inhibition observed with NADP+ versus NADPH as the varying substrate and saturating 1,4benzoquinone (Table S2) rules out a sequential Bi-Bi steady-state kinetic mechanism. If this were the case, uncompetitive or competitive inhibition would be expected depending upon whether NADP+ were the first or second product released from the PA1225-NADP+-quinol complex, respectively.35 During Ping-Pong Bi-Bi steady-state of PA1225 with saturating 1,4benzoquinone, the reduced form of the enzyme exists mostly in complex with the quinone substrate (Ered-S in Scheme 1) rather than in the free form that binds NADP+ (Ered in Scheme 1), thereby resulting in no inhibition of the enzyme by NADP+. A Ping-Pong Bi-Bi steady-state kinetic mechanism has been previously reported for all the NAD(P)H:quinone reductases characterized thus far,28 consistent with the reactive center of both the nicotinamide moiety of

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Biochemistry


NAD(P)H and the quinone substrates occupying the same or a similar space with respect to the N5 atom of the flavin to allow for the hydride transfers in the active site of the enzyme.28, 34, 36

Scheme 1. Proposed steady-state kinetic mechanism of PA1225. Eox, oxidized enzyme; Ered, reduced enzyme; S, 1,4-benzoquinone; P, 1,4-benzoquinol; and k3 = kred.

In a 2014 study of P. aeruginosa azoreductases it was proposed that since both NAD(P)H quinone oxidoreductases and azoreductases have related reaction mechanisms they may form an enzyme superfamily, and PA1225 was included in a proposed extended azoreductase family of enzymes.24 As shown here by the lack of turnover of PA1225 with NADPH and methyl red (Table 2), the enzyme does not have azoreductase activity. In this regard, PA1225 is similar to another enzyme originally included in the extended azoreductase family WrbA (PA0949), which also reduces quinones but does not show azo bond cleavage when it was tested against 11 azo compounds.32 PA1225 is not an NADPH:oxidase. Evidence to support this conclusion comes from the steady-state kinetics and the flavin-monitored turnover experiment, showing that the enzyme-

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bound flavin stayed in the reduced form for up to 4 min upon in the presence of limited NADPH and oxygen. In this regard, previous studies demonstrated that the FAD-dependent NQO136, 37 and Lot6p28,

38

from Eukaryotes also lack NAD(P)H oxidase activity. In contrast, the FMN-

dependent NADH:quinone reductase PA1024 from P. aeruginosa

has a small, but not

negligible, NADH oxidase activity of ~1 s-1.39 PA1225 binds the reducing substrate NADPH more tightly at low pH, suggesting that the enzyme may operate in an acidic physiological environment. In vivo, PA1225 is overexpressed in P. aeruginosa PAO1 along with nitronate monooxygenase, a flavin-dependent enzyme that oxidizes propionate 3-nitronate.13, 14 Propionate 3-nitronate is known to be a potent, irreversible inhibitor of succinate dehydrogenase.14,

40

Inhibition of succinate dehydrogenase results in

accumulation of succinate, a shift in metabolism from respiratory to fermentative, and a decrease in intracellular and extracellular pH due to excretion of the excess succinate.41 All these phenomena were observed in a deletion mutant of Staphylococcus aureus in which succinate dehydrogenase was knocked out.41 Moreover, increased intracellular NADH/NAD+ and NADPH/NADP+ ratios are associated with the inhibition of the respiratory metabolism. Thus, based on the available evidence one can speculate that PA1225 may play a role in restoring the NADPH/NADP+ ratio using the intracellular quinone pool under the acidic conditions ensuing in P. aeruginosa cells poisoned with P3N. In summary, we have cloned and expressed gene pa1225 from the genome of P. aeruginosa PAO1 and characterized biochemically the resulting purified enzyme. PA1225 is an FADdependent NADPH:quinone reductase that operates through a Ping-Pong Bi-Bi steady-state kinetic mechanism. NADPH is the preferred reducing substrate for the enzyme, although NADH can also be used with a 40-fold lower efficiency. The enzyme lacks azoreductase activity on

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Biochemistry


methyl red and shows a negligible NADPH:oxidase activity. Instead, single- and double-ring quinones are good oxidizing substrates with low Km values ≤2.5 µM. The hydride transfer from NADPH to enzyme-bound FAD is pH-independent and fully rate-limiting for the overall turnover of the enzyme at pH 6.0. Binding of the reducing substrate NADPH occurs more tightly at low pH, suggesting that the enzyme may operate physiologically in acidic conditions. Thus, this study elucidates the enzymatic function of PA1225, provides biochemical evidence for a validated annotation of the gene pa1225 as encoding for an NADPH:quinone reductase, and offers insights towards the role of this enzyme in P. aeruginosa PAO1.

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AUTHOR INFORMATION Corresponding Author Prof. Giovanni Gadda, Department of Chemistry, Georgia State University, PO Box 3965, Atlanta, GA 30302-3965, U.S.A., Telephone: 404-413-5537; E-mail: [email protected]. Funding This work was supported in part by Grant CHE-1506518 by the NSF (to G. G.) and Louis-Stokes Alliance for Minority Participation (to E. F.) Conflict of interest The Authors declare no conflict of interest.

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Biochemistry


Acknowledgements The authors would like to thank Renata Reis, Dabney Dixon, and Donald Hamelberg for their insightful discussions on PA1225, and Siming Wang for providing mass spectrometry services.

Supporting Information Supporting Information with the SDS-PAGE of the purified enzyme, stopped-flow traces with NADH, and steady-state kinetic data, is available free of charge on the ACS Publications website at …

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Biochemistry

PA1225, a novel FAD-containing NADPH:quinone Reductase TOC

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Kinetic Characterization of PA1225 from ... - ACS Publications

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