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Kinetic Investigation of a Presumed Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes a New Class of NAD(P)H:quinone Reductases Renata Almeida Garcia Reis, Francesca Salvi, Isabella WIlliams, and Giovanni Gadda Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00207 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 12, 2019

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Biochemistry

Kinetic Investigation of a Presumed Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes a New Class of NAD(P)H:quinone Reductases

Renata A. G. Reis#, Francesca Salvi#, Isabella Williams#§, Giovanni Gadda#$&‡(*),

#

Department of Chemistry, $Department of Biology, &Center for Biotechnology and Drug Design

and ‡Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 303023965. §The Lovett School, Atlanta, GA 30327-3009

(*)

Address correspondence to:

Dr. Giovanni Gadda Department of Chemistry, Georgia State University, P.O. Box 3965, Atlanta, GA 30302-3965; Phone: (404) 413-5537, Fax: (404) 413-5505, Email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT PA0660 from Pseudomonas aeruginosa PAO1 is currently classified as a hypothetical nitronate monooxygenase (NMO) but no evidence at transcript or protein level is present. In this study, PA0660 was purified and characterized in its biochemical and kinetic properties. Absorption spectroscopy and mass spectrometry demonstrated a tightly, non-covalently bound FMN in the active site of the enzyme. Analytical ultracentrifugation showed that the enzyme exists as a dimer in solution. Despite its annotation, PA0660 did not exhibit nitronate monooxygenase activity. The enzyme could be reduced with NADPH or NADH with a marked preference for NADPH, as indicated by ~30-fold larger kcat/Km and kred/Kd values. Turnover could be sustained with NAD(P)H and quinones, DCPIP, and to a lower extent molecular oxygen. However, PA0660 did not turn over with methyl red, consistent with lack of azoreductase activity. The enzyme turned over through a Ping-Pong Bi-Bi steady-state kinetic mechanism with NADPH and 1,4-benzoquinone showing a kcat value of 90 s-1. The rate constant for flavin reduction with saturating NADPH was 360 s-1, whereas that for flavin oxidation with 1,4-benzoquinone was 270 s-1, consistent with both hydride transfers from the pyridine nucleotide to the flavin and from the flavin to 1,4-benzoquinone being partially rate-limiting for enzyme turnover. A BlastP search and a multiple sequence alignment analysis of PA0660 highlighted the presence of six conserved motifs in >1,000 ORFs currently annotated as hypothetical NMOs. The results presented suggest that PA0660 should be classified as NAD(P)H:quinone reductase and serve as a paradigm enzyme for a new class of enzymes.


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Biochemistry

Table of contents


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INTRODUCTION Fueled by the rapid progress in sequencing technology, genome sequencing is advancing at an escalating pace1. Public databases like GenBank have rapidly increased their size, and have become a crucial tool for organizing available biological data2. However, the accuracy of the predicted functions is essentially unknown3, since most of the annotations are made only based on sequence similarities. Functional annotation of gene products that accounts for biochemical evidence is more accurate than bioinformatics alone but has lagged because it relies on more difficult and laborious experimental work. In the case of prokaryotes, for instance, the functional annotation based on biochemical evidence represents less than 0.4% of the microbial genomic genes4,5. Consequently, misannotation of molecular function in public databases continues to be a significant problem, with broad negative impact in biotechnology and medicine3. After the publication of the complete Pseudomonas aeruginosa PAO1 genome sequence in the year 20006, multidisciplinary efforts have focused on accurate annotation and the development of a continually updated database by the Pseudomonas Community Annotation Project (PseudoCAP)7,8. Despite the effort, more than 2000 P. aeruginosa PAO1 genes remained annotated as “hypothetical proteins” in 2016, showing that this is an ongoing process8. One example of an enzyme family consisting mostly of hypothetical proteins is the nitronate monooxygenase (NMO, E.C. 1.13.12.16), which includes more than 5000 genes in the GenBank. NMOs are members of the Group H flavin-dependent monooxygenases and catalyze the oxidation of the metabolic poison proprionate-3-nitronate (P3N)9 and other nitronate analogs. Three genes were originally annotated based on bioinformatics as hypothetical nitronate monooxygenase in P. aeruginosa PAO1, namely pa0660, pa1024 and pa4202. A recent structural and kinetic study on the gene product PA4202 established this enzyme as an NMO (PaNMO)10. The crystal structure of PaNMO solved to 1.44 Å resolution (PDB code 4Q4K), and the biochemical and kinetic investigation of the enzyme allowed the identification of four consensus motifs in the primary sequence of the protein. The consensus motifs established NMO Class I and II. Class I includes 500 hypothetical NMO belonging to bacteria, fungi, and two animals, and oxidizes anionic nitronates. Class II includes only 12 fungal enzymes and can oxidize both the neutral and anionic forms of the substrate and with different consensus motifs was also identified in that study10. The subsequent kinetic characterization of PA1024 demonstrated that the enzyme is not an NMO, and it uses NADH, but not NADPH, and quinones


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Biochemistry

as reducing and oxidizing substrates, respectively11. In 2018 PA1024 was reclassified as an NADH:quinone reductase (E.C. 1.6.5.11), which may participate in the maintenance of an appropriate [NAD+]/[NADH] ratio for the catabolism of fatty acids in P. aeruginosa PAO111. Six conserved motifs in the primary sequence of PA1024, along with the structural and biochemical data, define a new class of NADH:quinone reductases that includes more than 490 hypothetical proteins in the GenBank11,

12

. To date, no biochemical evidence at transcript or

protein level is available for PA0660, which is currently annotated as a hypothetical NMO based on bioinformatics. PA0660 shares only 27% and 29% sequence identity and modest overall sequence similarity of 38% and 42% with PaNMO and PA1024, respectively. Additionally, PA0660 does not possess the consensus motifs of Class I and Class II NMO, and PA1024, suggesting that the enzyme may have different catalytic activity and could potentially identify a new class of enzymes. In this study, we have cloned the gene pa0660 from the genomic DNA of P. aerugionosa, expressed and purified the His-tagged recombinant PA0660, and characterized the spectral and kinetic properties of the enzyme. We showed that PA0660 does not exhibit NMO activity and it reacts with both NADPH and NADH. Oxidizing substrates were investigated using steady-state kinetics. Both half-reactions catalyzed by PA0660 were studied using rapid kinetics to obtain information on the steps of flavin reduction and oxidation. Six conserved motifs were identified in the protein sequence of PA0660, which are different from the motifs identified for PaNMO and PA1024, and are conserved in more than 1000 proteins erroneously annotated as hypothetical NMO in the GenBank database. The results established the enzyme mechanism of PA0660 and support the conclusion that PA0660 is a novel, previously unknown NAD(P)H:quinone reductase.


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MATERIALS AND METHODS Materials. The genomic DNA of P. aeruginosa PAO1 was a kind gift from J. Spain (Georgia Institute of Technology, Atlanta, GA). The enzymes BamHI, NdeI, DpnI, calf intestinal alkaline phosphatase (CIP), and T4 DNA ligase were from New England BioLabs (Ipswich, MA), Pfu DNA polymerase was from Stratagene (La Jolla, CA), and oligonucleotides from Sigma Genosys (The Woodlands, TX). Escherichia coli strain DH5α was from Invitrogen Life Technologies (Grand Island, NY). E. coli Rosetta(DE3)pLysS and pET15b expression vector were from Novagen (Madison, WI). QIAprep Spin Miniprep kit and QIAquick PCR purification kit were from Qiagen (Valencia, CA). HiTrapTM Chelating HP 5 mL affinity column and prepacked PD10 desalting columns were purchased from GE Healthcare (Piscataway, NJ). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased from Promega (Madison, WI). Nitroalkanes and quinones were purchased from Sigma-Aldrich (St. Louis, MO). NADH and NADPH disodium salts were purchased from VWR (Radnor, PA). All other reagents used were of the highest purity commercially available. Cloning. The gene pa0660 was amplified from the genomic DNA of P. aeruginosa PAO1 by PCR in the presence of 3% DMSO. Initial denaturation was at 95 °C, followed by 20 cycles of denaturation for 45 s at 95 °C, annealing for 45 s at 56 °C (with the annealing temperature progressively decreasing by 0.2 °C at each cycle), extension for 3 min at 72 °C, and a final step for 10 min at 72 °C. The pa0660 gene amplified was purified by QIAquick PCR purification kit. The amplicon and the expression vector pET15b were independently digested at 37 °C with NdeI and BamHI for 2 h and purified with the QIAquick PCR purification kit. The pET15b plasmid was further dephosphorylated with 0.5 units of calf intestinal alkaline phosphatase for 30 min at 37 °C, purified using QIAquick PCR purification kit, and ligated to the insert with incubation for 15 h at 16 °C with T4 DNA ligase. E. coli strain DH5α was transformed with the ligation mixture and the resulting colonies grown at 37 °C on Luria-Bertani agar plates containing 50 μg/mL ampicillin were screened for the presence of the desired insert by DNA sequencing at the Cell, Protein and DNA core facility at Georgia State University. The DNA sequencing confirmed the correct insertion of the gene in the vector pET15b and the absence of undesired mutations. Recombinant

Expression

and

Purification.

E.

coli

expression

strain

Rosetta(DE3)pLysS transformed with the construct pET15b/pa0660 was used to inoculate 100


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Biochemistry

mL of Luria-Bertani Broth containing 100 μg/mL of ampicillin and 34 μg/mL of chloramphenicol, which was incubated at 37 °C overnight. One aliquot of 10 mL of this culture was used to inoculate 1 liter of Luria-Bertani Broth containing 100 μg/mL of ampicillin and 34 μg/mL of chloramphenicol, which was incubated at 37 °C until it reached an optical density at 600 nm of 0.6. Recombinant protein expression was induced by addition of IPTG to a final concentration of 400 μM and the culture incubated at 37 °C for 20 h. The wet cell paste of 5 g, recovered by centrifugation, was resuspended in 25 mL of lysis buffer containing 20 mM sodium phosphate, 10 mM imidazole, 300 mM NaCl, 10% v/v glycerol, 1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl2, 2 mg/mL lysozyme, 5 µg/mL DNase, and 5 µg/mL RNase, pH 7.6. The resuspended cells were subjected to several cycles of sonication. The cell free extract obtained after centrifugation at 12000 g for 20 min was loaded onto a HiTrapTM Chelating HP 5 mL affinity column equilibrated with buffer A [20 mM sodium phosphate, 10 mM imidazole, 300 mM NaCl, 10% v/v glycerol, pH 7.6]. After washing with 10 column volumes of buffer A, 5 column volumes of 10% buffer B (buffer A with 500 mM imidazole), 5 column volumes of 20% buffer B, PA0660 was eluted with 40% buffer B, dialyzed against 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, and 10% (v/v) glycerol and stored at -20 °C. Cofactor Identification. Cofactor identification was performed by mass spectrometry of the flavin extracted from a desalted sample of PA0660 in water treated at 100 °C for 20 min using a Waters Micromass Q-TOF micro (ESI-Q-TOF) in negative ion mode at the Mass Spectrometry Facility of Georgia State University. Spectroscopic Studies. Ultraviolet−visible (UV−visible) absorption spectra were recorded with an Agilent Technologies (Santa Clara, CA) model HP 8453 PC diode-array spectrophotometer equipped with a thermostated water bath in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, and 10% (v/v) glycerol at 25 °C. The molar ratio of FMN to PA0660 was determined by extracting FMN from PA0660 through heat denaturation at 100 °C for 15 min, removing precipitated protein by centrifugation, and spectroscopically estimating the total FMN concentration in the supernatant using the molar extinction coefficient of 12,500 M-1 cm-1 at 450 nm as previously described13. The concentration of flavin-bound active enzyme was determined by using the experimentally determined extinction coefficient ε447 = 11, 544 M-1 cm-1 (this study). The total protein concentration was determined using the Bradford method with bovine serum


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albumin as standard14. The concentration of NAD(P)H was determined spectroscopically15 by using an ε340 of 6220 M−1 cm−1. Sedimentation Velocity Analytical Ultracentrifugation (AUC) Analysis. For sedimentation velocity experiments, PA0660 was loaded into an AUC cell equipped with 12 mm double-sector Epon centerpieces and quartz windows and equilibrated at 20 °C in an AN50 Ti rotor for 1 h. The left and right sectors of the cell were loaded with 400 μL of 22 μM and 45 μM PA0660, respectively. All solutions were buffered in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, and 10% (v/v) glycerol. Sedimentation velocity data were collected using a Beckman Optima AUC analytical ultracentrifuge using a rotor speed of 40,000 rpm (i.e., 128,794 g) at 20 °C. Data were recorded by monitoring the sedimentation of the absorbance signal at 445 nm using a radial step size of 0.001 cm. The partial specific volume (Vbar) of 0.7349 was calculated from the amino acid sequence of His-tagged PA0660. The buffer viscosity of 0.01391 Poise and density of 1.03762 g/mL were calculated using UltraScan (www.ultrascan.uthscsa.edu). Sedimentation velocity data were analyzed using both SEDFIT (www.analyticalultracentrifugation.com) and UltraScan. Continuous sedimentation coefficient distribution c(s) analyses were restrained by maximum entropy regularization at P = 0.95 confidence interval. The baseline, meniscus, frictional coefficient, systematic time-invariant and radial invariant noise were fit. Resulting distribution data were plotted using Sigmaplot (Systat. Software) and the percentage of the oligomer present a teach concentration was determined by integrating the area under the peak. Nitronate monooxygenase activity assay. NMO activity was tested as previously described10,

16-18

, by monitoring the initial rate of oxygen consumption with a Hansatech

Instruments computer-interfaced Oxy32 oxygen-monitoring system at atmospheric oxygen, i.e., 230 µM oxygen, and 30 °C. Stock solutions of nitronates and nitroalkanes were prepared as previously described16, 17. Enzyme concentration was 0.5 µM and substrate concentration was 1 mM proprionate-3-nitronate (P3N) or 3-nitropropionate (3-NPA), or 20 mM for 2-nitropropane, propyl-2-nitronate,

nitroethane,

or

ethylnitronate.

A

positive

control

for

nitronate

monooxygenase activity was performed in parallel with purified PaNMO to a final concentration of 4 nM and 1 mM P3N as previously described10. NAD(P)H oxidase activity. NAD(P)H oxidase activity was monitored by following NAD(P)H oxidation at 340 nm (ε340 = 6,220 M-1 cm-1)15 in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, and atmospheric oxygen at 25 °C. Apparent kinetic parameters were determined by


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Biochemistry

varying the concentration of NAD(P)H from 6.25 to 100 µM. Enzyme concentration was 0.4 µM. Diaphorase activity assay. Turnover of PA0660 with NAD(P)H and the artificial electron acceptor 2,6-dichloroindophenol (DCPIP) was studied by monitoring the reduction of DCPIP at 600 nm (ε600 = 20,600 M-1 cm-1) in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, and atmospheric oxygen at

25 °C. Apparent kinetic parameters were obtained by varying the

concentration of NADH from 15 to 500 µM and NADPH from 9 to 360 µM at two fixed saturating concentrations of DCPIP, 60 and 80 µM. Enzyme concentration was 55 nM. Quinone reductase activity. Quinone reductase was tested by following NADPH oxidation at 340 nm (ε340 = 6,220 M-1 cm-1)15 in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, and atmospheric oxygen at 25 °C. NADH was kept at a constant, saturating concentration of 50 µM. Stock solutions of quinones were prepared in 100% ethanol, except 2,6-dimethoxy-1,4benzoquinone, which was dissolved in DMSO. Azoreductase activity assay. The azoreductase activity assay was performed by following the reduction of the azo dye methyl red at 430 nm (ε430 = 23,360 M-1 s-1)19, 20 in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, and atmospheric oxygen at 25 °C. The concentrations of enzyme, NAD(P)H, and methyl red were 0.5 µM, 100 µM, and 100 µM, respectively. Rapid Kinetics. Time-resolved absorbance spectroscopy of the reduction and oxidation of the enzyme-bound flavin with NAD(P)H and 1,4-bezoquinone were carried out with an SF61DX2 Hi-Tech KinetAsyst high-performance stopped-flow spectrophotometer in both doublemixing and single-mixing modes, thermostated at 25 °C, equipped with a photodiode array or a photomultiplier detector, under aerobic conditions. The enzyme solution was equilibrated by passing through a PD10 column against 20 mM Tris-Cl, pH 7.5, 150 mM NaCl. The reductive half-reaction was performed at varying concentrations of NADPH or NADH under pseudo-firstorder conditions where the concentration after mixing of the enzyme was ∼10 μM and that of the reducing substrate was between 0.2 and 2.4 mM (NADH) or 0.0625 and 1.0 mM (NADPH). Equal volumes of the enzyme and reducing substrate were mixed in the stopped-flow spectrophotometer single-mixing mode following established procedures. The instrument dead time was 2.2 ms. The concentration of the substrate NAD(P)H was determined spectrophotometrically at 340 nm with the extinction coefficient15 6,220 M-1cm-1. The oxidative half-reaction was measured by mixing equal volumes of the protein solution and NADPH at a


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concentration equal to 0.9 of that of the enzyme to avoid excess substrate, which aged in a loop for 0.5 s until complete reduction had been achieved, before mixing with an equal volume of buffer containing different concentrations of 1,4-benzoquinone. This yielded a solution containing ~5 µM reduced PA0660 and 1,4-benzoquinone (0.125 – 1 mM) in the desired buffer. Enzyme Monitored Single Turnover. The enzyme PA0660 at a final concentration of 10 μM was mixed with a stoichiometric amount of NADPH at atmospheric oxygen. Timeresolved absorption spectra for the detection of flavin intermediates were recorded for 750 s in the range from 340 to 600 nm with an SF-61DX2 Hi-Tech KinetAsyst high-performance stopped-flow spectrophotometer equipped with a photo-diode array detector, thermostated at 25 °C. The experiment was carried out in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl. The instrument dead time was 2.2 ms. Steady-state kinetic mechanism. The traditional approach to obtain the steady-state kinetic mechanism by varying the concentration of both NAD(P)H and 1,4-benzoquinone could not be carried out due to low Km values for NADPH and high Km values for NADH. At low concentrations of 1,4-benzoquinone, all the experimental points at different concentrations of NADPH were in the saturation area. On the other hand, with NADH, full saturation was not achieved at different concentrations of 1,4-benzoquinone. An alternative method21 was used in order to obtain information on the steady-state kinetic mechanism of PA0660 mechanism. Initial rates of reaction were measured as a function of the concentrations of the NADPH while keeping the ratio of the concentration of NADPH to that of 1,4-benzoquinone at a constant value. A ratio of 1/5 was kept constant with NADPH (6.25, 8, 10, 12.5, 18.75, 25 µM) and 1,4-benzoquinone (31.25, 40, 50, 62.5, 93.75, 125 µM). The experiment was carried out in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, at 25 °C. Data Analysis. Kinetic data were fit using Sigmaplot (Systat Software). The apparent steady-state kinetic parameters at varying concentrations of NAD(P)H and quinones were determined by fitting the initial rates of reactions to the Michaelis-Menten equation (Equation 1) or to Equation 2 in cases where substrate inhibition was seen. 𝑣# = 𝑒

𝑣# = 𝑒

'(( '((

𝑘)'* [𝐴] (1)

𝐾0 + [𝐴]

(2)

'((

𝑘)'* [𝐴] 𝐴2 𝐾' + 𝐴 + 𝐾3 ACS Paragon Plus Environment

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Biochemistry

When the ratio of the concentration of NAD(P)H to 1,4-benzoquinone was kept equal to a constant value α (i.e., A/B = α), Equations (3) and (4) were used to determine the steady-state kinetic mechanism. Here, a steady-state kinetic mechanism with formation of a ternary complex yields a polynomial curve, whereas that without formation of a ternary complex yields a straight line21. Both equations will have an intercept on the 𝑒⁄𝑣# axis of 1⁄𝑘678 . 𝑒 1 1 = M(𝐾7 + 𝛼𝐾P ) + 1R S T 𝑣# 𝐴 𝑘678

(3)

𝑒 1 1 1 = M𝛼𝐾]7 𝐾P 2 + (𝐾7 + 𝛼𝐾P ) + 1R S T 𝑣# 𝐴 𝐴 𝑘678

(4)

Stopped-flow traces were fit with the software KinetAsyst 3 (TgK-Scientific, Bradford on-Avon, U.K.) to Equation 5 or 6, each of which describes a double-exponential or singleexponential process, respectively. A represents the absorbance at 447 nm at time t, B1 and B2 represent the amplitudes of the decrease in absorbance, kobs1 and kobs2 represent the observed rate constants for the change in absorbance, and C is an offset value accounting for the nonzero absorbance of the enzyme-bound reduced flavin at infinite time. ;?@ *

+ 𝐵2

;?@ *

+𝐶

𝐴 = 𝐵: 𝐴 = 𝐵:

;?A *

+𝐶

(5) (6)

Concentration dependence of the observed rate constants for flavin reduction or oxidation was analyzed with Equation 7, which describes a hyperbolic trend. S represents the concentration of organic substrate, kred and kox are the rate constant for flavin reduction and oxidation, respectively, at saturating substrate concentration, and Kd is the apparent dissociation constant for substrate binding. 𝑘#CD =

50 µM (Figure 3A), and Km, kcat, kcat/Km, and Ki values were determined by using Eq. 2 (Table 1). When NADH was used instead of NADPH, substrate inhibition was not observed (Figure 3B), and Km, kcat, and kcat/Km values were determined (Table 1). The comparison of the kcat/Km values shows that PA0660 is 25-times more specific for NADPH as a substrate than NADH at pH 7.5. Table 1. Apparent Steady-State Kinetics Parameters of PA0660 for NADPH or NADH at fixed saturating concentrations of DCPIPa app app Reducing DCPIP (µM) app(kcat/Km) (M-1 s-1) kcat (s-1) appKm (µM) Ki Substrate NADPH 60 1,300,000 ± 200,00 15 ± 1 12 ± 2 200 ± 40 NADPH 80 1,000,000 ± 300,000 16 ± 2 16 ± 5 170 ± 50 NADH 60 50,000 ± 10,000 13 ± 1 240 ± 40 NAb NADH 80 50,000 ± 6,000 12 ± 1 230 ± 20 NAb a Conditions: 20 mM Tris-Cl, pH 7.5, and 150 mM NaCl, at 25 °C; [NADPH] from 9 to 360 µM and [NADH] from 15 to 500 µM. bNot applicable.


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Figure 3. Concentration dependence of the initial rate of reaction of PA0660 with (A) NADPH varied from 9 to 360 µM and (B) NADH varied from 15 to 500 µM at two fixed saturating concentrations of the electron acceptor DCPIP. Measurements were conducted in 20 mM TrisCl, pH 7.5, 150 mM NaCl, at 25 °C, atmospheric oxygen, in triplicate, with the average values and the associated standard errors shown.

Flavin oxidation by molecular oxygen. Time-resolved absorption spectroscopy was used to monitor the flavin state when 10 µM PA0660 was incubated with 10 µM NADPH and atmospheric oxygen, i.e., 0.23 mM, at pH 7.5 and 25 °C. The absorbance at 447 nm of the enzyme-bound flavin rapidly decreased and the enzyme was fully reduced to the hydroquinone state within ~0.1 s (Figure 4A). When 10 µM enzyme was reduced with an equimolar amount of NADPH before mixing with atmospheric oxygen the increase in absorbance at 447 nm became apparent after ~100 s (Figure 4B), yielding an observed rate constants for flavin oxidation of 0.08 s-1. Thus, flavin oxidation by molecular oxygen was >1,000-times slower than flavin reduction, allowing for the investigation of the reductive half-reaction in aerobic conditions. The lack of absorption changes in the 360-400 nm and the 500-800 nm regions of the UV-visible absorption spectrum are consistent with absence of any detectable C4a-(hydro)peroxyflavin27, 28, neutral flavin semiquinone, or charge-transfer complex between the flavin and the pyridine nucleotide.


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Biochemistry

Figure 4. Optically monitored reaction of the enzyme-bound flavin (10 µM after mixing) during turnover with atmospheric oxygen and NADPH for 1.5 s (A) and 750 s (B) at pH 7.5 and 25 °C. Insets show time resolved absorption spectra of 10 µM PA0660 mixed with 10 µM NADPH. Black line in both insets represents oxidized enzyme-bound flavin in the presence of NADPH after mixing (0.75 ms). Red line in the inset in panel A and B corresponds to the flavin hydroquinone, spectrum recorded after 1.5 s. Blue line in the inset in panel B corresponds to the spectrum of oxidized flavin after 300 s.

Reductive Half-Reaction with NAD(P)H. To investigate the hydride transfer reaction from NAD(P)H to the FMN cofactor in PA0660, the reduction of the enzyme was carried out in a stopped-flow spectrophotometer by monitoring the decrease in absorbance at 447 nm, pH 7.5 and 25 °C. The experiment was performed under aerobic conditions because the NADPH:oxidase activity of PA0660 is negligible (vide supra). PA0660, at final concentration of 10 μM, was fully reduced with NADPH in a biphasic pattern (Figure 5A), with a fast phase accounting for >90% of the total absorbance change, and a slow phase independent of the [NADPH] of 90% of the total absorbance change at 447 nm that was assigned to flavin reduction. Most of flavin reduction could be observed in the stopped-flow traces allowing for an accurate determination of the kobs values for flavin reduction with NADH (Figure 5B). Since saturation could not be achieved with concentrations of substrate as high as 2.4 mM (Figure 5C), only the kred/Kd could be estimated with NADH, with a value of 150,000 ± 10,000 M-1s-1 (Table 2). Thus, while PA0660 reacts with both NADPH and NADH, it is 35-times more specific for NADPH.

Table 2. Rapid kinetics parameters for the reaction catalyzed by PA0660 at pH 7.5a Reducing Substrate kred/Kd (M-1 s-1) kred (s-1) Kd (µM) NADPH ~5,500,000 ~360 61 ± 4 NADH 150,000 ± 10,000 ND ND Oxidizing Substrate kox/Kd (M-1 s-1) kox (s-1) Kd (µM) -1 1,4-benzoquinone 550,000 ± 60,000 270 ± 10 s 500 ± 50 µM a Conditions: 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, at 25 °C; [NADPH] from 0.0625 to 1 mM, [NADH] from 0.2 to 2.4 mM, and [1,4-benzoquinone] from 0.125 to 1 mM.


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Figure 5. Aerobic reduction of PA0660 with NAD(P)H in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, at 25 °C. (A) Stopped-flow traces at 447 nm with varying NADPH concentrations: 62.5 µM (black), 125 µM (red), 250 µM (blue), 500 µM (green), and 1000 µM (pink). (B) Stoppedflow traces at 447 nm with varying NADH concentrations: 200 µM (black), 400 µM (red), 800 µM (blue), 1200 µM (green), and 2400 µM (pink). The data were fit to Equation 5. For clarity, one out of five experimental points is shown (vertical lines). The instrument dead time is 2.2 ms. Arrows indicate the initial absorbance of the oxidized enzyme before mixing with reducing substrate. (C) Concentration dependence of the observed rate constants of flavin reduction with NADPH (●) and NADH (□). The solid curves were generated by fitting the data to Equation 6. Each data point was done in triplicate with the average values and the associated standard errors shown.


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Quinones as Oxidizing Substrate. Based on their powerful oxidizing ability, quinones were selected as potential oxidizing substrates. Turnover of PA0660 with different quinones as the oxidizing substrate was monitored at fixed saturating concentration of 50 µM NADPH at pH 7.5 and 25°C. PA0660 turned over with several quinones and the apparent steady-state kinetic parameters were determined. As shown in Table 3, the bicyclic 1,4-naphthoquinone and 5hydroxy-1,4-naphthoquinone were the best substrates for the enzyme based on the kcat/Km value, whereas 1,4-benzoquinone was the best monocyclic substrate. Table 3. Apparent Steady-State Kinetics Parameters of PA0660 for NADPH or NADH at pH 7.5a app app Electron acceptor (kcat/Km) (M-1 s-1) appkcat (s-1) Km (µM) 1,4-Benzoquinones 2,3,4,6-tetramethyl30,000 ± 7,000 15 ± 2 500 ± 100 2,6-dimethoxy16,000 ± 500 41 ± 1 2500 ± 50 benzoquinone 250,000 ± 50,000 87 ± 9 370 ± 70 2,3-dimethoxy-5-methyl- (Q0) 130,000 ± 30,000 66 ± 7 500 ± 100 1,4-Naphthoquinones naphthoquinone 1,250,000 ± 400,000 80 ± 10 100 ± 25 2-methyl300,000 ± 90,000 63 ± 9 230 ± 70 5-hydroxy1,100,000 ± 200,000 82 ± 7 74 ± 11 a Conditions: 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, at 25 °C; [NADPH] from 9 to 360 µM and [NADH] from 15 to 500 µM.

To investigate whether PA0660 show azoreductase activity, turnover of the enzyme was monitored with the azo dye methyl red and 100 µM NAD(P)H, pH 7.5, and 25 °C. The lack of absorbance changes at 430 nm was consistent with PA0660 having no azoreductase activity. In agreement with the slow rate for flavin oxidation by molecular oxygen (vide supra), PA0660 displayed negligible NAD(P)H:oxidase activity with an

app

kcat value of 0.12 ± 0.01 s-1 with

NADPH and 0.09 ± 0.01 s-1 with NADH at pH 7.5 and 25 °C (Figure S2 and Table S1). Oxidative Half-Reaction with 1,4-benzoquinone. The oxidative half-reaction was studied in a double-mixing stopped-flow spectrophotometer upon mixing the reduced enzyme with varying concentrations of 1.4-benzoquinone at 25 °C. The reduced enzyme was prepared by aging PA0660 after mixing with an equimolar concentration of NADPH, and flavin oxidation


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Biochemistry

was monitored by following the increase in absorbance at 447 nm at pH 7.5 and 25 oC. As shown in Figure 6A, the enzyme was fully oxidized in a monophasic pattern. The observed rate constants for flavin oxidation showed a hyperbolic dependence on the [1,4-benzoquinone] (Figure 6B), allowing for the determination of a kox value of 270 ± 10 s-1 and a Kd value of 500 ± 50 µM (Table 2).

Figure 6. Time-resolved absorbance spectroscopy of the oxidation of reduced PA0660 in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, at 25 °C. The enzyme was premixed with NADPH (0.9-fold the enzyme concentration) and allowed to age for 0.5 s before mixing with different concentrations of 1,4-benzoquinone. The [PA0660] was ~5 µM after double mixing. (A) Stopped-flow traces at 447 nm with varying 1,4-benzoquinone concentrations: 0.125 mM (black), 0.250 mM (red), 0.500 mM (blue), 0.750 mM (green), and 1.0 mM (pink). Due to an ε240nm value of 24.3 mM-1cm1 for 1,4-benzoquinone that interferes in the region around 450 nm the stopped-flow traces acquired between 0.125 and 1 mM 1,4-benzoquinone show different offsets. The data were fit to Equation 6. For clarity, one out of ten experimental points is shown (vertical lines). The instrument dead time is 2.2 ms. The arrow indicates the initial absorbance of the reduced enzyme before mixing with the oxidizing substrate. (B) Concentration dependence of the observed rate constants for flavin oxidation with 1,4-benzoquinone. The solid curve was generated by fitting the data to Equation 7. Each data point was done in triplicate with the average values and the associated standard errors shown. The inset displays spectra of the initial oxidized enzymebound flavin (black), reduced after mixing with NADPH (red) and final reoxidized enzymebound flavin (blue), with the red and blue arrows indicating the direction of the spectral changes. The offset in the reoxidized enzyme-bound flavin spectrum is due to the absorbance of the quinone with a ε240nm value of 24.3 mM-1cm-1.


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Steady-State Kinetic Mechanism. The steady-state kinetic mechanism of PA0660 was investigated with NADPH and 1,4-benzoquinone as substrates, at pH 7.5 and 25 °C. 1,4Benzoquinone was chosen over bicyclic quinone substrates because its absorption properties with peaks at 240 nm and 285 nm did not interfere with the spectrophotometric assay. 1,4Benzoquinone is also a good prototype because it is considered a parent for all types of quinones found in living tissues29. The traditional approach to obtain the steady-state kinetic mechanism by varying the concentration of both NAD(P)H and 1,4-benzoquinone could not be carried out because the Km values for NADPH or NADH were either to low or too high for accurate kinetic determination (data not shown). Consequently, the initial rates of reaction were measured as a function of the concentrations of the NADPH while keeping the ratio of the concentration of NADPH to that of 1,4-benzoquinone fixed at a constant value21. This alternative approach results in double reciprocal equations that are linear for a ping-pong mechanism (Equation 3) and polynomial for a sequential mechanism (Equation 4)21. As shown in Figure 7, PA0660 showed a linear pattern, consistent with a ping-pong steady-state kinetic mechanism. A kcat value of 90 ± 20 s-1 was estimated from the y-intercept in Figure 7.

Figure 7. Double-reciprocal plot of the initial rates of reaction as function of [NADPH] at a fixed ratio α = 0.2 of [NADPH]/[1,4-benzoquinone]. Initial rates were measured in 20 mM Tris-


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Biochemistry

Cl, pH 7.5, 150 mM NaCl, at 25 °C, in triplicate, with the average values and the associated standard errors shown. The line was obtained by fitting data to Equation 3.

Bioinformatics. A BlastP search and multiple sequence alignment of the protein sequence of PA0660 highlighted the presence of six conserved motifs (Table 4) among >1,000 sequences in the non-redundant protein database. The consensus motifs identified for PA0660 are different from the conserved motifs identified in PaNMO for Class I NMO10 and the NADH:quinone reductases identified from PA102411. Figure 8 shows an example of seven hypothetical NMOs with the conserved motifs identified in PA0660 boxed in green, while the protein sequences of PaNMO and PA1024 show the conserved motifs identified in previous studies10,

11

boxed in yellow and orange, respectively. The sequence identity and sequence

similarity shared by PA0660 and hypothetical nitronate monooxygenases carrying the conserved motifs identified in the protein sequence of PA0660 are shown in Table S2. Table 4. Conserved motifs in the protein sequence of PA0660 Motif I

Consensus sequence 14 P-X-h-X-[A/S]-P-[M/L]-F-[L/I]-X-S24

II

33

C-(X)4-h-[G/A]-[S/T/A]-h-P-A-L-N-X-R47

III

98

X-(h)2-I-[T/S]-S-[L/V]104

IV

115

H-X-[Y/W]-G-G-X-V-[F/L]-H-D124

V

139

[V/A]-D-G-(h)3-V-(X)2-G-A-G-G-H-A-G-(X)3-P158

VI

192

G-[A/C]-D-h-X-Y-h-G-[T/S]-X-F-[I/L]-(X)3-E207

The numbering of the residues refers to the protein sequence of PA0660; the brackets identify residues that can alternatively be present in that position, h represents position occupied by a hydrophobic residue, while X represents a position where any amino acid is accepted.


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Figure 8. Multiple sequence alignment of hypothetical nitronate monooxygenases carrying the conserved motifs identified in the protein sequence of PA0660. The numbering of the residues refers to PA0660 sequence. The conserved motifs of PA0660, listed in Table 4, are boxed in green (sequences 1 to 8). Sequences 9 and 10 belong to PA1024 and to PaNMO, with the


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different motifs described for the class of NADH:quinone reductases similar to PA1024 and for NMO Class 1 boxed in orange and yellow, respectively. The sequences identifiers used are: PA0660, P. aeruginosa PAO1 (NP_249351.1); C.algicola, Cellulophaga algicola (WP_013550013.1); B.parapertussis, Bordetella parapertussis (WP_015040637.1); S.silvestris, Solibacillus silvestris (WP_014823224.1); B.dendrobatidis, Batrachochytrium dendrobatidis JAM81 (XP_006680703.1); A.anophageffer, Aureococcus anophagefferens (XP_009037650.1); E.huxleyi, Emiliania huxleyi CCMP1516 (XP_005792771.1); P.hodgsonii, Pantholops hodgsonii (XP_005978881.1); PA1024, P. aeruginosa PAO1 (NP_249715.1); PaNMO, i.e. PA4202 P. aeruginosa PAO1 (NP_252891.1). A large-scale analysis of sequence-function relationships in NMO family was performed in order to investigate possible biological functions related to PA0660. A sequence similarity network (SSN) for the NMO family (Pfam family PF03060) was constructed using the Enzyme Functional Initiative Similarity Tool, EFI-EST (http://efi.igb.illinois.edu/efi-est/index.php)25 (Figure 9). The products of the three genes originally annotated as hypothetical NMOs in P. aeruginosa PAO1, namely pa0660, pa1024 and pa4202, are in different clusters in the sequence similarity network, consistent with the differences identified for these enzymes in terms of the conserved motifs present in the sequence. As expected, the cluster composed by the first neighbors of PA0660 is mostly associated with the incorrectly annotated NMO activity. However, some other functions are reported such as enoyl-acyl-carrier-protein reductase, Llactate dehydrogenase and N-acyl-D-glutamate deacylase.


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Figure 9. Sequence similarity network for sequences from the nitronate monooxygenase family (Pfam family PF03060) with an alignment score of 60. The network was generated using EFI-EST25 and represented using Cytoscape26. Groups of protein sequences with ≥ 50% pairwise identity are represented by individual dots. First neighbors of PA4202, PA1024 and PA0660 are colored in green, orange and purple, respectively.


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DISCUSSION The exponential increase in the determination of protein sequences via genome sequencing30 allows for rapid growth of public databases such as GenBank and UniProt/TrEMBL31. However, the majority of protein sequences have not been experimentally characterized. This results in a large number of hypothetical or erroneously annotated proteins in database4, 5. Although these data embrace vast promise for biological and medical discovery, the accuracy of these predictions is essentially unknown3. Quality of prediction can be improved by establishing rigorous reference standards of protein with experimentally determined functions and activities. An example of a family of enzymes with a significant number of hypothetical proteins in the data base is the NMOs, with over 5000 genes in the GenBank currently annotated as NMO. Two previous studies addressed the subject of the gene function prediction of hypothetical NMOs from P. aeruginosa PAO110,

11

and allowed the identification of two

different classes of enzymes represented by PaNMO (PA4202) and PA1024, which was reclassified as an NADH:quinone reductase. In this study, we have cloned, expressed, and purified the third hypothetical NMO from P. aeruginosa PAO1 encoded by the gene pa0660. Despite its annotation as hypothetical NMO, the biochemical and kinetic characterization of PA0660 presented here show that no enzymatic activity was detected with the physiological substrate P3N or the neutral form 3-NPA. The enzyme PA0660 was not able to use nitronates/nitroalkanes of 2 and 3 carbon chain length as substrates. The lack of activity with neutral nitroalkanes suggests that PA0660 is also not a nitroalkane oxidase32. The enzyme, instead, uses NAD(P)H and quinones as reducing and oxidizing substrates, respectively.

Therefore, PA0660 should be classified as an

NAD(P)H:quinone reductase (EC 1.6.5.2). FMN is the cofactor of PA0660 and the enzyme exists as a dimer in solution, as established via ESI-TOF spectrometry and analytical ultracentrifugation analysis, respectively. The UV-visible absorption spectrum of purified PA0660 displays the characteristic flavin signature, with maxima at 357 and 447 nm at pH 7.5. The high-energy band is significantly blue shifted (~ 18 nm) from the maxima of 375 nm of free flavin in solution33. This spectral perturbation of the enzyme-bound cofactor suggests a hydrophobic environment surrounding the flavin in PA066034. The low-energy band also presents a small hypsochromic shift from 450 to 447 nm, and a well-defined shoulder at 470 nm which are consistent with a hydrophobic


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environment of the pyrimidine ring of the FMN cofactor33. In this regard, FMN is the cofactor for most NAD(P)H:quinone reductases from prokaryotes characterized heretofore, excepting modulator of drug activity B (MdaB) from Helicobacter pylori35 and P. aeruginosa36 that use FAD. PA0660 is highly efficient in using NADPH and NADH as electron donors, with a preference for NADPH, as indicated by the steady-state kinetics and reductive half-reaction studies. The enzyme turns over with NAD(P)H and the electron acceptor DCPIP. The secondorder rate constant for substrate capture kcat/Km demonstrates a 25-fold preference of the enzyme for NADPH compared to that for NADH at pH 7.5 (Table 1). Additional evidence of the preference for NADPH comes from reductive half-reaction studies in a stopped-flow spectrophotometer at pH 7.5, as the second-order rate kred/Kd in the presence of NADPH is 35 times higher than with NADH as substrate. The enzyme preference for NADPH is mostly related to substrate binding in the active site rather than catalytic effects, as indicated by the steady-state kinetic of the enzyme showing a 20-fold smaller Km value for NADPH compared to that for NADH at pH 7.5. Due to the ability of using NADPH and NADH as reducing substrates, PA0660 can be considered also a diaphorase37. Example of FMN-containing enzymes with diaphorase activity are azoreductases20, oxidoreductases classified as old yellow enzymes (OYE)38, and flavin-dependent monooxygenases39. PA0660 thus is remarkably different from the gene product PA1024 from P. aeruginosa PAO1, which is specific for NADH11, and suggests a different function of PA0660 and PA1024 based on NAD(P)H preference. Enzymes specific for NADPH operates mainly as reducing power in anabolic reaction, whereas enzymes specific for NADH, are preferentially used in catabolic pathways40, 41. A recent study revealed that steric constrains control the strict substrate specificity of PA1024 for NADH42. In the case of PA0660, no structural information is available thus far, so that a rationale to explain the substrate specificity based on the substrate-binding site is currently unknown. Concerning the structure of the substrate, NADPH differs from NADH due to the presence of the ribose 2’-phosphate. It is possible to suggest that an enzyme group interacting with the 2’phosphate of NADPH could be responsible for the substrate specificity of the PA0660. However, the preference between NADPH and NADH can be not exclusively governed by the direct interaction with the ribose 2’-phosphate43. The nature of the substrate NADPH or NADH also is implicated in the substrate inhibition mechanism. At high concentrations of NADPH (> 50 µM)


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Biochemistry

substrate inhibition is observed, being the same not observed in the presence of NADH at concentration range of 15 to 500 µM. The substrate inhibition in this case could be related to the tighter binding observed for NADPH, as indicated by the steady-state kinetic of the enzyme showing a 20-fold smaller Km value for NADPH compared to that for NADH at pH 7.5. Different biological functions can be created by substrate inhibition in different biochemical contexts44. The precise mechanism by which both substrate inhibition and substrate specificity occur in PA0660 needs further structural investigation of the enzyme by itself and in complex with the products NAD(P)+. PA0660 can reduce a broad spectrum of quinone substrates, as demonstrated by the steady-state kinetic data (Table 3). The enzyme prefers bicyclic quinones, the best substrates being the 1,4-naphthoquinone and the 5-hydroxy-1,4-naphthoquinones, as indicated by the large kcat/Km values. 1,4-benzoquinone was the best substrate tested in the one ring quinone series. Addition of methyl groups in the 2 position, or methoxy groups in the 2 and 6 position results in a significant decrease in the kcat/Km value, possibly due to increased steric constrains for the formation of a catalytically competent enzyme-substrate complex. This is in agreement with what has been reported for the reactivity of the NADH:quinone reductase PA1024 from P. aeruginosa with different quinones. Other examples of flavin-dependent quinone reductases in P. aeruginosa are tryptophan [W] repressor-binding protein (WrbA) (PA0949), and modulator of drug activity B (MdaB) (PA2580)36. PA0660 obeys a Ping-Pong Bi-Bi steady-state mechanism. As suggested by the linear pattern observed in the double reciprocal plot when an alternative method was applied to evaluate the catalytic mechanism of PA0660 by carrying out experiments in which the concentrations of NADPH and 1,4-benzoquinone were varied in a constant ratio. The enzyme was able to turnover at 90 ± 20 s-1 in the presence of NADPH and 1,4-benzoquinone. All reported flavin-dependent NAD(P):quinone reductases were demonstrated to operate via a PingPong Bi-Bi steady-state kinetic mechanism35. This is consistent with both reducing and oxidizing substrates occupying a similar space oriented to the N5 atom of the flavin that promotes the transfer of hydride in both half reactions in the active site of the NAD(P)H:quinone reductases35, 42, 45, 46

. Comparison between steady-state and rapid kinetics parameters suggests that both

reductive and oxidative half-reactions are partially rate-limiting steps in PA0660 catalysis. The


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estimated kred and kox values of ~360 s-1 and ~270 s-1 for flavin reduction and oxidation (Table 2) are significantly larger than the kcat value of ~90 s-1 estimated for the enzyme overall turnover. The fact that the Michaelis-Menten constant Km for NADPH (Table 1) is lower that the binding constant Kd (Table 2) implies that product release is slow compared to the chemical step47, which would be consistent with product release being the rate-limiting step in turnover. PA0660 displays a negligible NAD(P)H:oxidase activity. Evidence to support this conclusion comes from the flavin-monitored turnover experiment and the steady-state kinetics. After flavin reduction, the enzyme stays in the reduced form for ~100 s in the presence of oxygen. At pH 7.5, the kobs obtained at 10 µM NADPH and atmospheric oxygen and the apparent kcat obtained at atmospheric oxygen and varied concentrations of NADPH are 0.08 s-1 and 0.12 s1

, respectively. Altogether, these findings indicate that in the presence of molecular oxygen, the

oxidative half-reaction is fully rate-limiting for the overall turnover of the enzyme at pH 7.5. Also, during the enzyme monitored single turnover no accumulation of a species with absorbance at 360-380 nm was detected, indicating no accumulation of the C4a(hydro)peroxyflavin intermediate which would support monooxygenase activity. Changes in absorbance at 500-600 nm which are indicative of the formation of charge-transfer complexes were also not detected. An NADPH:quinone reductase from P. aeruginosa recently characterized, PA1225, also lack NAD(P)H oxidase activity48. In contrast, the FMN-dependent NADH:quinone reductase from P. aeruginosa PA1024 has a small, but not negligible, NADH oxidase activity11 of ~1 s-1. Different bacterial flavin-dependent quinone reductases have been annotated as azoreductases to demonstrate their ability to detoxify azo dyes from the environment49-51. In a recent study on azoreductases from P. aeruginosa, it was proposed that several azoreductases can rapidly reduce a wide range of quinones. Based on their reaction mechanisms similarities, NAD(P)H quinone reductases and azoreductases were suggested to form an FMN-dependent superfamily of enzymes52. As shown here, PA0660 does not have azoreductase activity, as demonstrated by the lack of enzymatic activity with the azo dye methyl red. In this regard, WrbA36, PA102411 and PA122548 are other NAD(P)H:quinone reductases identified that do not show azoreductase activity beside PA0660. The conserved motifs identified in PA0660 contain critical differences to the other enzymes annotated as NMOs in P. aeruginosa PAO1, PA1024 and PaNMO, that discriminate


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Biochemistry

the proteins biological function. These three enzymes are in different clusters in the sequence similarity network constructed for the NMO family (Figure 9). The six conserved motifs identified in the protein sequence of PA0660 (Table 4) are conserved in >1000 sequences of hypothetical NMO in the non-redundant protein database (Figure 8). These sequences belong to bacteria, with the notable exceptions from eukaryotic sources of the fungus Batrachochytrium dendrobatidis JAM81, the alga Aureococcus anophagefferens, the unicellular phytoplankton Emiliania huxleyi CCMP1516, the starlet sea anemone Nematostella vectensis, and the Tibetan antelope Pantholops hodgsonii. This is remarkably different from the two classes of enzymes represented by PaNMO and NADH:quinone reductase PA1024, which are conserved in bacteria and fungi. Diverse species of bacteria possess hypothetical proteins carrying the six motifs identified in PA0660 and some of them are known pathogens such as Bordetella parapertussis, Bordetella bronchiseptica, and Leptospira santarosai. The fact that this class of enzymes appears to be so widely represented in bacteria and not strictly conserved in fungi and other eukaryotes can be interpreted in two ways: either the function of the enzymes represented by PA0660 is involved in a cellular pathway specific to bacteria metabolism53, such as peptidoglycan biosynthesis, or the corresponding fungal enzymes evolved to a different subclass of enzymes with different conserved motifs. Genomic context comparison does not provide information on the protein function prediction since the operon containing PA0660 includes only one more enzyme, PA0661, currently described as a conserved hypothetical protein. There is no crystal structure available for PA0660 and due to a protein sequence identity lower than 30% with templates in the PDB database a homology model of this protein would not be reliable. It is not possible therefore to locate the six conserved motifs identified in this study on the three-dimensional structure of PA0660. However, the multiple sequence alignment of hypothetical proteins carrying the motifs identified in PA0660, PaNMO (PA4202)10, and PA102411, 12, boxed in green, yellow, and orange in Figure 8, highlights that three conserved motifs occupy the same position in the alignment. Specifically, motifs I, the end of motif III, and motif V of PA0660 correspond to the following motifs (the numbering is given first for the motifs in PA1024 and second for PaNMO): motifs I and I, motifs VII and II, and motifs IV and III. These three locations, that will be referred as locations A, B, and C, with different consensus sequences in the multiple sequence alignment are marked with a red asterisk and labeled in Figure 8. In the case of PA1024 and PaNMO the comparison of the two crystal structures (PDB


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codes 4Q4K10 and 2GJL12) shows that the three locations A, B, and C overlay also in the crystal structure, specifically in the TIM barrel domain (FMN-binding domain). Location A contacts the face of the cofactor not exposed to the active site cavity, location B is near the pyrimidine moiety of the isoalloxazine ring, and location C is on the surface above the active site with a fully conserved histidine residue (H183 in PaNMO and H152 in PA1024) projecting in the active site cavity. Despite the lack of structural information on PA0660 it is possible to hypothesize that it possesses a TIM barrel domain and that motifs I, III, and V identified in this study are located similarly to PaNMO and PA1024 (locations A, B, and C). In this case the scaffold of the TIM barrel domain appears to be recycled by nature for three FMN-containing enzymes catalyzing different reactions and the side chains of residues in the positions A, B, and C are important for protein function. The consensus sequences identified for location A, B, and C in the three different classes of enzymes represented by PA0660, PA1024, and PaNMO are suggested to be relevant for both uses in multiple sequence alignments for gene function prediction and for enzyme design. This study complements the two previous studies on the improvement of the gene function prediction of hypothetical NMOs from P. aeruginosa10, 11 and identifies a new class of bacterial enzymes with NAD(P)H:quinone reductase activity. PA0660 is an FMN-dependent NAD(P)H:quinone reductase that operates through a ping-pong bi-bi steady-state kinetic mechanism with a preference for NADPH over NADH. A broad spectrum of quinone substrates can be reduced by PA0660. Both reductive and oxidative half-reactions are partially rate-limiting steps in PA0660 catalysis. Seven conserved motifs were identified in the protein sequence of PA0660, which are conserved in more than 1000 hypothetical NMOs belonging mostly to bacteria that need to be reclassified.


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ASSOCIATED CONTENT Supporting information The Supporting Information is available free of charge on the ACS Publications website. Protein purification of PA0660 visualized on a polyacrylamide gel (Figure S1), PA0660 NAD(P)H:oxidase activity investigation (Figure S2 and Table S1), Sequence identity and similarity shared by PA0660 and hypothetical nitronate monooxygenases carrying the conserved motifs identified in the protein sequence of PA0660 (Table S2). Accession codes PA0660, UniProtKB entry Q9I5R1.

AUTHOR INFORMATION Corresponding Author *Department of Chemistry, Georgia State University, P.O. Box 3965, Atlanta, GA 30302-3965. Telephone: 404-413-5537. Email: [email protected]. ORCID Giovanni Gadda: 0000-0002-7508-4195 Renata Almeida Garcia Reis: 0000-0003-2565-0889 Funding This work was supported by National Science Foundation Grant CHE-1506518 (to G.G.) and the Catch Them Young Program – Molecular Basis of Disease – from Georgia State University for High School students (to I.W.). Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS The authors thank Dan Su for the insightful discussions, Daniel Ouedraogo for assistance with the stopped-flow experiments, Siming Wang for providing mass spectrometry services, and John Mick Robbins for the sedimentation velocity analytical ultracentrifugation analysis.


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Kinetic Investigation of a Presumed Nitronate ... - ACS Publications

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