Insights into urease inhibition by N-(n-butyl) phosphoric triamide


Feb 8, 2019 - Luca Mazzei , Michele Cianci , Umberto Contaldo , and Stefano Ciurli. J. Agric. Food Chem. , Just Accepted Manuscript. DOI: 10.1021/acs...
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Agricultural and Environmental Chemistry

Insights into urease inhibition by N-(n-butyl) phosphoric triamide through an integrated structural and kinetic approach Luca Mazzei, Michele Cianci, Umberto Contaldo, and Stefano Ciurli J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04791 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019

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Insights into urease inhibition by N-(n-butyl) phosphoric triamide

2

through an integrated structural and kinetic approach

3

Luca Mazzei,† Michele Cianci,‡ Umberto Contaldo,† and Stefano Ciurli†,*

4

†Laboratory

5

of Bologna, Italy.

6

‡Department

7

Marche, Ancona, Italy

8

* Corresponding author; email:

[email protected]

9

ORCID for Luca Mazzei:

0000-0003-1335-9365

10

ORCID for Michele Cianci:

0000-0001-5607-6061

11

ORCID for Umberto Contaldo:

0000-0002-1700-170X

12

ORCID for Stefano Ciurli:

0000-0001-9557-926X

of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University

of Agricultural, Food and Environmental Sciences, Polytechnic University of

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ABSTRACT

2

The nickel-dependent enzyme urease represents a negative element for the efficiency of

3

soil nitrogen fertilization as well as a virulence factor for a large number of pathogenic

4

and antibiotic-resistant bacteria. The development of ever more efficient urease inhibitors

5

demands knowledge of their mode of action at the molecular level. N-(n-butyl)-

6

phosphoric tri-amide (NBPTO) is the oxo-derivative of N-(n-butyl)-thiophosphoric tri-

7

amide (NBPT), extensively employed in agriculture to increase the efficiency of urea-

8

based fertilizers. The 1.45 Å resolution structure of the enzyme-inhibitor complex

9

obtained upon incubation of Sporosarcina pasteurii urease (SPU) with NBPTO shows the

10

presence of di-amido phosphoric acid (DAP), generated upon enzymatic hydrolysis of

11

NBPTO with release of n-butyl amine. DAP is bound in a tridentate binding mode to the

12

two Ni(II) ions in the active site of urease using two O atoms and an amide NH2 group,

13

while the second amide group of DAP points away from the metal center into the active

14

site channel. The mobile flap modulating the size of the active site cavity is found in a

15

disordered closed/open conformation. A kinetic characterization of the NBPTO-based

16

inhibition of both bacterial (SPU) and plant (Canavalia ensiformis or jack bean, JBU)

17

ureases, carried out by calorimetric measurements, indicates the occurrence of a

18

reversible slow-inhibition mode of action. The latter is characterized by a very small value

19

of the equilibrium dissociation constant of the urease-DAP complex caused, in turn, by

20

the large rate constant for the formation of the enzyme-inhibitor complex. The much

21

greater capability of NBPTO to inhibit urease, as compared to NBPT, is thus not caused

22

by the presence of a P=O vs. a P=S moiety, as previously suggested, but rather by the

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readiness of NBPTO to react with urease without the need to convert one of the P-NH2

2

amide moieties to its P-OH acid derivative, as in the case of NBPT. The latter process is

3

indeed characterized by a very small equilibrium constant that reduces drastically the

4

concentration of the active form of the inhibitor in the case of NBPT. This indicates that

5

high-efficiency phosphoramide-based urease inhibitors must have at least one O atom

6

bound to the central P atom, in order for the molecule to efficiently and rapidly bind to

7

the dinickel center of the enzyme.

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INTRODUCTION

2 3

Urease (urea amidohydrolase, E.C. 3.5.1.5) is a nickel-dependent enzyme found in a

4

large variety of organisms such as plants, algae, fungi, and prokaryotes 1-6 that catalyzes

5

the rapid hydrolytic decomposition of urea (Scheme 1) 7-8:

6

7

Scheme 1

8 9

The enzymatic hydrolysis of urea occurs in two steps: the first, strictly requiring urease,

10

consists of the decomposition of urea to give ammonia and carbamate, followed by the

11

spontaneous reaction of carbamate to give a second molecule of ammonia and

12

bicarbonate (Scheme 2) 4-6. The enzymatic reaction has a half-time of a few microseconds,

13

with a kcat/KM that is 3 × 1015 times higher than the rate of the uncatalyzed reaction,

14

making urease the most efficient hydrolase known 9.

15

Scheme 2

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This reaction causes an overall pH increase that has negative consequences both on

2

human health 10 and agriculture 11. Indeed, a large amounts of urease is present in soils

3

as intra- or extra-cellular enzyme, respectively present in whole cells of living organisms

4

such as plants

5

Urease reaction in soils causes a substantial decrease in the efficiency of urea-based soil

6

fertilization because of i) fast ammonia volatilization process that leads to loss of N to the

7

atmosphere, ii) root damage to seedlings and young plants as a consequence of the pH

8

increase, iii) decrease of ammonium absorption by plant roots, iv) formation of airborne

9

particulate matter and v) atmospheric pollution and increase of the greenhouse effect 14.

10

Additionally, urease is the main virulence factor for a large variety of human pathogens:

11

among the twelve antibiotic-resistant priority pathogens listed in 2017 by the World

12

Health Organization

13

activity to survive in the host organism. These aspects render urease a critical target to

14

develop new drugs for both agricultural and medicinal applications.

12

or microbes 2, or adsorbed in organo-mineral colloidal complexes

15,

13.

ten are ureolytic bacteria that take advantage of the urease

15

Phosphoramides are a class of well-known and very potent urease inhibitors, acting

16

with a slow-binding inhibition mechanism 16-18. The inhibition process appears to involve

17

a hydrolytic event catalyzed in situ by urease, with the subsequent trapping of a

18

tetrahedral moiety that blocks the enzyme active site by mimicking the transition state

19

formed during the enzyme reaction of urea hydrolysis. In this context, significant

20

information on the structural basis of urease inhibition by phosphoramides has been

21

provided by the X-ray crystal structures of SPU in complex with di-amido phosphate

22

(DAP) after the treatment of the enzyme with phenyl-phosphoro-diamidate (PPD) 19, and

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in complex with mono-amido thiophosphate (MATP) after incubation of SPU with N-

2

diamino-phosphino-thioyl-butan-1-amine,

3

thiophosphoric triamide (NBPT hereafter) 18. NBPT is used in agriculture as a nitrogen

4

stabilizer to reduce ammonia volatilization 20 even though some negative aspects of this

5

protocol have been reported 21-29. Early studies were conducted on the efficacy of NBPT

6

in inhibiting ureases from different sources and in reducing ammonia volatilization upon

7

urea addition, either in vitro or in soils 30-35. These studies claimed that NBPT has little or

8

no effect as a urease inhibitor, while it must be converted to its oxo-analogue, N-(n-butyl)-

9

phosphoric triamide (NBPTO), in order for a strong inhibition to occur. The latter

10

conversion has been reported to take place predominantly in soils, rather than in solution

11

30-31, 34.

12

NBPT itself is able to inhibit plant urease without necessarily being converted to NBPTO,

13

which led to the suggestion that the moiety interacting with urease is DAP in the case of

14

NBPTO and the unaltered inhibitor in the case of NBPT 36. These controversies have been

15

recently solved through a multi-faceted investigation combining kinetic and

16

crystallographic evidence, and leading to the conclusion that urease inhibition in the

17

presence of NBPT entails the initial spontaneous hydrolysis of the inhibitor to its di-

18

amido derivative (NBPD), which then undergoes an enzyme-dependent transformation

19

to n-butyl amine and mono-amido thiophosphate (MATP), the latter binding to the Ni(II)

20

ions in the urease active site thus blocking catalysis. 18 The intrinsic capability of NBPT to

21

inhibit urease in vitro and in cell has been additionally demonstrated, 28-29 in time intervals

commonly

known

as

N-(n-butyl)-

This hypothesis was later partially challenged by evidences demonstrating that

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(minutes) that, according to the measured half-time of conversion of NBPT to NBPTO

2

(days),30-31, 34, 37 cannot implicate any role for NBPTO.

3

Within this framework, the causes for NBPTO being a much more efficient urease

4

inhibitor as compared to NBPT are still unclear. The present study reports the X-ray

5

crystal structure of urease from S. pasteurii, a widespread and highly ureolytic soil

6

bacterium, inhibited in the presence of NBPTO. The structure, determined at 1.45 Å

7

resolution, demonstrates that NBPTO interacts with the Ni(II) ions in the urease active

8

site and undergoes in situ hydrolysis that generates the di-amido phosphate derivative

9

(DAP). Extensive kinetic study of the inhibition of SPU and JBU in the presence of NBPTO

10

was then carried out to elucidate the nature of the urease inhibition mechanism and to

11

compare, under identical assay conditions, NBPT

12

reveal that the difference between these two inhibitors rests in the presence of an oxygen

13

atom in NBPTO, while in the case of NBPT the required formation of the di-amido

14

derivative NBPD is largely disfavored, with an equilibrium constant estimated to be in

15

the order of 10-4. This required pre-equilibrium significantly decreases the effective

16

concentration of the active form of the inhibitor (NBPD). These results clarify a long-

17

standing controversy and pave the way to improve and develop more efficient

18

derivatives of phosphoramides as general urease inhibitors for agricultural and medical

19

applications.

18

and NBPTO. The results obtained

20 21 22

MATERIALS AND METHODS

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Enzyme and inhibitor sources

3

SPU (Mr = 250 kDa) was expressed and purified from S. pasteurii following a previously

4

described protocol 38. JBU (Mr = 550 kDa) type C-3, powder (≥ 600,000 units/g) was purchased

5

from Sigma-Aldrich SRL, Milan (Italy). Protein quantification was carried out by measuring

6

enzymatic activity via a pH-STAT method at pH 7.0

7

equal to 2500 units mg-1 and 3500 units mg-1, respectively 17, 40. NBPTO was purchased from Santa

8

Cruz Biotechnology, Inc., Heidelberg (Germany).

9

Crystallization, data collection and structural determination

39,

and considering their specific activity

10

A 11 mg mL-1 SPU solution in 50 mM HEPES buffer at pH 7.5, containing 50 mM Na2SO3 and

11

2 mM EDTA, was incubated for 1 h with 50 mM NBPTO dissolved in the same buffer.

12

Subsequently, 2 µL of the SPU-NBPTO solution was diluted with 2 µL of a precipitant solution

13

containing 1.6-2.1 M ammonium sulfate (AMS) dissolved in 50 mM sodium citrate buffer at pH

14

6.3 and containing the same concentration of NBPTO. The final pH of the drop was pH 7.0.

15

Crystallization trials were performed at 293 K using the hanging-drop vapor-diffusion

16

method, equilibrating the drop against 1 mL of the precipitant solution using 24-well XRL Plates

17

(Molecular Dimensions, Suffolk, UK). Rice-shaped protein crystals (0.15 x 0.15 x 0.4 mm3) grew

18

in the presence of 1.9-2.1 M AMS after few days. They were cryoprotected by transferring them

19

in a solution of 100 mM citrate buffer at pH 6.3, containing also 20% (v/v) ethylene glycol, 2.4 M

20

ammonium sulfate and the same concentration of NBPTO present in the crystallization drop. The

21

crystals were then flash-cooled and stored in liquid nitrogen.

22

Diffraction data were collected at 100 K using synchrotron radiation at the ID30-A beamline of

23

the ESRF storage ring, Grenoble (France) 41-43 by performing helical scans along the crystal

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to achieve higher resolution by minimizing radiation damage. Data were processed using

2

XDS

3

the crystal structures of SPU determined so far, and diffracted to a resolution of 1.45 Å. The

4

structure was determined by rigid body refinement using REFMAC 46 and, as a starting model, the

5

crystal structure of SPU in complex with MATP (PDB code 5OL4, 1.28 Å resolution) 18 devoid

6

of solvent molecules and ligands, and randomized in order to remove any possible phase bias.

7

Model building and water or ligand addition/inspection were manually conducted using COOT

44

and AIMLESS 45. The crystals belonged to space group P6322, isomorphous with all

8

47-48.

9

and then, at the last round, anisotropically refined to final Rfactor and Rfree of 12.53% and 14.31%,

The structure was isotropically refined, including the hydrogen atoms in the riding positions,

10

respectively. The model was checked with the PDB REDO web server

11

refinement statistics are reported in Table 1. Figures were generated using PyMol (The PyMOL

12

Molecular

13

(http://www.crystalmaker.com). Structure coordinates and structure factors were deposited in the

14

PDB with the accession code 6H8J.

Graphics

System,

Version

1.8

Schrödinger,

49.

LLC.),

Data collection and

and

CrystalMaker

15 16

### Table 1 here ###

17 18

Calorimetric studies on the enzymatic urea hydrolysis by urease

19

The determination of the kinetic parameters for the inhibition of SPU and JBU by NBPTO was

20

carried out by calorimetry using a high-sensitivity VP-ITC (ITC: Isothermal Titration Calorimetry)

21

micro-calorimeter (MicroCal LLC, Northampton, MA, USA), following an experimental set up

22

previously described

23

water, and the temperature of the reference and sample cells was set and stabilized at 298

18.

For all experiments, the reference cell was filled with deionized

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K. Stirring speed was 300 rpm, and thermal power was monitored every 2 s using high

2

instrumental feedback. All reagents used were buffered using 50 mM HEPES, pH 8.0,

3

containing 2 mM EDTA.

4

Progress-curves experiments were performed by setting up a so-called reverse M1 experiment

5

in which 15 µL of an enzyme solution (2-3 nM SPU and 1-1.5 nM JBU) were injected into a 100

6

mM urea solution (final volume = 1.50 mL) in the presence of increasing concentrations of

7

NBPTO (in the range 20 – 120 nM). The final enzyme concentration in the sample cell was 20-30

8

pM for SPU and 10-15 pM for JBU. The recorded thermal power (TP, µcal s-1) was integrated

9

over a 3600 s time period, starting from the time at which it reached its minimum. The resulting

10

total heat (µcal) was converted to the corresponding concentration of urea consumed during the

11

reaction according to the ∆𝐻app (cal mol-1) of the reaction. The values of the apparent molar

12

enthalpy (∆𝐻app) and of 𝐾M and 𝑘cat relative to the reaction of urea hydrolysis catalyzed by either

13

SPU or JBU, required to correlate the amount of heat produced by the reaction per mole of

14

hydrolyzed substrate, were taken from previous published data. 18, 38

15 16 17

RESULTS

18 19

Structural study of SPU inhibited in the presence of NBPTO

20

The structure of SPU co-crystallized in the presence of NBPTO was determined at 1.45

21

Å resolution by X-ray diffraction crystallography. The refined model shows the typical

22

quaternary structure of SPU, made of an (αβγ)3 assembly closely matching that of native

23

urease (PDB code 4CEU)

50

with a backbone RMSD of 0.17, 0.18 and 0.10 Å for the α, β

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and γ subunits, respectively. A complete evaluation of pairwise backbone RMSD per

2

residue between the two models is given in Figure 1-SI and reveals a substantial

3

superimposition, except for the residues in the range 310-340 of the  subunit. This

4

protein portion consists of an highly flexible helix-loop-helix motif, also known as mobile

5

flap, which is located over the active site cavity and modulates its aperture (Figure 1) 5.

6

This mobile flap has been almost invariably found in an open conformation 19, 38, 50-57 with

7

the exception of the structures of SPU inhibited by di-amido phosphate (DAP)

8

mono-amido thiophosphate (MATP)

9

urea hydrolysis enzymatic reaction, produced in situ when urease is inhibited in the

10

presence of PPD and NBPT, respectively. Indeed, in the latter two cases the mobile flap

11

is found in a closed conformation, suggested to stabilize the reaction intermediate during

12

the enzymatic reaction 3, 5. In the case of the final refined model of SPU inhibited in the

13

presence of NBPTO, the mobile flap is found in a double conformation, with a relative

14

occupancy of 60% (closed flap, blue ribbon in Figure 1) and 40% (open flap, red ribbon in

15

Figure 1). Furthermore, the region corresponding to residues 327-330 in the open

16

conformation, which includes a portion of the loop between the two helices in the flap, is

17

highly disordered so that a backbone trace could not be obtained.

18,

19

and

two analogues of the intermediate state of the

18 19

### Figure 1 here ###

20 21 22

The electron density around the Ni(II) ions in the active site region is clearly defined at the achieved 1.45 Å resolution (Figure 2A).

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### Figure 2 here ###

3 4

The overall structural layout of the amino acid side chains directly involved in the

5

nickel-binding is fully conserved with respect to all previous crystal structures of SPU. In

6

particular, the two Ni(II) ions are bridged by the carboxylate group of the carbamylated

7

αLys220*, which is bound to Ni(1) by Oθ1 (at 2.0 Å) and to Ni(2) by Oθ2 (at 2.1 Å). Ni(1)

8

is further coordinated by αHis249 Nδ (at 2.0 Å) and by αHis275 Nε (at 2.0 Å), whereas

9

Ni(2) is bound to αHis137 Nε (at 2.1 Å), αHis139 Nε (at 2.1 Å) and αAsp363 Oδ1(at 2.2

10

Å). The Ni(II) ions are well ordered (B-factor values of 15.9 and 14.6 Å2 for Ni(1) and

11

Ni(2), respectively) and separated by 3.8 Å, a value consistent with that found in native

12

SPU (3.7 Å, PDB codes 2UBP 19 and 4CEU 50), as well as in the case of the enzyme inhibited

13

in the presence of PPD and binding DAP in the active site (3.8 Å, PDB code 3UBP 19) or

14

with NBPT and binding MATP in the active site (3.7 Å, PDB code 5OL4 18).

15

The unbiased omit electron density map (shown in orange in Figure 2A and calculated

16

with Fourier coefficients Fo-Fc and phases from the last cycle of REFMAC refinement

17

before ligand addition) reveals the presence of an electron density around the two Ni(II)

18

ions that does not match the solvent molecules usually located in the active site of native

19

SPU. This electron density has a tetrahedral shape and indicates the presence of a non-

20

protein ligand that completes the coordination environment of the metal ions by binding

21

to the two Ni(II) centers through three atoms, with a fourth atom pointing away from the

22

bimetallic center. This tetrahedral arrangement exactly replaces the cluster of four water

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molecules existing in the active site of enzyme in its resting state. The calculation of a

2

composite omit map only confirmed the presence of the tetrahedral electron density,

3

unrelated to protein residues, following a bias-free procedure (Figure 2-SI).

4

Attempts to model a sulfate ion in this region yielded an Fo-Fc map that showed large

5

inconsistencies of electron density (see Supplementary Information for details). This

6

result, together with the absence of sulfate bound to the two Ni(II) ions in the active site

7

of native SPU crystallized under the same conditions

8

anion bound to the dinuclear Ni cluster at the active site of the protein under study.

19, 50

excludes the presence of this

9

Attempts to fit NBPTO into the electron density corresponding to the omit map(s)

10

failed, because of the absence of the n-butyl group, which must be due to the release of

11

n-butyl amine following a hydrolytic event. 1H NMR spectra on samples of NBPTO in

12

HEPES buffer at pH 7.0 kept at room temperature for four weeks ruled out a spontaneous

13

hydrolytic process producing n-butyl amine, indicating that the reaction must be

14

catalyzed by urease itself through a nucleophilic attack of the Ni-bridging hydroxide on

15

the P atom, as previously observed in the case of SPU inactivated by PPD 19 and NBPT 18.

16

Consequently, the electron density observed in the bridging position (LB) between the

17

Ni(II) ions can be ascribed to an O atom. The large scattering factor of the phosphorus

18

atom, as well as the anomalous signal shown in Figure 2B, allowed to unequivocally

19

locate its position as the central atom in the tetrahedral electron density. The identity of

20

the remaining functional groups around the P atom (L1 and L2 bound to Ni(1) and Ni(2),

21

respectively, and LD as the distant group) was then investigated by analyzing the H-

22

bonding network around the tetrahedral moiety. In particular, L1 must have at least two

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non-bonding electron pairs available, one deputed to coordinate Ni(1) and the other to

2

receive a hydrogen bond from αHis222 Nε (at 2.7 Å), which is protonated as inferred by

3

the interaction of αHis222 Nδ with the peptide NH group of αAsp224 (at 2.9 Å). In such

4

a situation, a hypothetical P-NH2 group would not satisfy this requirement, because its

5

only lone pair would be needed to coordinate Ni(1); on the other hand, an oxygen atom

6

bound to Ni(1) would satisfy this criterion. A similar analysis suggests the presence of

7

two hydrogen bonding acceptor atoms in the vicinity of L2, namely the backbone

8

carbonyl O atoms of αAla170 and αAla366 (both at 2.9 Å). This implies that L2 must be a

9

double H-bonding donor, supporting the presence of a P-NH2 group in this position. The

10

analysis of the H-bonding network around LD is less straightforward: the presence of two

11

potential H-bonding acceptor atoms, namely the backbone carbonyl O atoms of αGly280

12

and αAla366 (at 3.2 and 3.1 Å, respectively), together with the presence, in the case of the

13

closed flap conformation, of His323 N2 (at 3.2 Å), a potential H-bond donor or acceptor

14

depending on its protonation state, renders ambiguous the identification of this group if

15

based only on criteria that use H-bonding network capability. In order to resolve this

16

issue, and to support the assignment of L1 and L2 as described above, three independent

17

refinement procedures were carried out that involved placement of i) di-amido

18

phosphoric acid (DAP), ii) mono-amido phosphoric acid (MAP) or iii) phosphoric acid

19

(PA) into the electron density corresponding to the omit map. The results of these

20

refinements are provided in Table 2.

21 22

### Table 2 here ###

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Refining the ligand in different orientations within the active site yielded different sets

3

of B-factor values, with significant discrepancies between atoms either belonging to the

4

ligand or to the surrounding ions and residues. Minimizing and rationalizing these

5

discrepancies allowed the discrimination of the correct orientation of DAP within the

6

urease active site. B-factor value for the L1 ligand is consistent with its identity as an O

7

atom. The value of the B-factor obtained for the LD atom is consistent with those of the

8

surrounding atoms when refined as a N atom, while it is significantly larger when refined

9

as an O atom as in the case of MAP or PA. On the other hand, the B-factor value obtained

10

for the L2 atom is incongruently large when refined as an O atom (as in the case of PA) as

11

opposed to a N atom as in the case of DAP or MAP. Overall, this analysis is consistent

12

with the presence of DAP as the inhibitor found in the active site of urease crystallized in

13

the presence of NBPTO. The final refinement procedure was therefore performed by

14

modeling DAP in the active site of the inhibited SPU crystal structure, and the obtained

15

2Fo-Fc electron density map is shown in Figure 3A. In particular, DAP binds to Ni(1) and

16

Ni(2) by its OL1 and NL2 atoms, respectively. The second DAP oxygen atom (OB)

17

symmetrically bridges the two Ni(II) ions, while the second N atom points away from the

18

binuclear metallo-center towards the active site cavity opening (Figure 3B). Refined

19

selected distances and angles are given in Table 3.

20 21

### Figure 3 here ###

22

### Table 3 here ###

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1 2

Kinetic study of SPU inhibited in the presence of NBPTO.

3

The determination of the inhibition mechanism of SPU and JBU by NBPTO was achieved by

4

carrying out measurements of reaction kinetics using microcalorimetry. This technique was used

5

to carry out progress curve experiments, following a previously established protocol 18.

6 7

8

Progress curve experiments monitor the consumption of substrate (∆[S]) as a function of time ( Δ[𝑆] 𝑡), and permit to discern among different inhibition modes (Scheme 3):

Scheme 3

9

i) a fast enzyme inhibition mechanism that considers an equilibrium between an enzyme

10

(E), an inhibitor (I) and their complex (EI) regulated by values of the kinetic constants 𝑘3

11

and 𝑘 ―3 that are comparable to those of 𝑘1 and 𝑘 ―1, the latter regulating the equilibrium

12

between the enzyme (E), the substrate (S), and their complex (ES); the ES complex then

13

further evolves to products (P) through the 𝑘cat constant; ii) a slow-binding reversible

14

inhibition by which the formation of the EI complex occurs through a single equilibrium

15

regulated by smaller 𝑘3 and 𝑘 ―3 rate constants as compared to 𝑘1 and 𝑘 ―1, and iii) a slow-

16

binding inhibition with the initial formation of an EI complex through a rapid

17

equilibrium regulated by 𝑘3 and 𝑘 ―3, followed by a slow isomerization step, governed by

18

𝑘4 and 𝑘 ―4, to form a different and more stable complex, E*I 18. In the latter two cases, the

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inhibition strength varies with time, and therefore they are also referred to as time-

2

dependent inhibitors 58-59. In the case of a fast inhibition mechanism, Δ[𝑆] 𝑡 would be a straight

3

line, while in the presence of a slow-binding inhibition, Δ[𝑆] 𝑡 is expected to be linear at the

4

beginning of the reaction (with a velocity 𝑣𝑖) but subsequently to deviate from linearity until it

5

reaches a linear steady state in the later phases of the reaction (with a velocity 𝑣𝑠) 18.

6

The progress curves showing the consumption of urea as a function of time, determined at

7

increasing concentrations of NBPTO are shown in Figure 4. All obtained traces for SPU (Figure

8

4A) and JBU (Figure 4B) in the presence of NBPTO clearly deviate from linearity, indicating a

9

slow-binding inhibition mode for both bacterial and plant urease.

10 11

### Figure 4 here ###

12 13

Data analysis was performed as described previously for the inhibition of SPU and JBU in the

14

presence of NBPT 18. In particular, the obtained experimental progress curves were fitted to Eq. 1

15

58-59:

16 17

∆[S] = 𝑣𝑠𝑡 +

(𝑣𝑖 ― 𝑣𝑠) 𝑘𝑜𝑏𝑠

[1 ― 𝑒𝑥𝑝( ― 𝑘𝑜𝑏𝑠𝑡)]

Eq. 1

18 19

The fits provided the values for 𝑣i and 𝑣s, together with those for the pseudo first-order constant

20

𝑘obs that controls the transition from the initial phase to the steady-state phase for each

21

concentration of NBPTO tested (Table 4).

22

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### Table 4 here ###

2 3

The large invariance of initial velocities 𝑣i with respect to the concentration of inhibitor, as well

4

as the linear dependence of the 𝑘obs on the concentration of NBPTO used for both SPU (Figure

5

4C) and JBU (Figure 4D) support an inhibition mechanism that involves the formation of a

6

EI complex through a slow equilibrium (regulated by smaller 𝑘3 and 𝑘 ―3 rate constants)

7

that competes with the fast equilibrium to form the ES complex (regulated by larger 𝑘1

8

and 𝑘 ―1 rate constants) 58-59. The values determined for 𝑣i, 𝑣s and 𝑘obs at each concentration of

9

NBPTO were used to determine the kinetic constant 𝑘 ―3 (Table 4) for the dissociation of the EI

10

complex by using Eq. 2 58-59:

11 12

𝑣𝑠

Eq. 2

𝑘 ―3 = 𝑘𝑜𝑏𝑠 𝑣𝑖

13 14

The similar values of 𝑘 ―3 found for SPU and JBU support the hypothesis that a similar molecular

15

mechanism for the dissociation of the EI complex is operative for bacterial and plant urease in the

16

case of their inhibition by NBPTO. This underlines the presence of a common inhibiting species

17

that dissociates from the active site of urease, established in the present crystallographic study to

18

be diamidophosphate (DAP).

19 20

The value of the kinetic constant 𝑘3 (Table 4), which controls the formation of the EI complex, was estimated using the known values of [I], [S], 𝐾M and 𝑘 ―3 by means of Eq. 3 58-59:

21 22

𝑘3[I]

Eq. 3

𝑘𝑜𝑏𝑠 = 𝑘 ―3 + (1 + [S]/𝐾M)

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The value of 𝑘3 found for JBU is similar to that obtained for SPU, supporting the hypothesis that

3

a similar molecular mechanism for the formation of the EI complex is operative for both bacterial

4

and plant urease. This, together with the similar values of 𝑘 ―3, confirms the hypothesis of a shared

5

mechanism of catalysis and inhibition regardless of the biological source of the enzyme, consistent

6

with the largely conserved architectural structure of ureases among diverse natural kingdoms 5.

7

Finally, the equilibrium constant 𝐾𝑖∗ for the dissociation of the EI complex (Table 4) was

8

estimated as the 𝑘 ―3/𝑘3 ratio. The values of 𝐾𝑖∗ for SPU and JBU are again of the same order of

9

magnitude. The somewhat stronger inhibition by NBPTO observed for JBU can be ascribed both

10

to the smaller value of 𝑘 ―3 and to the larger value of 𝑘3, differences that could derive from small

11

changes in the microenvironment of the two active sites.

12 13 14

DISCUSSION

15 16

The present study combines kinetic and structural evidence to clarify the mode of action of

17

NBPTO as a urease inhibitor. The results thus obtained, together with previous analogous data

18

obtained using the thio-derivative of NBPTO, namely NBPT, allow us to shed new light on the

19

general aspects of urease inhibition by phosphoramides. It is also of interest to notice that purple

20

acid phosphatases (PAP), like urease, are hydrolases containing a binuclear metal center made of

21

Fe and/or Zn, which additionally share with urease an important role in both agriculture and human

22

health

60.

Understanding the mode of action of inhibitors targeting urease could thus help

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developing new lead compounds not only for this Ni-dependent enzyme but also for Fe,Zn-

2

dependent PAPs.

3

The available crystal structures of ureases from several bacteria and higher plants show

4

a typical trimeric assembly 4-6. Each assembly is in turn composed by a single chain [(α)3]

5

in higher plants, as in the cases of jack bean (Canavalia ensiformis) urease (JBU)

6

pigeon pea (Cajanus cajan) urease 62, by two chains [(αβ)3] in the case of Helicobacter pylori

7

63,

8

aerogenes 4-6. The minimal trimer eventually forms [(α)3]2 dimers in higher plants or nearly

9

spherical [(αβ)3]4 tetramers in H. pylori.63 Each trimeric assembly hosts three conserved

10

61

and

and by three chains [(αβγ)3] in the cases of Sporosarcina pasteurii (SPU) and Klebsiella

active sites, containing two Ni(II) ions 4-6.

11

The structure of SPU inhibited in the presence of NBPTO reveals a substantial conservation of

12

all tertiary and quaternary features of the protein structure, and the presence of DAP in the active

13

site, and all evidences indicate that the NBPTO-to-DAP conversion is catalyzed by urease, with

14

NBPTO acting as a suicide substrate. This situation is analogous to that observed for the inhibition

15

of SPU in the presence of PPD 19. NBPTO and PPD share therefore the same inhibition mechanism,

16

differing only for the group leaving the phosphoro-diamide molecular framework, namely phenol

17

for PPD and n-butyl amine for NBPTO (Figure 5). On the other hand, it has recently been shown

18

by protein crystallography that NBPT must undergo a preliminary spontaneous hydrolytic step to

19

the corresponding diamide (NBPD, Figure 5) in order to be processed by the enzyme, with NBPD

20

acting as a suicide substrate undergoing enzymatic hydrolysis to yield the final product, namely

21

the mono-amido thiophosphate (MATP) moiety bound to the Ni(II) ions in the active site of urease

22

18.

23

O atom is bound to the central P atom: this requirement is fulfilled in the case of PPD and NBPTO,

This analysis indicates that phosphoramides are good urease inhibitors only when at least one

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while for NBPT the preliminary loss of an amide group is necessary. This step thus implicates the

2

presence of a pre-equilibrium, whose constant can be estimated using the following analysis of the

3

rate constants for the inhibition of SPU and JBU in the presence of NBPT and NBPTO.

4

A comparison of the values of 𝑘 ―3 obtained for SPU and JBU inhibited in the presence of

5

NBPTO (Table 4) with the values of 𝑘 ―3 found for the same enzymes inhibited in the presence of

6

NBPT using an identical experimental protocol [(13.7 ± 1.4) x 10-5 s-1 for SPU and (2.70 ± 0.80)

7

x 10-5 s-1 for JBU] 18, indicates that the presence of the P=O or the P=S group does not significantly

8

influence the dissociation rate of the moiety bound to the active site. Similar values were also

9

determined in the case of PPD [(9.57 ± 1.76) x 10-5 s-1 for SPU and (2.12 ± 0.77) x 10-5 s-1 for

10

JBU, unpublished calorimetric-based data from our laboratory], consistently with previously

11

published data

12

moiety is MATP, sharing the same tetrahedral geometry, differing for the identity of the distal

13

group LD (a N atom for DAP and a S atom for MATP) but using the same ligands to bind the

14

cluster of two Ni(II) ions in the active site [a bridging O atom, an O atom bound to Ni(1) and a N

15

atom bound to Ni(2) (Figure 5)]. It can be concluded that the similar values for the dissociation

16

constants is due to the identical coordination mode of the actual inhibiting species.

36, 64.

In the case of NBPTO this moiety is DAP, while in the case of NBPT the

17

On the other hand, a comparison of the values of 𝑘3 obtained for SPU and JBU inhibited in the

18

presence of NBPTO (Table 4) with the values of 𝑘3 found for SPU and JBU inhibited in the

19

presence of NBPT using the same experimental protocol [(21.4 ± 2.2) M-1 s-1 and (28.7 ± 2.0) M-

20

1

21

NBPTO is four orders of magnitude faster than in the case of NBPT. Similar values were also

22

determined for PPD [(3.87 ± 0.41) x 105 M-1 s-1 for SPU and (1.33 ± 0.19) x 105 M-1 s-1,

23

unpublished data from our laboratory], consistently with previously published data 64.

s-1, respectively]

18,

reveals that the association step involving plant or bacterial urease and

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Overall, these effects are combined in the much larger values of the equilibrium dissociation

2

constants 𝐾𝑖∗ for the enzyme-inhibitor complex in the case of NBPT [(6.4 ± 0.9) µM for SPU and

3

(0.94 ± 0.27) µM for JBU 18] as compared to NBPTO [(0.62 ± 0.11) nM for SPU and (0.13 ± 0.02)

4

nM for JBU]. This remarkable four orders of magnitude larger inhibition strength of NBPTO as

5

compared to NBPT must be ascribed to the need to generate the diamide NBPD prior to urease

6

inhibition (Figure 5). The small value of 𝑘3 found for SPU and JBU inhibited in the presence of

7

NBPT must thus reflect the smaller (ca. four orders of magnitude) concentration of the active

8

species (NBPD) as compared to the nominal concentration of NBPT used in the experiment.

9

These considerations, in conclusion, suggest that the equilibrium constant of the fast NBPT-to-

10

NBPD hydrolysis reaction is ca. 10-4. This analysis indicates that the significantly larger inhibition

11

capability of NBPTO vs. NBPT is not due to the conversion of the P=S to the P=O as previously

12

suggested 33, but rather to the conversion of a phosphoro-triamide to a phosphoro-diamide. The

13

possibility that NBPTO is partially converted to n-butyl-phosphoro-diamide (NBPDO) in analogy

14

to NBPT cannot be excluded, but this has not been observed probably because of the rapid reaction

15

of NBPTO itself with urease regardless of its conversion to NBPDO, the latter most likely being

16

largely unfavorable in analogy to the NBPT-to-NBPD conversion.

17

The requirement for the presence of a P-O bond in order for the phosphoramide to act as an

18

efficient urease inhibitor is a strong indication that the initial step of the interaction of the inhibitor

19

with urease is the binding of this O atom to Ni(1), less coordinatively saturated than Ni(2). This

20

step would then be followed by a nucleophilic attack by the bridging hydroxide, leading to the loss

21

of n-butyl amine (for NBPT and NBPD) or phenol (for PPD) preliminary to, or in concert with,

22

further coordination of the remaining amido group to Ni(2). In all three cases, the Ni(II) ions are

23

bound to the same set of ligands, the difference being the identity of the distal group (amide N for

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DAP or a thiol S for MATP (Figure 5)), which then justifies the similar dissociation rate constants

2

observed for all the tested phosphoramides. The results of the kinetic study described here for the

3

inhibition of urease with NBPTO parallels previous results obtained on a number of different

4

phosphoramides.16 In particular, the large diversity of the rate constant for the formation of the

5

enzyme-inhibitor complex as a function of the inhibitor, and the very similar values of the rate

6

constant for the dissociation of the same adduct, supports the hypothesis that the enzyme-inhibitor

7

complex invariably involves DAP regardless of the phosphoramide used. On the other hand, when

8

thio-phosphoramides are used, the enzyme-inhibitor complex involves MATP

9

addition, the values of koff for DAP in the case of SPU and JBU are very similar to those observed

10

for MATP 18, consistently with the interaction of the same ligand atoms with the Ni(II) ions in

11

both cases (Figure 5).

18

(Figure 5). In

12

The kinetics of urease inhibition by NBPTO, together with the structure of the complex between

13

the enzyme and DAP, generated in situ by a urease-catalyzed hydrolysis of the primal inhibitor

14

and with structural and kinetic studies of the inhibition of urease with NBPT and PPD, clarify

15

previous misconceptions for the mechanism by which phosphoramides act as urease inactivators.

16

In particular, this set of studies established that the reason for NBPTO and PPD acting as much

17

superior urease inhibitors than NBPT, by four orders of magnitude, is not due to the substitution

18

of a P=S (for NBPT) with a P=O (for NBPTO and PPD) moiety, but rather to the need for NBPT

19

(and not for NBPTO nor PPD) to undergo an unfavorable pre-equilibrium leading to the formation

20

of NBPD, the diamide of the parent phosphoro-triamide. This result notwithstanding, NBPT is a

21

more effective soil urease inhibitor than NBPTO because of its higher stability toward hydrolysis

22

65

and longer persistence in the environment 66.

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These results are key to the purpose of developing new and more efficient inhibitors for urease, as well as for purple acid phosphatases, for applications in agriculture and medicine.

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ACKNOWLEDGMENTS

2

X-ray diffraction data were collected under the beam time award number MX-1949 from the

3

European Synchrotron Radiation Source (ESRF, Grenoble, France). Luca Mazzei is a postdoctoral

4

research assistant supported by the University of Bologna. Umberto Contaldo is a M.Sc. student

5

at the University of Bologna.

6 7

ABBREVIATIONS

8

NBPTO: N-(n-butyl)-phosphoric tri-amide; NBPT: N-(n-butyl)-thiophosphoric tri-

9

amide; SPU: Sporosarcina pasteurii urease; DAP: di-amido phosphoric acid; JBU:

10

Canavalia ensiformis or jack bean urease; MATP: mono-amido thiophosphate; NBPD: N-

11

(n-butyl)-thiophosphoric di-amide; EDTA: ethylene diamine tetra-acetic acid; AMS:

12

ammonium sulfate; PDB: Protein Data Bank; ITC: Isothermal Titration Calorimetry; HEPES: 4-

13

(2-hydroxyethyl)-1-piperazineethanesulfonic acid; RMSD: root mean square deviation; PPD:

14

phenyl phosphoro diamidate; MAP: mono-amido phosphoric acid; PA: phosphoric

15

acid; ES: enzyme-substrate; EI: enzyme-inhibitor; NBPDO: N-(n-butyl)-phosphoric di-

16

amide.

17

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2 3 4 5 6 7 8 9 10

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Table 1. Data collection, processing and refinement statistics Data collection Wavelength (Å) 0.9677 Detector DECTRIS EIGER X 4M Crystal-to-Detector distance (mm) 101.3 Oscillation angle (degrees) 0.1 Number of images 1800 Space group P6322 Unit cell (a, b, c, Å) 131.4, 131.4, 188.7 Resolution range (Å) a 48.74 – 1.45 Total number of reflections a 2317155 (103149) a Unique reflections 168094 (8062) Multiplicitya 13.8 (12.8) a Completeness (%) 99.6 (97.9) Rsym a,b (%) 10.7 (194.0) a,c Rpim (%) 3.1 (57.8) Mean I half-set correlation CC(1/2) a 0.999 (0.655) a Mean I/σ(I) 16.2 (1.5) Refinement statistics No. of monomers in the asymmetric unit 3 Rfactord (%) 12.53 d Rfree (%) 14.31 Cruickshank’s DPI for coordinate error e based on Rfactor (Å) 0.05 Wilson plot B-factor (Å2) 14.3 Average all atom B-factor f (Å2) 19.7 RMS (bonds) d 0.08 d RMS (angles) 1.40 Total number of atoms 7241 Total number of water molecules 605 Solvent content (%) 55.2 3 Matthews Coefficient (Å /Da) 2.75 Ramachandran plotg Most favored regions (%) 90.4 Additionally allowed regions (%) 8.8 Generously allowed regions (%) 0.6 Disallowed regions (%) 0.2 aHighest resolution bin in parentheses; bR ∑ ∑ ∑ ∑ | | sym = hkl j Ij ― 〈I〉 / hkl jIj, where I is the intensity of a reflection, and 〈I〉 is the mean intensity of all symmetry related reflections j; c𝑅𝑝𝑖𝑚 = ∑hkl{[1/(N ― 1)]1/2∑j|Ij ― 〈I〉|}/∑hkl∑jIj, where I is the intensity of a reflection, and 〈I〉 is the mean intensity of all symmetry related reflections j, and N is the multiplicity 67; dTaken from REFMAC 46; Rfree is calculated using 5% of the total reflections that were randomly selected and excluded from refinement; eDPI

= 𝑅𝑓𝑎𝑐𝑡𝑜𝑟 ∙ 𝐷𝑚𝑎𝑥 ∙ 𝑐𝑜𝑚𝑝𝑙 ―⅓

𝑁𝑎𝑡𝑜𝑚𝑠

(𝑁𝑟𝑒𝑓𝑙 ― 𝑁𝑝𝑎𝑟𝑎𝑚𝑠),

where Natoms is the number of the atoms included in the

refinement, Nrefl is the number of the reflections included in the refinement, Dmax is the maximum resolution of reflections included in the refinement, compl is the completeness of the observed data, and for isotropic refinement, Nparams  4Natoms 68; fTaken from BAVERAGE 69; gTaken from PROCHECK 69.

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Table 2. Isotropic B-factor values (Å2) for the independently refined diamido phosphate (DAP), monoamido phosphate (MAP) and phosphoric acid (PA) at 1.45 Å Ligand Ni(1) Ni(2) P LD L1 L2 LB

DAP 15.7 14.4 16.0 16.3 15.5 15.4 15.9

MAP 15.7 14.4 15.6 18.4 15.7 15.6 16.0

PA 15.7 14.4 15.5 18.7 15.9 18.0 16.2

4

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Table 3. Selected distances and angles around the Ni(II) ions in the crystal structure of native

2

SPU (PDB code 4CEU), SPU inhibited in the presence of PPD and binding DAP in

3

the active site (PDB code 3UBP), SPU inhibited in the presence of NBPT and binding

4

MATP in the active site (PDB code 5OL4), and SPU inhibited in the presence of

5

NBPTO and binding DAP in the active site (PDB code 6H8J)

Ni - L Distances (Å) Ni(1) - αLys220* Oθ1 Ni(1) - LB Ni(1) - L1 Ni(1) - αHis249 Nδ Ni(1) - αHis275 Nε Ni(2) - αLys220* Oθ2 Ni(2) - LB Ni(2) - L2 Ni(2) - αHis137 Nε Ni(2) - αHis139 Nε Ni(2) - αAsp363 Oδ1 Ni(1) ••• Ni(2) L1 ••• L2 L - Ni - L Angles (°) αLys220* Oθ1 - Ni(1) - αHis249 Nδ αLys220* Oθ1 - Ni(1) - αHis275 Nε αLys220* Oθ1 - Ni(1) - LB αLys220* Oθ1 - Ni(1) - L1 αHis249 Nδ - Ni(1) - αHis275 Nε αHis275 Nε - Ni(1) - LB LB - Ni(1) - L1 L1 - Ni(1) - αHis249 Nδ αHis249 Nδ - Ni(1) - LB αHis275 Nε - Ni(1) - L1 αLys220* Oθ2 - Ni(2) - αHis137 Nε αLys220* Oθ2 - Ni(2) - αHis139 Nε αLys220* Oθ2 - Ni(2) - L2 αLys220* Oθ2 - Ni(2) - LB αAsp363 Oδ1 - Ni(2) - αHis137 Nε

4CEU 3UBP 5OL4 6H8J @ 1.50 Å @ 2.00 Å @ 1.28 Å @ 1.45 Å 1.9 2.1 2.0 2.0 2.1 2.3 2.2 2.2 2.2 2.2 2.2 2.1 2.0 2.0 2.0 2.0 2.0 2.1 2.1 2.0 2.1 1.9 2.0 2.1 2.1 2.3 2.2 2.3 2.1 2.3 2.2 2.3 2.1 2.1 2.1 2.1 2.1 2.3 2.1 2.1 2.1 2.1 2.1 2.2 3.7 3.8 3.7 3.8 2.4 2.5 2.6 2.7 100.4 107.2 96.6 108.2 98.6 94.6 67.0 89.3 154.2 141.6 90.8 91.7 92.9 95.6 82.8

111.0 105.6 95.4 102.7 103.0 87.4 66.9 87.9 147.2 143.2 90.0 94.0 99.4 99.8 79.7

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101.5 104.0 96.8 105.3 99.0 94.9 69.4 87.2 153.5 148.0 90.4 91.5 98.7 96.9 81.5

105.3 105.9 95.9 107.1 99.6 93.5 69.3 85.2 150.8 144.1 90.8 92.0 97.7 96.8 81.0

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αAsp363 Oδ1 - Ni(2) - αHis139 Nε αAsp363 Oδ1 - Ni(2) - L(2) αAsp363 Oδ1 - Ni (2) - LB L2 - Ni(2) – LB LB - Ni(2) - αHis137 Nε αHis137 Nε- Ni(2) - αHis139 Nε αHis139 Nε- Ni(2) - L(2) αLys220* Oθ2 - Ni(2) - αAsp363 Oδ1 LB - Ni(2) - αHis139 Nε L2- Ni(2) - αHis137 Nε Ni(1) - LB - Ni(2)

86.4 94.5 89.1 67.7 95.0 108.5 88.4 172.4 155.3 162.6 122.1

82.4 92.3 88.0 70.6 92.6 107.9 86.9 167.5 155.2 161.9 110.2

83.4 91.4 91.6 69.3 93.4 107.6 88.7 168.6 157.4 161.2 116.6

85.7 91.6 89.3 69.5 93.7 109.8 86.0 170.2 154.9 161.9 114.4

1 2

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Table 4. Initial and steady-state rates, and pseudo first-order constant of SPU and JBU, inhibited

2

in the presence of NBPTO, determined by calorimetry (pH 8.0, 298 K). SPU [NBPTO] (nM) 𝒗𝐢 (x 104 mM s-1) 104

s-1)

𝒗𝐬 (x mM 𝒌𝐨𝐛𝐬 (x 104 s-1) 𝒌 ―𝟑 (x 105 s-1) 𝒌𝟑 (x 10-5 M-1 s-1) 𝑲𝒊∗ (nM) [NBPTO] (nM) 𝒗𝐢 (x 104 mM s-1)

0.0 7.35 ± 0.01 -

0.0 8.78 ± 0.01

𝒗𝐬 (x 104 mM s-1)

-

𝒌𝐨𝐛𝐬 (x 104 s-1) 𝒌 ―𝟑 (x 105 s-1) 𝒌𝟑 (x 10-5 M-1 s-1) 𝑲𝒊∗ (nM)

-

20.0

40.0

7.57 ± 0.01

7.09 ± 0.01

1.70 ± 0.01 5.45 ± 0.02

60.0

100.0

6.70 ± 0.01

6.74 ± 0.01

1.05 ± 0.01 0.80 ± 0.01 9.08 ± 0.02 12.94 ± 0.01 12.19 ± 2.18 1.95 ± 0.09 0.62 ± 0.11 JBU 60.0 80.0

0.37 ± 0.01 15.93 ± 0.04

0.39 ± 0.01 20.35 ± 0.02

100.0

120.0

8.55 ± 0.01

8.56 ± 0.02

8.48 ± 0.01

8.18 ± 0.01

7.98 ± 0.01

0.910 ± 0.008 4.06 ± 0.01

0.587 ± 0.342 ± 0.008 0.005 5.82 ± 0.01 7.68 ± 0.01 3.28 ± 0.50 2.47 ± 0.03 0.13 ± 0.02

0.380 ± 0.001 9.31 ± 0.02

0.284 ± 0.001 11.14 ± 0.04

40.0

7.04 ± 0.01

80.0

3 4

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Figure 1.

2

conformations of the active site flap, which modulates the access to the active site Ni(II) ions

3

(green spheres), are shown in red (open conformation) or blue (closed conformation). The side

4

chains of αLys220*, αCys322, and αHis323 are also shown.

Ribbon diagram of SPU inhibited in the presence of NBPTO. The two

5

6 7

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Figure 2.

2

The nickel coordination environment is shown with the final 2Fo − Fc electron density map

3

displayed at 1.0 σ (cyan), while the unbiased Fo − Fc omit map corresponding to the ligand is

4

shown at 3.0 σ (orange). Carbon, nitrogen, oxygen, and nickel atoms are colored gray, blue, red,

5

and green, respectively. (B) The same atomic model and nickel coordination environment are

6

shown (rotated by ca. 45° along the horizontal axis) with the anomalous map contoured at 3.0 σ.

(A) Atomic model of the SPU active site inhibited in the presence of NBPTO.

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Figure 3.

2

Nickel coordination environment shown superimposed to the final 2Fo − Fc electron density map

3

contoured at 1.0 σ. The electron density of the inhibitor is colored blue. (B) Crystallographic

4

structure of the same environment. Putative hydrogen bonds are shown as thin blue lines.

5

Spheres are drawn using the relative atomic radius values in CrystalMaker. Carbon, nitrogen,

6

oxygen, phosphorus, and nickel atoms are colored gray, blue, red, orange, and green,

7

respectively.

Atomic model of the SPU active site inhibited in the presence of NBPTO. (A)

8

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Figure 4.

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experiments. (A and B) Progress curve plots representing the consumption of urea over time at

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increasing concentrations of NBPTO, for SPU (A) and JBU (B). (C and D) Linear plots of kobs as

4

a function of NBPTO concentration for SPU and JBU, respectively. In both panels, the lines

5

represent the results of an exponential or linear fit of the data.

Inhibition of ureases in the presence of NBPTO determined by progress curve

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Figure 5.

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PPD (top panel), NBPTO (middle panel), and NBPT (bottom panel). In all cases, the three atoms

3

involved in directly binding the Ni(II) ions in the active site of urease are shown in blue and

4

displayed with the same orientation, while the fourth atom, which occupies the distal position, is

5

shown in red.

Reaction schemes describing urease inhibition by the phosphoramide derivatives

6

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Ribbon diagram for the structure of the region close to the active site Ni(II) ions (green spheres) of SPU inhibited in the presence of NBPTO. The active site flap is shown in red (open conformation) or blue (closed conformation). The side chains of αLys220*, αCys322, and αHis323 are also shown. 496x437mm (300 x 300 DPI)

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(A) Atomic model of the SPU active site inhibited in the presence of NBPTO. The nickel coordination environment is shown superimposed to the final 2Fo − Fc electron density map contoured at 1.0 σ (cyan), while the unbiased Fo − Fc omit map corresponding to the ligand is shown contoured at 3.0 σ (orange). Carbon, nitrogen, oxygen, and nickel atoms are colored gray, blue, red, and green, respectively. (B) The same atomic model and nickel coordination environment are shown (rotated by ca. 45° along the horizontal axis) superimposed to the anomalous map contoured at 3.0 σ. 147x243mm (300 x 300 DPI)

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Atomic model of the SPU active site inhibited in the presence of NBPTO. (A) Nickel coordination environment shown superimposed to the final 2Fo − Fc electron density map contoured at 1.0 σ. The electron density of the inhibitor is colored blue. (B) Crystallographic structure of the same environment. Putative hydrogen bonds are shown as thin blue lines. Spheres are drawn using the relative atomic radius values in CrystalMaker. Carbon, nitrogen, oxygen, phosphorus, and nickel atoms are colored gray, blue, red, yellow, orange, and green, respectively. 147x271mm (300 x 300 DPI)

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Inhibition of ureases in the presence of NBPTO determined by progress curve experiments. (A and B) Progress curve plots representing the consumption of urea over time at increasing concentrations of NBPTO, for SPU (A) and JBU (B). (C and D) Linear plots of kobs as a function of NBPTO concentration for SPU and JBU, respectively. In both panels, the lines represent the results of an exponential or linear fit of the data. 254x173mm (300 x 300 DPI)

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Reaction schemes describing urease inhibition by the phosphoramide derivatives PPD (top panel), NBPTO (middle panel), and NBPT (bottom panel). In all cases, the three atoms involved in directly binding the Ni(II) ions in the active site of urease are shown in blue and displayed with the same orientation, while the fourth atom, which occupies the distal position, is shown in red. 124x96mm (300 x 300 DPI)

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