Kinetic analysis and probing with substrate analogues of the reaction pathway of the nitrile reductase QueF from Escherichia coli

JBC Papers in Press. Published on October 17, 2016 as Manuscript M116.747014

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.747014

The reaction pathway of the nitrile reductase QueF from E. coli

Kinetic analysis and probing with substrate analogues of the reaction pathway of the nitrile reductase

QueF from Escherichia coli

Jihye Jung^1,2, Tibor Czabany², Birgit Wilding^1,3, Norbert Klempier³and Bernd Nidetzky²

¹Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria

²Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse

12/1, 8010 Graz, Austria

³Institute of Organic Chemistry, Stremayrgasse 9, A-8010 Graz, Austria

Running title: The reaction pathway of the nitrile reductase QueF from E. coli

* To whom correspondence should be addressed: Institute of Biotechnology and Biochemical Engineering,

Graz University of Technology, Petersgasse 12/1, 8010 Graz, Austria. Tel.: +43-316-873-8400; Fax: +43-

316-873-8434; E-mail: bernd.nidetzky@tugraz.at

Keywords: Nitrile reductase, substrate-binding recognition, ITC, thermodynamics, kinetics, reaction

pathway analysis

ABSTRACT

The enzyme QueF catalyzes a four-

(∆H = -2.3 kJ/mol; -T∆S = -19.5 kJ/mol) but not

as substrate for reduction or oxidation. A model

of QueF substrate recognition and a catalytic

pathway for the enzyme are proposed based on

these data.

electron reduction of a nitrile group into an

amine, the only reaction of this kind known in

biology. In nature, QueF converts 7-cyano-7-

deazaguanine (preQ₀) into 7-aminomethyl-7-

deazaguanine (preQ₁) for the biosynthesis of the

tRNA-inserted nucleoside queuosine. The

proposed QueF mechanism involves a covalent

thioimide adduct between preQ₀and a cysteine

nucleophile in the enzyme, and this adduct is

subsequently converted into preQ₁in two

NADPH-dependent reduction steps. Here, we

show that the Escherichia coli QueF binds preQ₀

in a strongly exothermic process (∆H = -80.3

kJ/mol; -T∆S = 37.9 kJ/mol, K_d= 39 nM)

whereby the thioimide adduct is formed with

half-of-the-sites reactivity in the homodimeric

enzyme. Both steps of preQ₀reduction involve

transfer of the 4-proR-hydrogen from NADPH.

They proceed about 4- to 7-fold more slowly

than trapping of the enzyme-bound preQ₀as

covalent thioimide (1.63 s^-1) and are thus mainly

rate limiting for the enzyme's k_cat(= 0.12 s^-1).

Kinetic studies combined with simulation reveal

a large primary deuterium kinetic isotope effect

(KIE) of 3.3 on the covalent thioimide reduction

and a smaller KIE of 1.8 on the imine reduction

to preQ₁. 7-Formyl-7-deazaguanine, a carbonyl

analogue of the imine intermediate, was

synthesized chemically and is shown to be

recognized by QueF as weak ligand for binding

Reduction of a nitrile group to a primary

amine is a transformation well known and

important to synthetic chemistry (1–4). Its

unique biological equivalent is the reaction

catalyzed by QueF enzymes (5). QueF was

discovered from the biosynthetic pathway of the

tRNA-modified nucleobase queuosine (Q)

where it was shown to utilize NADPH for the

conversion of 7-cyano-7-deazaguanine (preQ₀)

into 7-aminomethyl-7-deazaguanine (preQ₁)

(Figure 1). Until today, QueF remains the only

enzyme known to promote the intricate nitrile-

to-amine chemistry. The catalytic mechanism of

QueF is therefore of significant fundamental

interest. In addition, it also has potential

application relevance. Q is a nucleoside from the

wobble position of the tRNAs for Asn, Asp, His

and Tyr, and it is thus required for translation

efficiency and fidelity (6). Despite its ubiquitous

occurrence in nature, Q is synthesized de novo

only in bacteria (7–9). The enzymes of the Q

pathway, QueF in particular, due to its apparent

uniqueness, therefore represent promising drug

targets to combat bacterial infections selectively.

Moreover, due to the hazardous conditions

required in the chemical reaction (1–4),

1

The reaction pathway of the nitrile reductase QueF from E. coli

"greener" routes of nitrile reduction are highly

Despite these preceding insights into

QueF structure and function, fundamental issues

of the enzymatic mechanism remain. Which

interactions between QueF and preQ₀are

required in the induced-fit binding of the

substrate or in the formation of a competent

thioimide intermediate? How does NADPH bind

and which of its diastereotopic hydrogens at the

nicotinamide C4 is used for hydride transfer in

each reduction step? Is the very small k_catof

QueF really limited by one of the hydride

transfer steps, as QM/MM calculations have

suggested (14), or are rather the associated

physical steps (which were not analyzed

computationally) slow? To address these

questions, we prepared tailored analogues of

preQ₀, in which specific recognition sites for

QueF binding were removed or altered by

targeted organic synthesis (Figure 2). Together

with the native preQ₀, we used these analogues

to characterize the kinetics and thermodynamics

of substrate binding by the QueF enzyme from E.

coli (ecQueF). We applied transient and steady

state kinetic analysis in combination with kinetic

simulations to characterize, and determine rate

constants for, the individual steps of the

enzymatic reaction pathway. We also developed

a synthesis of 7-formyl-7-deazaguanine (Figure

2) as a carbonyl surrogate of the imine

intermediate of the QueF reaction. We

characterized the binding of this analogue to

ecQueF and examined its reactivity as substrate

for enzymatic reduction or oxidation was

demanded. A biocatalytic route going by the

QueF mechanism presents an interesting option.

QueF enzymes have been characterized

structurally from unimodular and bimodular

classes (10, 11), and the herein studied QueF

from Escherichia coli is a bimodular protein.

The bimodular QueF is a functional homodimer

whereas the unimodular QueF folds into a

homodecamer (a dimer of pentamers) (5, 10, 11).

In both types of QueF, the active site is

positioned at a structural interface, created from

different subunits in the unimodular QueF and

from the tandem tunneling-fold domains of the

same subunit in the bimodular QueF. The

arrangement of catalytic groups is highly

conserved in both enzymes (10–12).

The basic QueF mechanism was

delineated in biochemical and structural studies

of the unimodular enzyme from Bacillus subtilis

(11, 13) (Figure 1). The binding of preQ₀

involves a large induced fit, resulting in the

functional active site to be formed and to

become completely secluded from solvent. Cys⁵⁵

then attacks the nitrile group to build a covalent

thioimide, which it was possible to trap both in

the crystal and in solution. QM/MM calculations

on the bimodular QueF from Vibrio cholerae

suggested a role for Asp (Asp⁶²; B. subtilis

numbering) as a proton shuttle during the

thioimide formation (14) (Figure 1). Hydride

reduction of the thioimide by NADPH, also

under protonic assistance by Asp, subsequently

gives a covalent hemithioaminal. NADP⁺is

exchanged by NADPH and the C-S bond

examined.

A

model of substrate binding

recognition by ecQueF is suggested based on the

evidence presented. We show that each

reduction step by the enzyme involves transfer

of the pro-4-R hydrogen of NADPH. We also

show that the hydride transfer steps are rate

determining in the overall reaction.

cleaved to yield

a

noncovalent imine

intermediate, which is finally reduced to preQ₁.

The QM/MM calculations suggested that Asp²⁰¹

protonates the Cys¹⁹⁴(V.cholerae numbering) on

hemithioaminal breakdown and provides

electrostatic stabilization during the subsequent

hydride transfer (14). Free energy barrier

analysis indicated that the imine reduction is

slowest among the chemical steps of the

computed reaction pathway for the V. cholerae

QueF. In B. subtilis QueF, the k_cat(0.011 s^-1) (5,

13) is much smaller than the rate constant of

thioimide formation (2.78 s^-1) (11). Therefore,

this also locates the rate-determining step in the

sequence of steps involved in the NADPH-

dependent reductions.

RESULTS

ITC Study of Substrate and Coenzyme

Binding to ecQueF―ITC experiments were

conducted to determine the thermodynamic

characteristics of substrate and NADPH binding

to ecQueF (25 °C, pH 7.5; 100 mM sodium

phosphate buffer). The results are shown in

Figure 3 and the parameters calculated from the

data are summarized in Table 1. The binding of

preQ₀was a strongly exergonic process (ΔG < 0)

2

The reaction pathway of the nitrile reductase QueF from E. coli

unable to obtain a binding isotherm due to the

low water solubility of this compound.

in which a highly favorable enthalpy term (ΔH)

overcompensated the nonfavorable contribution

from the binding entropy (−TΔS). The 39 nM

dissociation constant (K_d) thus determined was

in good agreement with the K_destimate of 36

nM obtained from a gel filtration analysis of the

unimodular QueF from B. subtilis (11). The

number of preQ₀binding sites occupied in the

ecQueF homodimer was determined from the

ITC data to be unity (± 5%). Binding models

comprising two equivalent or distinct binding

sites/enzyme homodimer were also examined

but were inconsistent with the experimental

results. The half-of-the-sites reactivity of

ecQueF implied by these findings was in line

with the structural suggestion that the minimal

catalytic unit of V. cholerae QueF should be the

protein homodimer (10).

Contrary to the binding of preQ₀and 2-

deamino preQ₀that both were enthalpy-driven

processes, the binding of 7-formyl preQ₀was

hardly exothermic. It therefore relied completely

on a favorable entropy contribution to become a

spontaneous process characterized by a negative

ΔG. Indeed, the −TΔS term exhibited sign

change from positive to negative, hence turned

from being non-favorable to favorable for

binding, when the binding of preQ₀(or 2-

deamino preQ₀) was compared to that of 7-

formyl preQ₀. Despite the completely different

thermodynamic signatures of the binding of 2-

deamino preQ₀and 7-formyl preQ_0,the

dissociation constants of these two ligands were

similar (Table 1).

Removal of the active-site nucleophile

Besides analyzing preQ₀binding in the

in an inactive C190A variant of ecQueF

weakened the binding of preQ₀(K_d= 5.5 µM)

about 141-fold compared to preQ₀binding to the

wildtype enzyme. This reflected a drastically

lowered enthalpy contribution to the binding

(ΔΔH = +35.6 kJ/mol) in the enzyme variant as

compared to wildtype ecQueF. The entropy term

of preQ₀binding to the C190A variant was

positive (−TΔS = +14.6 kJ/mol). The results are

summarized in Table 1. As in the wildtype

enzyme, the ∆H of preQ₀binding to the C190A

variant was strongly dependent on the buffer

used (phosphate, HEPES, Tris). The ∆G of

binding was however hardly affected by the

buffer (Table 2). We determined from the data

that 0.46 ± 0.27 protons were taken up by the

C190A variant on binding of preQ₀(pH 7.5;

Table 1).

standard phosphate buffer, we also determined

the ΔH of preQ₀binding in Tris and HEPES

buffer (Table 2, Figure 3H). Because the three

buffers differ strongly in ionization enthalpy

(ΔH_ion), the proton release or uptake in

conjunction with ligand binding can be

determined from the difference in the ΔH of

binding. We showed that in terms of ΔG, the

overall preQ₀binding to ecQueF was hardly

affected by the buffer used (Table 1 and 2). The

binding stoichiometry was also unchanged on

variation of the buffer. However, there was a

large buffer effect on ΔH (Table 2), and we

determined that 0.78 ± 0.09 protons were taken

up by each ecQueF dimer on binding of preQ₀

under the conditions used (pH 7.5, Table 1).

The 2-deamination of preQ₀resulted in a

drastic weakening of the substrate binding,

reflected in a 21.5 kJ/mol loss in the ΔG

stabilizing the ecQueF-bound state, comparing

2-deamino preQ₀to preQ₀. This is equivalent to

a 6 × 10³-fold increase in the K_dof the 2-

deaminated substrate. The effect in ΔG was due

to a strongly lowered enthalpy contribution to

the binding of 2-deamino preQ₀as compared to

preQ₀(ΔΔH = +51.4 kJ/mol). The entropy term

was however less unfavorable for the binding of

2-deamino preQ₀than it was for the binding of

preQ₀. It thus compensated to some degree the

very large binding enthalpy change due to

removal of the 2-amino group. We also

examined the binding of 6-deoxo preQ₀but were

The binding of NADPH by QueF is

generally not well understood. We show here

(Figure 3; Table 1) that NADPH was bound by

the free forms of wildtype ecQueF and the

C190A variant thereof. With both enzymes the

binding occurred in an enthalpy-driven fashion.

The overall binding was highly exergonic and

characterized by a 16.0 µM and 18.1 µM

dissociation constants for the wildtype enzyme

and the C190A variant, respectively. These

thermodynamic characteristics appear consistent

with a binding process involving biological

recognition and are unlikely to reflect a merely

adventitious binding of NADPH. If so, therefore,

3

The reaction pathway of the nitrile reductase QueF from E. coli

the binding of NADPH to ecQueF would not

appearance of an absorbance band with

maximum absorption at 380 nm (Figure 4C). A

molar extinction coefficient of 10.02 ± 0.14 mM^-

¹cm^-1was determined. With this we could show

that the preQ₀was linked to each ecQueF dimer

in a 1:1 stoichiometry. Results of protein mass

analysis (Figure 4D) were in good agreement

with these findings, showing that the sample

from incubation of ecQueF with preQ₀was

composed in about equal amounts of the

monomeric enzyme-preQ₀adduct (57%) and the

also monomeric apo-enzyme (43%). Half-of-the-

sites reactivity in the ecQueF dimer was strongly

supported thus. Time-resolved titration analysis

in stopped-flow experiments also revealed that a

molar preQ₀concentration equivalent to that of

the ecQueF dimer was sufficient for complete

covalent conversion of the available active sites

(Figure 6).

require preQ₀to have been bound to the enzyme

before. Additionally, we determined that

NADPH binds to the binary complex of C190A

and preQ₀with about 5-fold enhanced affinity

(K_d= 3.6 µM) compared to its binding to the

free enzyme. The binding stoichiometry was 1

NADPH/enzyme dimer. Despite the relatively

small difference in ∆G of binding (∆∆G = -4

kJ/mol), the thermodynamic signature of

NADPH binding to the C190A-preQ₀complex

was quite distinct from that of NADPH binding

to the free enzyme. The binding of NADPH to

the C190A-preQ₀complex lacked a contribution

from entropy (−T∆S = +0.07 kJ/mol) and the

enthalpy contribution was lowered (∆∆H =

+16.2 kJ/mol) in comparison to NADPH binding

to the free enzyme. Note: NADPH binding to the

covalent adduct between wildtype enzyme and

preQ₀does not give a stable complex because

catalytic reduction takes place. It was therefore

not examined with ITC.

Incubation of ecQueF in the presence of

2-deamino preQ₀also gave rise to a new

absorbance band but compared to the preQ₀

thioimide adduct, its maximum absorption was

at a lower wavelength of around 340 nm (Figure

4C). A molar extinction coefficient of 10.50 ±

0.04 mM^-1cm^-1was determined. Contrary to

preQ₀, a substrate/enzyme molar ratio of about

13 was required so that covalent conversion of

ecQueF with 2-deamino preQ₀was complete

(Figure 4E). Protein mass analysis confirmed the

presence of the covalent adduct, and revealed

furthermore that the portion of total ecQueF

covalently modified (maximum: ∼40 - 50% of

dimer) was strongly dependent on the amount of

2-deamino preQ₀offered (Figure 4D). A K_dof

155 µM was determined for 2-deamino preQ₀

binding to ecQueF (Figure 4F), in useful

accordance with the results of the ITC

experiments (Table 1). No evidence of covalent

linkage formation between ecQueF and 6-deoxo

preQ₀or ecQueF and 7-formyl was found in

spectrophotometric titration analysis. Likewise,

the C190A variant did not show evidence of

covalent binding of preQ₀, as expected.

Substrate Binding and Covalent

Thioimide Formation―As in B. subtilis QueF

(11), the substrate binding in ecQueF was

traceable by both quenching and blue shift of the

protein's Trp fluorescence (Figure 4A and B).

Trp⁵⁷and Trp¹⁹⁸in the substrate-binding pocket

(Figure 5) are the likely reporter groups. On

addition of preQ₀, the ecQueF fluorescence

(excitation: 280 nm) was quenched and a slight

blue shift in the maximum wavelength of

emission (λ_max: 334 nm → 330 nm) was

observed. The quenching yield increased

dependent on the molar ratio of preQ₀and

enzyme homodimer used. It reached a maximum

of about 0.7 when this ratio was approximately

unity (Figure 4A). The titration of ecQueF with

2-deamino preQ₀and 6-deoxo preQ₀also

produced a fluorescence quenching, but the

corresponding yields were lower, 0.21 and 0.36,

respectively (Figure 4B), than for preQ₀. The 7-

formyl preQ₀behaved differently. Its incubation

with ecQueF caused a slight enhancement of the

protein fluorescence (∼10%) and the λ_maxwas

up-shifted (334 nm → 336 nm), as shown in

Figure 4B. Binding of preQ₀to the C190A

variant of ecQueF caused only a small amount

of fluorescence quenching (∼15%).

Addition of preQ₀to a pre-incubated

mixture of ecQueF with any of the three preQ₀

analogues reinstated in each case the effect on

fluorescence

quenching

and

"thioimide

absorbance band" formation that preQ₀had

alone. Therefore, this showed effective

competition of preQ₀with the other ligands for

binding to ecQueF and also demonstrated the

Formation of the covalent thioimide

between ecQueF and preQ₀was detectable by

4

The reaction pathway of the nitrile reductase QueF from E. coli

reversibility of thioimide covalent linkage

formation by 2-deamino preQ₀(Figure 4C).

Stereospecificity of ecQueF

preQ₀in water, we added 7-formyl preQ₀

(dissolved in DMSO) directly to the enzyme

solution containing NADPH so that enzymatic

reduction could precede substrate precipitation.

However, no reaction was observed, despite the

use of ecQueF concentrations (7.6 – 30.0 µM)

that would have allowed detection of just 1/10

enzyme turnover. Just to note, oxidation by

NADP⁺might have involved the 7-formyl group

in native or hydrated form, requiring

nucleophilic catalysis from active-site cysteine

in the former but not the latter (15–18).

Enzymatic conversion of 7-formyl-preQ₀was

also analyzed in the presence of NH₄SO₄or

NH₄Cl (each at 50 mM), considering that

addition of the ammonium ions might facilitate

the formation of an incipient imine to be reduced

by QueF. However, no reaction was observed

under these conditions.

for

Hydrogen Transfer from NADPH―This central

characteristic of the QueF mechanism has not

been elucidated in previous biochemical and

structural studies of the enzyme. We used

NADPH,

(4R)-[²H]NADPH,

or

(4S)-

[²H]NADPH for the reduction of preQ₀and

1

analyzed with H-NMR the products formed,

NADP⁺and preQ₁. Results are shown in Figure

7. In all reactions, the conversion of preQ₀into

preQ₁was traced by monitoring the singlet

signal of the deazaguanine C8 hydrogen, which

appears at 7.42 ppm in substrate and at 6.76 ppm

in product. When NADPH was used, the proton

spectra of the product exhibited a singlet peak at

4.08 ppm, which is assigned to the hydrogens of

the aminomethyl carbon of preQ₁(Figure 7A).

This peak was absent from the spectra of the

preQ₁released in the reaction with (4R)-

[²H]NADPH (Figure 7B). Besides, the product

of the oxidation of (4R)-[²H]NADPH was

NADP⁺not 4-[²H]NADP⁺. The hydrogen at the

position C4 on the nicotinamide ring was

identified unequivocally from the doublet signal

at 8.67-8.68 ppm (Figure 7B). When the

We furthermore examined the reverse

reaction of ecQueF, measuring reduction of

NADP⁺(100 µM), and product formation

associated with it, in the presence of preQ₁(40

µM). Using the standard pH of 7.5 but also

elevated pH values of up to 9.0 in order to

facilitate deprotonation of the 7-amino group,

there was no conversion of preQ₁above the

detection limit, which was roughly 1/10 turnover

of the molar enzyme concentration used (20 - 80

µM). To prevent product attached to the enzyme

from escaping detection, we denatured the

protein with methanol and repeated the HPLC

analysis, thereby confirming the absence of an

oxidation of preQ₁under the conditions used.

Initial rate analysis showed that the K_M

for NADPH was not affected by variation of the

preQ₀concentration in the range 5 - 100 µM.

The lower end limit of the substrate

concentration was defined by the time resolution

and sensitivity of the spectrophotometric assay.

Considering the 39 nM K_dfor preQ₀, only a

negligible portion of free enzyme could have

existed at steady state under these conditions.

alternatively

deuterium-labeled

(4S)-

[²H]NADPH was used, only the hydrogen was

transferred in the enzymatic reaction of preQ₀

and authentic preQ₁was obtained together with

4-[²H]NADP⁺(Figure 7D). These results are

clear in showing that ecQueF is specific for

transferring the 4-proR-hydrogen from NADPH;

and it does so in both hydride reduction steps of

the catalytic conversion of preQ₀to preQ₁.

Steady-state

Kinetic

Study

of

ecQueF―Based on the measurement of both

NADPH consumption and amine product

formation (Figure 8), we showed that besides

preQ₀the 2-deamino derivative was also a

workable substrate of ecQueF (vide infra). The

6-deoxo preQ₀was inactive within the detection

limits of the assay used. The 7-formyl preQ₀

(100 µM) was not reduced by ecQueF in the

presence of NADPH (150 or 300 µM), nor was

it oxidized in the presence of NADP⁺(300 µM).

Absence of activity with this substrate was

shown at different pH values in the range 5.5 –

7.5 (100 mM Ammonium acetate buffer, pH 5.5

or pH 6.67; 100 mM Tris buffer, pH 7.5).

Considering the poor solubility of 7-formyl

Therefore, this explains

a

coenzyme K_M

apparently independent of the substrate

concentration. Using 2-deamino preQ₀, the

substrate and coenzyme K_Mboth were dependent

on the concentration of each other in the assay.

Results are summarized in Table 1. In terms of

k_cat, 2-deamino preQ₀was an equally good nitrile

substrate as was preQ₀. The NADPH K_Mwas

however elevated about 4-fold in the reaction

5

The reaction pathway of the nitrile reductase QueF from E. coli

with 2-deamino preQ₀(Table 1). A huge

conditions were used. Fitting the initial increase

in absorbance with an exponential function, the

difference existed in the substrate K_M, consistent

with results of the ITC binding study of preQ₀

and 2-deamino-preQ₀.

corresponding k_obsshowed

a

hyperbolic

dependence on the NADPH concentration, with

a K_dof 3.0 (± 1.6) µM and a maximum value of

7.32 (± 0.46) s^-1(Figure 9E). The presence of

Transient Kinetic Study and Kinetic

Simulation―To characterize individual steps of

the ecQueF reaction a transient kinetic analysis

in stopped-flow experiments was performed.

Figure 6 shows representative time courses of

thioimide formation upon mixing the enzyme

with preQ₀in the absence of NADPH. The

absorbance increased exponentionally with an

amplitude determined by the limiting

concentration of ecQueF dimer or preQ₀. The

rate constants k_obs(1.35 ± 0.15 s^-1) were

independent of the initial concentrations of

enzyme and preQ₀. Using global fitting with

COPASI, a two-step binding-reaction model, E

+ S D E•S " E-S, was useful to describe the

data recorded at varied enzyme and substrate

concentrations (Figure 6A and B). It involved a

NADPH therefore resulted in

a

4.5-fold

speeding up of the formation of the thioimide

intermediate. The decrease in absorbance at both

340 nm and 380 nm was best fit with a single

exponential (single-turnover reactions) or a

straight line (multiple turnover reactions). The

rate constants calculated from these fits were

hyperbolically dependent on the NADPH

concentration, with a K_dof 10 (± 2) µM and a

maximum value of 0.15 (± 0.01) s^-1, which is

similar to the k_cat(Figure 9F).

The kinetic model in Scheme 1 was used

to fit the data from all experiments, comprising a

large set of averaged stopped-flow time traces

from 28 independent reaction conditions,

involving variation in NADPH concentration

between 10 and 150 µM. Figure 9 (panels A-C)

compares the experimental progress curves to

the corresponding fitting results, which reveals

useful agreement between the two. Table 3

summarizes the rate and binding constants

determined thus. The kinetic steps of thioimide

and imine reduction were the slowest in the

enzymatic pathway and their corresponding rate

constants were similar. Note that based on

decrease in NADPH absorbance (at 340 nm)

alone it would not have been possible to

distinguish the two steps. However, because the

thioimide reduction also involves concomitant

decrease in absorbance at 380 nm, the first

hydride transfer step has a spectral signature

different from the second. This could be used to

determine the rate constant associated with each

step. The rate constants of the microscopic

reaction steps (Table 3) were consistent with the

k_catmeasured at steady state (Table 1) and also

with the rate constants from stopped-flow

experiments. Figure 9G shows the distribution of

different enzyme forms in a single-turnover

stopped-flow reaction. The enzyme complexes

with preQ₀prevail during the reaction. Due to

the comparably weak binding of NADPH to the

covalent enzyme-preQ₀complex, NADPH that

was initially bound to enzyme prior to formation

of the covalent adduct was partly released from

the enzyme once the thioimide intermediate had

relatively fast binding of preQ₀with

a

dissociation constant (K_d_kinetic) of 2.63 (±

0.01) µM that was followed by a slower and,

within the limit of accuracy of the method used,

effectively irreversible transformation of the

noncovalent (E•S) into the covalent complex (E-

S) with a rate constant (k₃) of 1.63 (± 0.01) s^-1

(Table 3). The obtained k₃was in a good

agreement with the experimentally determined

k_obs. The relationship between the overall 39 nM

dissociation constant determined by ITC and the

kinetically determined parameters, that is,

K_d_overall = K_d_kinetic/(1 + k₃/k₄), can be used

to calculate an approximate value of 0.024 s^-1for

the thioimide release rate constant (k₄).

Figure 9A shows absorbance time traces

at 340 nm and 380 nm for a reaction under

conditions of a single-turnover of the enzyme

present. Time traces for reactions under

multiple-turnover conditions in which a limiting

enzyme concentration (5 µM) was used are

shown additionally in Figure 9B-D. The single-

turnover time traces were characterized by a fast

initial increase in absorbance, at 380 nm in

particular, which reflected formation of the

covalent intermediate (Figure 9A). The

subsequent decrease in absorbance was due to

NADPH consumption mainly, but also included

the effect of the concomitant decay of the

intermediate, especially when single-turnover

6

The reaction pathway of the nitrile reductase QueF from E. coli

been formed. This can be seen in the very early

phase of the reaction shown in Figure 9H.

Stopped-flow progress curves of preQ₀

reduction by (4R)-[²H]NADPH under multiple-

turnover reaction conditions are shown in Figure

10. The corresponding single-turnover reactions

are also presented there. Exponential fit of the

initial absorbance increase revealed that the

deuteration of NADPH did not cause slowdown

of the formation of the thiomide intermediate

(k_obs= 6.4 ± 0.3 s^-1), as expected. The

dependence of k_obson the NADPH concentration

was also unaffected (K_d= 3.0 ± 1.5 µM), as

shown in Figure 10D and E. Rate constants

determined from fits of the absorbance decrease

in individual stopped-flow traces are shown on

Figure 10F. From the hyperbolic dependence of

k_obson the NADPH concentration, a maximum

rate constant of 0.055 (± 0.003) s^-1was obtained

and the corresponding K_dwas 14 (± 3) µM.

Therefore, the deuteration of NADPH affected

strongly the enzymatic rate while the apparent

binding was hardly influenced.

Three features of the kinetic model in

Scheme 1 are to be emphasized. First of all,

there are three binding steps for NADPH (K₁-

K₃). Simpler models, in which NADPH binding

for thioimide reduction occurred only to the

covalent enzyme-preQ₀adduct or NADPH

binding occurred to the noncovalent ternary

complex and NADPH dissociation from the

covalent adduct was not allowed, failed to

describe the experimental time courses properly.

Secondly, the extremely small dissociation

constant for NADPH binding to the

hemithioaminal/imine intermediate is worth

noting. It suggests that NADPH was bound

almost irreversibly by ecQueF at this step.

Unless having this small value of K₃, the

dependence of the reaction on the NADPH

concentration could not be properly explained.

Thirdly, the covalent ternary complex showed a

much lower absorbance both at 340 nm (∼54%)

and 380 nm (∼60%) than expected from the sum

of the corresponding individual absorbances of

NADPH and thioimide adduct. Note that the

absorbance of NADPH was not affected by

binding to ecQueF. The result is interesting as it

suggests that the thioimide adduct and NADPH

perturb each other electronically in the ternary

complex. More interestingly even, the extent of

this electronic perturbation was attenuated

strongly when the ternary complex was formed

from (4R)-[²H]NADPH instead of NADPH. The

electronic effect and its dependence on the

isotopic substitution in NADPH might be

Results of a global fit of Scheme 1 to the

data are shown in the figures and reaction

constants determined thus are summarized in

Table 3. Deuteration of the coenzyme caused

slowdown of both reduction steps but the effect

was by far more pronounced on the thioimide

conversion. The corresponding KIEs were

determined as 3.3 ± 0.1 (thioimide reduction)

and 1.8 ± 0.1 (imine reduction). The value of

^DV_maxcan be explained from these two KIEs

(Table 3).

DISCUSSION

Groups of preQ₀Required for Binding

Recognition and Covalent Adduct Formation by

explained by

a

"near-attack" ground-state

ecQueF―Structural

and

sequence-based

conformer that involves close alignment, and

perhaps even partial bonding, between the

thioimide carbon and the C4 nicotinamide

comparisons reveal the bimodular QueFs (from

V. cholerae and E. coli) to exhibit substrate

binding pockets highly similar to that of their

unimodular counterpart (from B. subtilis) for

which a preQ₀complex structure has been

determined (10–12). Evidence characterizing the

substrate binding in ecQueF is therefore

interpreted on the basis of a high degree of

functional residue conservation in these enzymes.

Putting into relation the changes in protein

fluorescence, the heat released and the amount

of covalent thioimide adduct formed in titrations

of ecQueF with preQ₀(or analogs thereof), the

overall substrate binding is found comprised of

three main elements. The initial accommodation

hydrogen

of

NADPH.

Electronic

preorganization of the ternary complex for

efficient hydride reduction is therefore suggested.

KIE Study―Substitution of the reactive

4-proR hydrogen of NADPH by deuterium

caused slowdown of the enzymatic preQ₀

reduction. The effect was expressed in a

substantial primary KIE on both the maximum

rate (^DV_max= 2.40 ± 0.12) and the catalytic

efficiency for coenzyme (^DV_max/K_M= 2.68 ±

0.41).

7

The reaction pathway of the nitrile reductase QueF from E. coli

of the substrate in the binding pocket lacks a

as result of the induced-fit conformational

change, which in turn, according to our

interpretation, also explains the nonfavorable

−TΔS term in terms of a more compact

macromolecular structure. We also note the

good agreement between the dissociation

constants for noncovalent preQ₀complexes of

the wildtype enzyme (K_d_kinetic; Table 3) and

the C190A variant (Table 1). Most likely in

virtue of their effect on substrate positioning, to

align precisely the cyano group of the preQ₀

with the thiol group of Cys¹⁹⁰, these noncovalent

interactions are key to promote the covalent

thioimide adduct, as shown by the relative

inefficacy in that regard of preQ₀analogs

lacking the 2-amino or the 6-oxo group. The

overall thermodynamic signature of preQ₀

binding to ecQueF is strongly shaped by the

energetics of covalent bond formation, which

almost completely masks the effects of the

noncovalent interactions.

spectroscopic signature in absorbance and

fluorescence but, analyzing the binding of the

nonreactive substrate analog 7-formyl preQ₀, it

becomes traceable with ITC. Results show that

this first step in the binding is driven exclusively

by loss in entropy, which probably reflects the

desolvation of the substrate, the binding site in

ecQueF or both. The complete lack of an

enthalpic contribution to the binding of 7-formyl

preQ₀(Table 1) suggests that specific

interactions are not developed between this

ligand and the enzyme. The second step involves

a

protein conformational rearrangement,

detectable mainly by ITC but also by

fluorescence, which in analogy to the induced-fit

binding of preQ₀in the B. subtilis enzyme (11) is

assumed to preorganize and close up the ecQueF

active site. Trp¹⁹⁸of ecQueF (like the

corresponding Trp¹¹⁹of B. subtilis QueF) would

thus be placed in

a

more hydrophobic

microenvironment, with consequent effects on

their fluorescence properties. The preQ₀

complex structure of the C55A variant of B.

subtilis QueF adopts the "closed" conformation

just as the wildtype enzyme does (11), indicating

that thioimide formation is not essential for the

induced fit. Evidence from the current study

adds to the picture, showing that the cyano

group of preQ₀is crucial in substrate-binding

recognition, for its substitution by a formyl

group disrupts entirely the ligand-induced fit in

the enzyme. The finding appears relevant

biologically in that it suggests a mechanism by

which QueF discriminates between its substrate

and other metabolites of the queuosine

biosynthetic pathway that differ from preQ₀only

in the chemical group at position 7 of the

deazaguanine core.

On binding of preQ₀at pH 7.5 each

ecQueF dimer was shown by ITC to take up

0.78 ± 0.09 protons from the bulk solvent.

Preliminary evidence from time-resolved

experiments performed in the stopped-flow

apparatus and measuring proton uptake with a

pH indicator further reveals that the proton

uptake occurred concurrently with formation of

the thioimide intermediate. A proton is required

during conversion of the nitrile to the thioimide

group. In the case that the observable proton

uptake by ecQueF was an immediate

consequence of this reaction, binding of preQ₀

by an enzyme variant that is incapable of

thioimide adduct formation would accordingly

not involve proton uptake. Clear evidence of

proton uptake also by the C190A variant

therefore eliminated this mechanistic possibility.

An alternative mode of protonation, occurring in

consequence of the induced-fit protein

conformational change in preQ₀binding, is

therefore suggested. Identification of the groups

involved in the protontation requires further

study.

The overall ΔG of preQ₀binding to the

catalytic Cys variant (C190A) of ecQueF

comprises

a

substantial contribution from

enthalpy (ΔH = −44.7 kJ/mol; Table 1), which

favors binding. Unlike the basal contribution

from "desolvation" entropy registered for the

binding of 7-formyl preQ₀, the contribution from

entropy for preQ₀binding to the C190A variant

is nonfavorable (−TΔS > 0; Table 1) in this case.

The favorable ΔH term probably arises from

specific noncovalent interactions between the

C190A variant and preQ₀(cf. Figure 5), formed

The Kinetic Pathway of ecQueF Involves

Thioimide Reduction as the Rate-limiting

Step―Because preQ₀and NADPH both bind to

free ecQueF, the kinetic mechanism appears to

be random in principle. However, in the global

simulation-fitting analysis with COPASI,

binding of NADPH to the free enzyme appeared

8

The reaction pathway of the nitrile reductase QueF from E. coli

to be not kinetically significant, at least under

requires comment; however, we believe there

might be biological significance to it. Under the

premise of ecQueF being present intracellularly

in a molar concentration comparable to that of

preQ₀, thioimide adduct formation from an

enzyme-preQ₀-NADPH complex, in which the

NADPH is bound tightly (K₂), might serve to

ensure that the available preQ₀is trapped fast

and completely in a covalently enzyme-bound

form. Availability of NADPH in relation to K₁

would determine the portion of total enzyme

adduct able to undergo conversion into preQ₁.

The remaining portion, despite being unreactive

in the absence of bound NADPH, might still be

useful, for it could temporarily "store" part of

the preQ₀until reduction occurs. Because flux

the conditions used. In addition, although preQ₀

forms a catalytically competent thioimide adduct

with ecQueF even in the absence of NADPH,

the preferred reaction path when neither preQ₀

nor NADPH is limiting appears to be through

the noncovalent ternary complex (Scheme 1).

Formation of the thioimide adduct is accelerated

about 3.6-fold when NADPH is present and it is

relatively slow compared to the actual substrate

binding steps (Table 3). The preQ₀substrate

binds in a stoichiometry of 1 per ecQueF dimer.

The possibility of half-of-the-sites reactivity was

also hinted at by the structure of V. cholerae

QueF (10), suggesting that only a single

NADPH would be bound in two possible

orientations to load one of the two active sites.

Based on the structural model of ecQueF,

Wilding et al. arrived at the same conclusion

(12), suggesting however that only one of two

catalytic centers in the protein dimer was

accessible whereas the other was buried deeply

inside the protein. Evidence from the study of

the inactive C190A variant of ecQueF showed

that 1 NADPH was bound to each noncovalent

complex between enzyme dimer and preQ₀.

through the "normal" reaction pathway (k_cat

=

0.15 s^-1, Table 3) exceeds the "off-pathway"

thioimide cleavage, loss of adduct to

dissociation could thus be prevented effectively.

The very small K₃for the binding of NADPH for

imine reduction would help to ensure that any

preQ₀that enters the reduction pathway is taken

completely to the preQ₁product. Considering

that the preQ₀-derived imine might immediately

hydrolyze to the corresponding aldehyde on

being exposed to water, an important task of the

enzyme should be to avoid any partial reduction

of the nitrile substrate.

According to Scheme 1, the observable

kinetic

parameters

from

initial

rate

measurements are related to rate constants as

shown in Equations 1 and 2 (19). Due to the

slow breakage of the covalent thioimide linkage

(Table 3), the preQ₀K_Mtends to a value by far

too low to be experimentally measurable.

Evidence that in each step of the

NADPH dependent reduction by ecQueF the

hydride transfer took place by 4-proR

stereoselectivity provided the basis for

employing primary deuterium KIEs as selective

probes of the enzymatic reaction. There are two

isotope-sensitive steps in the enzymatic

mechanism (Scheme 1). Results of stopped-flow

kinetic analysis suggested a much larger KIE on

the thioimide reduction than on imine reduction.

The directly measured KIE on V_maxis explicable

as the combined result of the two individual

KIEs. According to Scheme 1, the V_max/K_Mfor

NADPH includes in principle all steps from the

binding of NADPH up to the product release

after the imine reduction. However, because K₃

is much smaller than K₁, the actual V_max/K_Mfor

ecQueF reflects the thioimide reduction and also

the KIE is therefore primarily from this step.

Note that V_max/K_Mdetermined at a saturating

preQ₀concentration implies reaction via binding

E/V_max= 1/k₉+ 1/k₁₁+ 1/k₁₄

K_M(NADPH) = K₁' + K₂' + K₃' = (k₆+ k₁₁)/k₅+

(k₈+ k₉)/k₇+ (k₁₃+ k₁₄)/k₁₂(Eq. 2)

(Eq. 1)

The results in Table 3 show that V_maxwas

limited, to a similar degree, by the rate constants

of thioimide and imine reduction. Rate limitation

solely by imine reduction, as suggested by

Ribeiro et al. from their computational study of

the reaction of V. cholerae QueF (14), was ruled

out for ecQueF based on the kinetic evidence.

The stopped-flow progress curves were not

consistent with a kinetic mechanism in which

the first hydride transfer was faster than the

second. The NADPH K_Mmainly reflects the

binding of NADPH to the covalent adduct of

ecQueF and preQ₀(K₁). A reaction pathway in

which the enzyme "loosens its grip" on NADPH

while moving from the noncovalent to the

covalent complex with the preQ₀substrate

of NADPH to the covalent thioimide adduct.

D

The V_max/K_Mcontained

a relatively high

9

The reaction pathway of the nitrile reductase QueF from E. coli

standard error but its value of 2.68 (± 0.41)

appears largely consistent with the calculated

KIE of 3.3 on the thioimide reduction step.

were of the highest purity available from Carl

Roth and Sigma-Aldrich. preQ₀and its

analogues, 7-cyano-2-deamino-7-deazaguanine

(2-deamino preQ₀) and 7-cyano-6-deoxo-7-

deazaguanine (6-deoxo preQ₀), were synthesized

as described previously (20).

An interesting feature of the preQ₀

conversion by ecQueF was that a reverse

reaction of preQ₁and NADP⁺was not

experimentally detectable, despite the use of a

broad range of pH conditions to evaluate the

reactivity of preQ₁protonated and unprotonated.

Note: the pK_aof the 7-amino group of preQ₁was

estimated to be around 8.6. One possibility is

that the oxidation of preQ₁was disfavored

thermodynamically to an extent that prevented

products (e.g., NADPH) to accumulate above

the detection limit; another is that incorrect

protonation of preQ₁, enzyme or both caused the

Synthesis of 7-Formyl-7-deazaguanine

(7-formyl preQ₀)―A new synthesis of 7-formyl

preQ₀

(systematic

name:

2-amino-5-

which

formylpyrrolo[2,3-d]pyrimidin-4-one)

contrary to literature known procedures (21–23)

does not require protecting group chemistry was

developed. preQ₀(808 mg, 4.61 mmol) and

sodium hypophosphite dihydrate (1.44 g, 13.6

mmol) were dissolved in the solvent mixture (15

mL of pyridine/acetic acid/deionized water in a

ratio of 2/1/1) and stirred under inert

atmosphere. Raney nickel catalyst (slurry in

water, approximately 500 mg) was added to the

stirred reaction mixture. Safety precaution: dry

Raney nickel is pyrophoric and should always be

handled under inert atmosphere. The resulting

reaction mixture was heated to 50 °C for five

hours. Subsequently, the reaction mixture was

cooled to room temperature and filtered over a

pad of celite. The filtrate was reduced in vacuo.

The remaining brown residue was dissolved in 6

reaction rate to decrease below

appreciable with the assays used.

a

level

The complete lack of activity of ecQueF

towards reducing 7-formyl preQ₀was explicable

from the evidence of binding studies that this

compound did not elicit a proper substrate-

binding recognition in the enzyme. Exclusion

already at the level of binding therefore

superseded the possible use of 7-formyl preQ₀as

probe of the imine reduction step in the

enzymatic mechanism.

In conclusion, the multistep catalytic

M

aqueous potassium hydroxide solution.

reaction pathway of ecQueF was characterized

using kinetic analysis and probing with substrate

analogues. The key role of the nitrile group for

substrate-binding recognition was elucidated and

an energetic fingerprint for the different steps of

substrate binding was obtained. Hydride

reduction of the covalent thioimide intermediate

is shown to be the slowest reaction step overall.

NADPH binds extremely tightly to the

hemithioaminal/imine intermediate compared to

its binding to the thioimide intermediate.

Therefore, this property of ecQueF may be

relevant physiologically, as it ensures that nitrile

reduction is always taken completely to the

amine when escape of the imine is prevented

effectively.

Remaining solids were filtered off. The filtrate

was cooled to 0 °C and neutralized by addition

of concentrated aqueous HCl. The product

precipitated from the solution and was isolated

by filtration and washed with copious amounts

of ice water and acetone (brown solid, 770 mg,

1

94%). H NMR (DMSO-d₆) δ 6.35 (bs, 2H,

NH₂), 7.49 (s, 1H, H-8), 10.04 (s, 1H, CHO),

10.72 (bs, 1H, H-9), 11.96 (bs, 1H, H-1); ¹³C

NMR (DMSO-d₆) δ 98.36 (C-7), 120.34 (C-8),

124.50 (C-5), 153.40 (C-2), 153.83 (C-4),

159.02 (C-6), 185.47 (CHO). NMR data were

recorded on a Bruker AVANCE III with

autosampler (¹H-NMR 300.36 MHz, ¹³C-NMR

75.53 MHz).

Enzyme Preparation―Wildtype ecQueF

and its C190A variant were obtained as N-

terminally His-tagged proteins using expression

in E. coli BL21-DE3 (12). Cells induced with

isopropyl β-D-1-thiogalactopyranoside (1 mM;

25°C, 20 h) were suspended in storage buffer

(100 mM Tris, 50 mM KCl, 1 mM tris(2-

carboxyethyl)phosphine, and 1% glycerol, pH

7.5) and sonicated. The enzymes were purified

EXPERIMENTAL PROCEDURES

Chemicals―NADPH (purity >98%) and

NADP⁺(purity >97%) were from Carl Roth

(Karlsruhe, Germany). 2-Propanol-d₈(99.5 atom%

D)

and

alcohol

dehydrogenase

from

Thermoanaerobium brockii were from Sigma-

Aldrich (St. Louis, United States). Materials

10

The reaction pathway of the nitrile reductase QueF from E. coli

from cell extract on a HisTrap^TMFF column (GE

protons involved in the binding and ∆H_ionis the

Healthcare, Buckinghamshire, UK) using a

linear gradient of imidazole (10-500 mM) in 50

mM Tris buffer (pH 7.4, 100 mM NaCl, 1 mM

DDT, 3 mM EDTA). Fractions containing the

enzyme were gel-filtered on a Hiprep^TM26/10

column or PD-10 Desalting columns (GE

Healthcare) equilibrated with storage buffer. The

purified enzyme was concentrated with Amicon

Ultra – 15 Centrifugal Filters (Merck Millipore,

Billerica, Massachusetts, United States) to about

75 - 80 mg/mL. The protein concentration was

measured with a Pierce BCA protein assay kit

ionization enthalpy of the buffer.

n_H+= (∆H_buffer1-∆H_buffer2)/(∆H_ion,buffer1-∆H_ion,buffer2

(Eq. 3)

The ∆H_ionof phosphate, HEPES and Tris at pH

7.5 and 25°C was obtained from literature as

3.60 kJ/mol, 20.40 kJ/mol and 47.45 kJ/mol,

respectively (27). The number of protons

calculated from three independent sets of

experiments was averaged.

)

The preQ₀-C190A complex was

prepared by mixing enzyme (105 µM) with

preQ₀(548 µM) and incubating for 40 minutes

at 25 °C. The solution of the enzyme complex

was then used for titration into the NADPH

solution. Using the 5.5 µM K_dfrom Table 1, the

final concentration of preQ₀-C190A complex

was calculated as 104 µM.

(Thermo

Fisher

Scientific,

Germering,

Germany). Protein stock solutions were stored at

-20 ºC and used up within 3 weeks due to

enzyme stability.

Isothermal

Titration

Calorimetry

(ITC)―A VP-ITC Micro Calorimeter from

Microcal (Malvern Instruments Ltd, Malvern,

UK) was used at 25 °C. The enzyme was gel-

filtered twice to sodium phosphate buffer (100

mM NaH₂PO₄-Na₂HPO₄, pH 7.5; 50 mM KCl)

using illustra NAP 5 columns (GE Healthcare).

Each experiment consisted of an initial 2 µL

injection of ligand solution into enzyme solution,

followed by 25 - 29 injections of 6 – 10 µL of

this solution, leaving 300 – 330 sec between

each injection. To avoid heat changes due to the

DMSO added from the ligand solution, DMSO

was also added to the enzyme solution (≤ 2.5%,

v/v). Data were normalized and evaluated using

ORIGIN with a single binding site model as

described previously (24). The enzyme molar

concentration was based on the protein

concentration assuming a functional ecQueF

homodimer with a molecular mass of 71772 Da

(wildtype enzyme) or 71708 Da (C190A variant).

Note that 3-deoxo preQ₀was not used for ITC

measurement because of its prohibitively low

water solubility.

Spectrofluorometric

and

Spectrophotometric Analysis of Substrate

Binding in ecQueF―Quenching of the intrinsic

Trp fluorescence was shown for B. subtilis QueF

to serve as reporter of the formation of the

noncovalent complex between enzyme and

preQ₀(11). Fluorescence titrations to study with

ecQueF in the binding of preQ₀and analogues

thereof were performed using a fluorescence

spectrophotometer F-4500 (Hitachi, Ltd., Tokyo,

Japan). Emission spectra were recorded in the

range 300 - 500 nm at 1200 nm/min with the

excitation wavelength at 280 nm. The quenching

yield was determined as the ratio (F₀-F_s)/F₀

where F₀and F_sare the protein fluorescence

intensities in the absence and presence of

substrate recorded at the same wavelength of

emission.

Formation of the thioimide intermediate

is traceable in B. subtilis QueF by the

appearance of a new absorbance band at 370 nm

(11, 13). Using ecQueF, we therefore analyzed

substrate

binding

also

by

absorbance

DU 800

ITC measurements were also performed

measurements.

A

Beckman

in HEPES and Tris buffer, each 100 mM, at pH

7.5 and 25 °C. The buffers additionally

contained 50 mM KCl. Data were acquired and

processed as described above. It was shown

previously that a comparison of the binding

enthalpies (ΔH) in HEPES and Tris is applicable

to determine the proton uptake or release in

conjunction with ligand binding (25, 26).

Equation 3 was used, where n_H+is the number of

spectrophotometer (Beckman Coulter, Inc.,

California, United States) was used. Before and

after each addition, a wavelength scan in the

range 300 - 800 nm was performed. The total

volume change due to multiple substrate

additions was less than 5% and the final DMSO

concentration in the enzyme solution did not

exceed 2%. The substrate concentration range

used was dependent on the binding affinity (see

the Results). In both fluorescent and

11

The reaction pathway of the nitrile reductase QueF from E. coli

spectrophotometric measurements, Tris buffer

were deconvoluted by Data analysis software,

using the MaxEnt2 algorithm.

(100 mM, pH 7.5) containing 50 mM KCl and

1.15 mM tris(2-carboxyethyl)phosphine) was

used. The fluorescence and spectrophotometric

data were averaged in triplicate measurements. It

was confirmed that the unbound substrates, in

the concentrations used, did not interfere with

the fluorescence measurements. Also, all

substrates had negligible absorbance in the

wavelength range of the experiment.

¹H-NMR

Measurement

of

the

Stereochemical Course of Hydrogen Transfer

from NADPH―NADPH and (4R)-[²H]NADPH

were prepared by using Thermoanaerobium

brockii alcohol dehydrogenase to reduce NADP⁺

(2.9 mM) from 2-propanol and 2-propanol-d₈

(each 80 mM), respectively (28). Tris buffer (25

mM, pD 9.0; pD = pH meter reading + 0.4) in

D₂O (99.8% D) was used. The reaction was

stopped after 30 min when about 2 mM NADP⁺

had been reduced. Enzyme was filtered off

(Amicon Ultra – 15 Centrifugal Filter), and 2-

propanol and acetone were evaporated at 40 °C

and about 20 mbar (Laborota 4000 efficient,

Heidolph, Schwabach, Germany). Enzyme and

2-propanol were added freshly to the mixture to

reduce all of the remaining NADP⁺. The

NADPH was recovered as described. (4S)-

[²H]NADPH was obtained via reduction of

NADP⁺(3 mM) by 1-[²H]-D-glucose (3 mM),

catalyzed by glucose dehydrogenase from

Bacillus megaterium (0.017 mg/mL) (29) in 25

mM Tris buffer (pH 8.5) containing 125 mM

KCl. The pH was controlled at pH 8.5 during the

reaction (30 °C, 600 rpm in a Thermomixer

Comfort, Eppendorf, Hamburg, Germany). The

deuterium content of (4R)-[²H]NADPH and

(4S)-[²H]NADPH thus obtained was determined

Protein Mass Analysis―The ecQueF

solution (130 µM, 0.6 mL) containing preQ₀

(130 µM) or 2-deamino preQ₀(130 µM, 520 µM,

and 2.6 mM) was incubated at room temperature

in 1 hour and then it was desalted using Amicon

Ultra 0.5 mL Centrifugal filters (Merck

Millipore). A final protein concentration of 30

pmol/µL was obtained in water containing 5%

acetonitrile and 0.1% trifluoroacetic acid. The

samples with preQ₀were separated on a

capillary HPLC system (1200 Agilent, Santa

Clara, United States) equipped with a PepSwift

RP monolithic column (500 µm × 50 mm,

Thermo Fisher Scientific) at a flow rate of 20

µL/min using a gradient prepared from solvent

A (0.05% trifluoroacetic acid in water) and

solvent

B

(0.05% trifluoroacetic acid in

acetonitrile): 0-5 min 10% B, 5-55 min 10-100%

B, 55-56 min 100-10% B, and 56-71 min 10% B.

The injection volume was 5 µL. The column

temperature was 60 ºC. Mass analysis was

1

by H-NMR and LC-MS to be 98% or greater.

performed in

a

Thermo LTQ-FT mass

The coenzyme preparations were used for

reduction of preQ₀with ecQueF. Enzyme was

removed by precipitation with methanol (10%,

by volume) and the sample analyzed with H-

NMR spectroscopy (Figure 7).

spectrometer (Thermo Fisher Scientific)

operated with an ESI source in positive mode

with mass range of 300 – 2000 m/z. The protein

mass spectra were deconvoluted (Protein

Deconvolution 2.0 software, Thermo Fisher

Scientific), using the Xtract algorithm. Enzyme

samples incubated with 2-deamino preQ₀were

not detectable on a Thermo LTQ-FT mass

spectrometer. These samples were therefore

separated on a capillary HPLC system (Dionex

Ultimate 3000, Thermo Fisher Scientific) with

the protocol described above, unless stated

otherwise. The flow rate was 15 µL/min. Solvent

A was 0.3% trifluoroacetic acid in water and

solvent B was 0.3% trifluoroacetic acid in

acetonitrile. The sample was analyzed in maXis

II ETD mass spectrometer (Bruker, Bremen,

Germany) operated with the captive spray source

in positive mode with a mass range of 250 –

3000 m/z. The obtained protein mass spectra

1

Spectra were recorded at 499.98 MHz

and 30 °C using a Varian INOVA 500-MHz

spectrometer (Agilent Technologies). VNMRJ

2.2D software was used to process the spectra.

The spectra of coenzymes, preQ₀and preQ₁

were compared with the literature known spectra

of these compounds (20, 30, 31). Considering

the possibility of deuterium labeling at the C4 of

NADP⁺, reported in the literature to occur in

reactions

of

the

T. brockii

alcohol

dehydrogenase under the conditions used here

(32, 33), we checked carefully with LC-MS the

(4R)-[²H]NADPH preparation used. Relevant

masses for doubly deuterated forms of NADPH

(1H⁺, 746; 2H⁺, 373; K⁺, 784) were not detected.

12

The reaction pathway of the nitrile reductase QueF from E. coli

Kinetic

Studies

at

Steady

Kinetic isotope effects (KIEs) on

enzymatic preQ₀reduction due to deuteration of

the coenzyme were obtained by comparing

initial rates measured with NADPH and (4R)-

[²H]NADPH, both synthesized as described

above. The coenzyme concentration was varied

(2.0 – 36 µM) at a constant saturating

concentration of preQ₀(10 µM). KIEs were

calculated by fitting Equation 4 to the data. E_V/K

and E_Vare the KIEs minus 1 on V_max/K_Mand

V_max, respectively. F is the deuterium fraction in

S. Superscript D is used to indicate a primary

deuterium KIE (e.g. ^DV_max).

State―Experiments were performed in 100 mM

Tris-HCl buffer, pH 7.5, containing 50 mM KCl

and 1.15 mM tris(2-carboxyethyl)phosphine.

BSA (0.2 g/L) was added to stabilize ecQueF

(0.5 µM). DMSO (1-2%, by volume) was used

to increase substrate solubility in all reactions.

The assay volume was 0.5 mL, and reactions

were started by adding enzyme (10 µL) to the

substrate (490 µL). Initial rates were determined

from the NADPH consumed in the enzymatic

reaction at 25 °C, by absorbance at 340 nm

(ε_NADPH= 6.22 mM^-1cm^-1). It was confirmed by

HPLC that the nitrile substrate was converted

into the amine product (Figure 8). Using

cuvettes with 1 cm light path in the Beckman

DU 800 spectrophotometer, the lowest substrate

or NADPH concentration usable in the assay

was about 2 µM. Averaged initial rates from

triplicate determinations were used. Kinetic

parameters were determined from nonlinear fits

of the Michaelis Menten equation to the data.

When the preQ₀concentration was constant (5.0,

10.0, 20.0, 50.0, and 100 µM), the NADPH

concentration was varied between 2.0 – 330 µM.

When the NADPH concentration was constant

(30.0, 50.0, 100, 200, or 340 µM), the preQ₀

concentration was varied (1.10 - 200 µM). Using

2-deamino preQ₀, the substrate concentration

(10.0 – 1000 µM) was varied at different

constant NADPH concentrations (30.0, 50.0,

100, 200, 300, or 380 µM). Under the conditions

used, the decrease in absorbance was linear over

1 - 10 minutes.

V = V_max[S]/{K_M(1+F·E_V/K)+[S](1+F·E_V)} (Eq.

4)

Stopped-flow Kinetic Studies, and Time

Course Fitting and Simulation―Rapid-mixing

kinetic analysis was done at 25 °C using a SX.18

MV Stopped-flow spectrophotometer from

Applied Photophysics (Leatherhead, UK).

Enzyme and substrate solutions were mixed in

equal volumes. In a multiple-turnover reaction,

the concentration of enzyme (5 µM) and preQ₀

(30, 40, and 60 µM) were constant while

NADPH or (4R)-[²H]NADPH (10 – 150 µM)

was varied. Reaction progress was monitored by

absorbance at 340 nm and 380 nm recorded

simultaneously. A multiwavelength detector was

used. The absorbance data were averaged in

triplicate measurements. Time courses were

simulated and reaction models fitted to them

using the program COPASI (version 4.11_build

65) (34). To translate absorbances and molar

concentrations into one another between

experiment and simulation, partial overlap in the

absorbance spectra of the reaction components

had to be taken into account. The ε_340nmvalues

(mM^-1cm^-1) used in analyzing the results were

5.7 (NADPH), 4.5 (thioimide adduct), 0.126

(preQ₀), 0.087 (NADP⁺), 0.63 (ecQueF), and 6.0

or 8.0 (thioimide adduct with NADPH or 4R-

[²H]NADPH, respectively). The corresponding

ε_380nmvalues (mM^-1cm^-1) were 1.4 (NADPH), 11

(thioimide adduct), 0.07 (preQ₀), 0.023 (NADP⁺),

0.33 (ecQueF), and 7.5 or 10 (thioimide adduct

with NADPH or 4R-[²H]NADPH, respectively).

The enzymatic reaction model was

For the enzymatic conversion of 7-

formyl preQ₀, Tris buffer (25 mM, pH 7.5)

containing 125 mM KCl was used. The 7-formyl

preQ₀(0.5 mM) was mixed with NH₄SO₄or

NH₄Cl (each at 50 mM) prior to attempted

reduction (NADPH, 300 µM or 1 mM) or

oxidation (NADP⁺, 1 mM) in the presence of

ecQueF (30 µM) at 30 ºC for 16 hours.

For the oxidation of preQ₁with ecQueF,

preQ₁was prepared enzymatic reduction of

preQ₀and then purified from the reaction

mixture by HPLC. After removing acetonitrile

and ammonium acetate buffer by vacuum

evaporation, the preQ₁was dissolved in DMSO

(≧ 98% purity). Reactions were started with

NADP⁺and lasted for 90 min. Tris buffer (pH

7.5, 8.0, 8.5, or 9.0) was used.

developed based on literature (10, 11, 13) and

the evidence from the current study, as described

under Results. Since the reaction steps of the

enzymatic mechanism are all relatively slow, we

assumed binding of preQ₀and NADPH in rapid

13

The reaction pathway of the nitrile reductase QueF from E. coli

equilibrium. It was furthermore assumed that the

stable and well defined parameter estimates. We

also showed that the fitting results were

relatively insensitive to change in the initial

parameter values by about 5-fold. From

covariance analysis it was found that each rate

constant was determined largely independently,

that is, statistical correlation with other rate

constants was low. In addition, we used different

algorithm (particle swarm and simulated

annealing) but the rate constants shown in Table

3 were hardly changed.

dissociation steps are irreversible under the

conditions used. Finally, reaction steps were also

assumed to be irreversible. A similar approach

of reducing the number of model parameters to

facilitate convergence of the fitting, also using

COPASI, has been applied to enzymatic

pathway analysis before (35). Robust parameter

estimates with standard deviation ≤ 5% were

obtained.

Reaction rate constants and binding

constants were determined by least-squares

model fitting that employed the evolutionary

programming algorithm with standard setting in

COPASI. The units of concentration and time

used in the model were µM and s, respectively.

The least sum of squares as objective function

was minimized, as described previously (34, 35).

Models were evaluated by comparing the

objective value of the estimation between the

different models. The model described in

Scheme 1 was obtained with the lowest

objective value, and the best fitting to the

experimental data was observed. Unconstrained

fitting was used for all parameters. Start values

of the parameters were those determined from

direct fits of the data. For example, the K_dvalues

for preQ₀(3 µM) and NADPH (10 µM) were

used and the rate constant of thioimide

formation in the absence and presence of

NADPH was 1.4 s^-1and 7.3 s^-1, respectively.

The hydride transfer rate constants were set to

k_cat. Using these start conditions, the fitting

converged readily to a unique solution with

HPLC analytics―The samples were analyzed

using an Agilent 1200 HPLC system equipped

with a 5 µm SeQuant ZIC-HILIC column (200

Å, 250 × 4.6 mm; Merck) and a UV detector (λ

= 262 and 340 nm). A linear gradient of 90 – 60%

buffer B (acetonitrile) in buffer A (100 mM

ammonium acetate pH 6.67) over 15 min was

used. The column was washed with 60% B for 5

min and 90% B for 7 min after each analysis.

The flow rate was 2.0 mL/min. The column

temperature was 30 °C. Optionally, a mass

detector (Agilent 6120 Quadrupole) was coupled

to the HPLC system equipped with a 2.7 µm

Poroshell SB-C18 column (120 Å, 100 × 3 mm;

Agilent) and a UV detector (λ = 210, 254 and

340 nm) to analyze the synthesized coenzyme.

The mass of the synthesized coenzymes was

scanned in a range of 50-1000 with negative

mode. A linear gradient of 2-12% buffer B

(acetonitrile) in buffer A (5 mM ammonium

acetate pH 6.0 and 7.0) over 12 min was used.

The post time was 2 min. The flow rate was 0.7

mL/min. The column temperature was 30 °C.

Acknowledgements: This work has been supported by the Federal Ministry of Economy, Family and

Youth (BMWFW), the Federal Ministry for Transport, Innovation and Technology (BMVIT), the Styrian

Business Promotion Agency SFG, the Vienna Business Agency, the Standortagentur Tirol, the

Government of Lower Austria, and Styrian Provincial Government Economic Affairs and Innovation

through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. We

thank Prof. Hansjoerg Weber (Graz University of Technology) for NMR analysis, Dr. Silvia Wallner

(Graz University of Technology) for helpful discussions on ITC measurements and Prof. Ruth Birner-

Grünberger and B. Darnhofer (Medical University of Graz) for protein-mass analysis.

Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this

article.

Author’s Contributions - JJ and BN designed the research. JJ and TC performed biochemical experiments

and analyzed data together with BN. BW and NK synthesized the substrates used. JJ and BN wrote the

paper. All authors commented and agreed on the final version of the paper.

14

The reaction pathway of the nitrile reductase QueF from E. coli

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The reaction pathway of the nitrile reductase QueF from E. coli

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FOOTNOTES:

The abbreviations used are: Q, queuosine; preQ₀, 7-cyano-7-deazaguanine; preQ₁, 7-aminomethyl-7-

deazaguanine; QM/MM, quantum mechanics/molecular mechanics; 7-formyl preQ₀, 7-formyl-7-

deazaguanine; ecQueF, Escherichia coli QueF; 2-deamino preQ₀, 7-cyano-2-deamino-7-deazaguanine; 6-

deoxo preQ₀, 7-cyano-6-deoxo-7-deazaguanine; LTQ, linear ion trap; FT, Fourier transform; ETD,

electron transfer dissociation; KIE, kinetic isotope effect.

16

The reaction pathway of the nitrile reductase QueF from E. coli

Table 1. Kinetic and thermodynamic parameters for ecQueF

preQ₀

0.120 ± 0.002

< 1

2-deamino preQ₀6-deoxo preQ₀7-formyl preQ₀

NADPH

k_cat,app(s^-1)

0.140 ± 0.014

50 ± 12

N.D.

7.80 ± 0.48^a,

35 ± 18^b

K_m,app(µM)

16.0 ± 0.5

0.039 ± 0.004 (2.29 ± 0.04) ×

c

K_d(µM)

(1.6 ± 0.3) × 10²(18.1 ± 1.1^f,

(5.5 ± 0.1)^d

10²

3.6 ± 0.6^g)

-38.9 ± 0.4

-80.3 ± 0.5

∆H (kJ/mol)

-TΔS (kJ/mol)

∆G (kJ/mol)

-28.9 ± 0.2

-2.3 ± 0.2

-19.5

(-47.5 ± 1.2^f,

-31.3 ± 1.2^g)

(-44.7 ± 0.2)^d

11.5

37.9 (14.6)^d

8.1

(20.3^f, 0.07^g)

-27.5 ± 0.4

(-27.2 ± 1.2^f,

-31.2 ± 1.2^g)

-42.4 ± 0.5

-20.9 ± 0.2

N.M.

-21.8 ± 0.2

N.D

(-30.1 ± 0.2)^d

0.78 ± 0.09

e

n_H+

N.M.

(0.46 ± 0.27)^d

a

b

^a,bData were obtained with preQ₀and 2-deamino preQ₀.

^cDissociation constants were obtained from ITC measurements.

^dData of preQ₀binding were obtained with the C190A variant.

^eThe number of protons (n_H+) taken up by the enzyme on preQ₀binding was obtained from the measured

enthalpies in sodium phosphate, HEPES, and Tris buffers at pH 7.5 (for the thermodynamic data of preQ₀

binding in these buffers, see Table 2).

^f,gData were obtained with the substrate-free C190A variant^fand the noncovalent C190A-preQ₀complex^g.

N.D., not detectable; N.M., not measured.

17

The reaction pathway of the nitrile reductase QueF from E. coli

Table 2. Thermodynamic parameters of preQ₀binding to wildtype and C190A ecQueF in HEPES

and Tris buffers

K_d(µM)

∆H (kJ/mol) -T∆S (kJ/mol) ∆G (kJ/mol) ∆H_ion^a(kJ/mol)

Wildtype^b

HEPES

Tris

0.034 ± 0.003

0.054 ± 0.005

-65.7 ± 0.4

-47.7 ± 0.3

22.9

5.1

-42.8 ± 0.4

-41.6 ± 0.3

20.40

47.45

C190A^c

HEPES

Tris

4.26 ± 0.11

4.05 ± 0.16

-42.3 ± 0.2

-23.2 ± 0.2

11.5

-7.7

-30.8 ± 0.2

-30.9 ± 0.2

20.40

47.45

^aThe ionization enthalpy of the buffer was obtained from literature (27).

b

The preQ₀solution (250 µM) was titrated to the wildtype enzyme solution (10 µM). DMSO

concentration was 1.25% in both solutions. The c-values were 294 and 184 in HEPES and Tris,

respectively.

c

The preQ₀solution (800 µM) was titrated to the C190A variant solution (60 µM). DMSO concentration

was 2% in both solutions. The c-values were 8.9 and 9.4 in HEPES and Tris, respectively.

18

The reaction pathway of the nitrile reductase QueF from E. coli

Table 3. Kinetic constants obtained by using the kinetic model of the ecQueF reaction shown in

Scheme 1

Kinetic constants

K_dk₂/k₁

k₃(k₄)^a

(2.63 ± 0.01) × 10^-6M

1.63 ± 0.01 s^-1(0.024 s^-1)^a

(20.4 ± 2.0) × 10^-6M

K₁k₆/k₅

K₂k₈/k₇

(3.23 ± 0.02) × 10^-6M

k₉(k₁₀)^b

5.9 ± 0.2 s^-1(0.56 s^-1)^b

k₁₁

K₃k₁₃/k₁₂

k₁₄

0.234 ± 0.002 s^-1(3.3 ± 0.1)^c

≦ 0.1 × 10^-6M

0.447 ± 0.006 s^-1(1.8 ± 0.1)^c

0.150 ± 0.001 s^-1(0.053 ± 0.001 s^-1)^c

k_cat

K_M(NADPH) (23.7 ± 2.0) × 10^-6M

^ak₄was obtained from the relationship K_d_overall = K_d_kinetic/(1 + k₃/k₄). The K_d_overall is 39 nM. k₄

was not included in the global fitting in COPASI. If it was included, its value was close to zero. Therefore,

the k₄value shown probably gives an upper bound for the dissociation rate constant.

^bk₁₀was obtained from the relationship k₃k₅/k₄k₆= k₇k₉/k₈k₁₀. Note that k₁₀was not included in the global

fitting in COPASI. If it was included, its value was close to zero. The k₁₀value shown probably gives an

upper bound for the dissociation rate constant.

^cKIEs were obtained by comparing rate constants of preQ₀reduction by NADPH, as shown in the table,

and 4R-[²H]NADPH (k₁₁= 0.069 ± 0.001 s^-1, k₁₄= 0.25 ± 0.01 s^-1).

19

The reaction pathway of the nitrile reductase QueF from E. coli

Scheme 1. The proposed kinetic mechanism of preQ₀reduction by ecQueF is shown. E is the free

ecQueF and the enzyme-bound preQ₀and NADPH are indicated by subscript and superscript, respectively.

An asterisk (*) on E indicates the covalent thioimide linkage between preQ₀and enzyme. E_imineindicates

enzyme with bound imine intermediate. The steps included in kinetic simulations are indicated in black

color. NADPH binding to E was excluded from the kinetic simulation and is indicated in light gray. The

kinetic constants from simulation and fitting of reaction time courses are shown in Table 3. The following

rate constants were obtained only as ratios to give the corresponding dissociation constants in rapid

equilibrium: K_d= k₂/k₁(green); K₁= k₆/k₅(light blue); K₂= k₈/k₇(blue) and K₃= k₁₃/k₁₂(purple). The

chemical and isotope-sensitive steps are thioimide reduction (k₁₁) and imine reduction (k₁₄). These steps

were assumed to be effectively irreversible under the reaction conditions used. Likewise, formation of the

thioimide intermediate (k₃, k₉) was assumed to be irreversible in the fitting because the rate constants of

the reverse reaction (k₄, k₁₀) are very small in comparison.

20

The reaction pathway of the nitrile reductase QueF from E. coli

Figure Legends

Figure 1. The proposed mechanism of QueF in the reduction of 7-cyano-7-deazaguanine (preQ₀) to

7-aminomethyl-7-deazaguanine (preQ₁). The dashed line indicates an electrostatic stabilization.

Figure 2. The structures of 7-cyano-2-deamino-7-deazaguanine (2-deamino preQ₀, A), 7-cyano-6-

deoxo-7-deazaguanine (6-deoxo preQ₀, B), and 7-formyl-7-deazaguanine (7-formyl preQ₀, C).

Figure 3. ITC analysis for the binding of substrates and NADPH to wildtype ecQueF (A-D) and to

the C190A variant (E-G) is shown. Each enzyme and ligand solution was prepared in the sodium

phosphate buffer. The obtained thermodynamic parameters are summarized in Table 1. (A-D) The preQ₀

(0.25 mM, A), 2-deamino preQ₀(5 mM, B), 7-formyl preQ₀(3 mM, C) and NADPH (2.9 mM, D) were

titrated to wildtype ecQueF solution (10 µM for preQ₀, 200 µM for 2-deamino preQ₀and NADPH, and

150 µM for 7-formyl preQ₀). The DMSO concentration of the enzyme solutions varied: 1.25% for preQ₀,

2.5% for 2-deamino and 7-formyl preQ₀and no DMSO for NADPH. The c-values (c = [protein

concentration]/K_d) obtained from the experiments were 259 for preQ₀and 12.5 for NADPH. Due to the

relatively low c-values for 2-deamino preQ₀and 7-formyl preQ₀(0.9 and 1, respectively), thermodynamic

parameters for the two substrates were calculated under a fixed molar stoichiometry (ligand

bound/ecQueF homodimer) of 1 that was obtained from the preQ₀binding measurement. (E-F) The preQ₀

(0.8 mM, E) and NADPH (1.2 mM, F) were titrated to a solution of the C190A variant (60 µM, E; 92 µM,

F). The thermodynamic characteristics on NADPH binding to the C190A variant were not affected by the

presence of DMSO. (G) The solution of the preQ₀-C190A complex (104 µM) was titrated into the

NADPH solution (11.3 µM). The DMSO concentration was 2% for preQ₀and 1.375% (C190A) or 1.37%

(the preQ₀-C190A complex) for NADPH. The c-values were 11 for preQ₀and 5.1 (C190A) or 3.5 (the

preQ₀-C190A complex) for NADPH. (H) The dependence of the apparent binding enthalpies (∆H_app) of

preQ₀binding to the wildtype (closed circles) and C190A variant (open circles) as a function of buffer

ionization enthalpy (∆H_ion) are shown. The slope is + 0.76 for the wildtype enzyme (R²= 0.996) and +

0.51 for C190A variant (R²= 0.921). The thermodynamic parameters are summarized in Table 1 and 2.

Figure 4. The nitrile substrates binding to ecQueF was analyzed by fluorescence titration (A, B),

spectrophotometric titration measuring covalent thioimide formation (C, E, F), and protein mass

analysis of ecQueF after incubation with preQ₀or 2-deamino preQ₀(D). The DMSO concentration in

the enzyme solution was 1%. (A) Protein fluorescence intensities of ecQueF (2.8 ± 0.4 µM) decreased

gradually until the ratio of ecQueF to preQ₀reached a value of 1 (top to bottom; 0.0 - 10 µM, [preQ₀]). (B)

Fluorescence intensities of ecQueF (black dashed line, 2.8 ± 0.4 µM) were changed by addition of

substrates (black, preQ₀; red, 2-deamino preQ₀; blue, 3-deoxo preQ₀; green, 7-formyl preQ₀; each 3 µM).

(C) Absorbance spectra for the covalent thioimide adduct of ecQueF (60 µM) with 2-deamino preQ₀(a,

500 µM and b, 1.00 mM) and the shifted spectrum of the enzyme adduct with 2-deamino preQ₀(500 µM)

upon addition of preQ₀(c, 60 µM) are shown. (D) The sample of ecQueF (130 µM) incubated with preQ₀

(top, 130 µM) is shown to contain preQ₀-free monomeric ecQueF (a, 35732.1 ± 0.5 Da) and covalent

adduct of monomeric ecQueF with preQ₀(b, 35906.0 ± 1.7 Da). The sample of ecQueF (130 µM)

incubated with 2-deamino preQ₀(bottom, 2.6 mM) is shown to contain substrate-free monomeric ecQueF

(c, 35732.7 ± 0.1 Da) and monomeric adduct with 2-deamino preQ₀(d, 35892.7 ± 0.1 Da). (E)

Absorbance spectra of covalent thioimide (ecQueF, 50 µM) with 2-deamino preQ₀(bottom to top; 50 –

1000 µM, [2-deamino preQ₀]) were obtained. The spectra of enzyme and adduct were represented by

dotted and solid line, respectively. (F) The binding stoichiometry ([B]/[E₀], closed circle) dependent on

the 2-demino preQ₀concentration is shown. The data are taken from panel E. [B] and [E₀] are the

concentrations of 2-deamino preQ₀bound to the enzyme and the initial concentration of free enzyme,

respectively. The black line is a hyperbolic fit of the data.

Figure 5. Overall structure (A) of ecQueF with preQ₀and NADPH and close-up view of Trp⁵⁷(B)

and Trp¹⁹⁸(C). Tryptophan is shown in magenta. Other amino acids in the active site and NADPH are

21

The reaction pathway of the nitrile reductase QueF from E. coli

shown in blue and yellow, respectively. Cys¹⁹⁰, preQ₀, and amino acids having hydrophilic interaction

with Trp¹⁹⁸are indicated by element-based colors.

Figure 6. Stopped-flow progress curves of preQ₀binding to ecQueF are shown together with results

of model fitting. Thioimide adduct formation in the absence of NADPH is shown, recording absorbance

at 380 nm (A) and at 340 nm (B). The ecQueF (30 µM) was mixed with preQ₀(bottom to top; 15, 30, 45,

60, and 120 µM). Black dashed lines show the averaged data in triplicate measurements. Orange solid

lines are the fit of a two-step binding mechanism, described in Scheme 1 and text, to the data.

1

Figure 7. H-NMR analysis of enzymatic reduction of preQ₀by NADPH (A), 4R-[²H]NADPH (B) or

4S-[²H]NADPH (D). Panel C shows a control comprising NADP⁺(475 µM) and preQ₀(238 µM). (A-C)

Tris buffer (25 mM, D₂O, 99.8% D; pD 7.5) containing 50 mM KCl and 1.15 mM tris(2-

carboxyethyl)phosphine was used. NADPH or (4R)-[²H]NADPH (475 µM) was incubated with ecQueF

(29 µM) and preQ₀(238 µM) at 25 ºC. The DMSO concentration was 0.5%. When about 90% of the

coenzyme had been consumed, the enzyme was filtered off and the filtrate was analyzed by ¹H-NMR. (D)

The NADPH solution (1.9 mM, 1.0 mL) was mixed with preQ₀(800 µM) and ecQueF (10 µM). The

DMSO concentration was 4%. When 1.6 mM of the NADPH was consumed, enzyme was removed. D₂O

1

(10%, by volume) was added to the solution prior to H-NMR analysis. Note: all peaks were broadened

and shifted by 0.05-0.10 ppm in the spectra of the ecQueF reaction with 4S-[²H]NADPH due to the

presence of H₂O.

Figure 8. Enzymatic reduction of ecQueF with preQ₀(A) or 2-deamino preQ₀(B) was analyzed by

HPLC. (A) The enzyme (21 µM) was mixed with preQ₀(1 mM) and NADPH (3 mM). (B) The enzyme

(11 µM) was mixed with 2-deamino preQ₀(100 µM) and NADPH (300 µM). Black lines indicate control

sample containing NADPH and nitrile substrate while gray lines indicate the enzymatic reduction.

Incubation was at 25 ºC for 10 – 20 min.

Figure 9. Stopped-flow progress curves of enzymatic reactions under single-turnover and multiple-

turnover conditions are shown together with results of kinetic simulation. (A) The single-turnover

reaction of preQ₀reduction is shown. The gray shaded area indicates the phase of covalent thioimide

formation and a close-up view of this phase is shown in the inset (top, 340 nm; bottom, 380 nm). The

ecQueF (10 µM) was mixed with preQ₀(10 µM) and NADPH (20 µM). (B) Multiple-turnover reaction

was recorded at 340 nm. The concentrations of enzyme and preQ₀were constant at 5 µM and 60 µM,

respectively. The NADPH concentration varied (bottom to top; 10, 20, 30, 40, 60, 80, and 100 µM). (C, D)

Close-up views of covalent thioimide formation in multiple-turnover reactions are shown at 340 nm (C)

and at 380 nm (D). In panel A-D, black dashed lines show the experimental data. Orange solid lines in

panel A-C present fit of the data to the kinetic mechanism described in Scheme 1 and blue solid lines in

panel D indicate single exponential fit of the data. (E, F) The dependence of stopped-flow rate constants

(k_obs) on the NADPH concentration is shown. The circles are the data and the solid line is a hyperbolic fit.

k_obsof formation of the thioimide adduct is shown in panel E. It was obtained from nonlinear fits of the

data in panel D. k_obsof the NADPH consumption is shown in panel (F). It was obtained from linear fits on

the data in panel B (1 - 10 s range). (G, H) Reaction simulation under single-turnover conditions applying

the kinetic parameters from Table 3 is shown. (G) The enzyme forms are represented as in Scheme 1. The

time-dependent concentrations of enzyme forms bound to preQ₀(E_preQ0), present as imine intermediate

(E_imine), and bound to NADPH (E^NADPH) were negligible and are therefore not shown. The gray shaded area

in panel G indicates the phase of covalent thioimide formation also shown in panel A. (H) Simulated

curves of NADPH consumption and of formation of NADP⁺and preQ₁are shown. The disappearance of

free preQ₀was too fast to be represented here (≤ 1 s).

Figure 10. Stopped-flow progress curves of enzymatic reactions under single-turnover and multiple-

turnover conditions with 4R-[²H]NADPH are shown together with results of kinetic simulation. (A-

D) Black dotted lines are the experimental data averaged from triplicate measurements. Orange solid lines

22

The reaction pathway of the nitrile reductase QueF from E. coli

in panel A-C present the fit of the data to the two-step binding mechanism described in Scheme 1. The

blue solid lines in panel D indicate single exponential fits of the data. (A) The single-turnover reaction of

preQ₀reduction is shown, recording absorbance. The gray shaded area in panel A indicates the phase of

covalent thioimide formation and a close-up view of this phase is shown in the inset (top, 340 nm; bottom,

380 nm). The ecQueF (10 µM) was mixed with preQ₀(10 µM) and (4R)-[²H]NADPH (20 µM). (B)

Multiple-turnover reaction was recorded at 340 nm. The concentration of enzyme and preQ₀were constant

at 5 µM and 15 µM, respectively. (4R)-[²H]NADPH concentration varied (bottom to top; 10, 20, 30, 40,

60, 80, and 100 µM). (C, D) A close-up view (C, at 340 nm; D, at 380 nm) of covalent thioimide

formation in multiple-turnover reaction is shown. (E, F) The dependence of k_obson the NADPH

concentration is shown. The circles are the data and the solid line is a hyperbolic fit. The k_obsof thioimide

formation (E) was obtained from the best fits shown in panel D and the k_obsof NADPH consumption (F)

was obtained from the linear fits on the data (1 - 20 s range) shown in panel B.

23

The reaction pathway of the nitrile reductase QueF from E. coli

Figure 1

24

The reaction pathway of the nitrile reductase QueF from E. coli

Figure 2

25

The reaction pathway of the nitrile reductase QueF from E. coli

Figure 3

26

The reaction pathway of the nitrile reductase QueF from E. coli

Figure 4

27

The reaction pathway of the nitrile reductase QueF from E. coli

Figure 5

28

The reaction pathway of the nitrile reductase QueF from E. coli

Figure 6

29

The reaction pathway of the nitrile reductase QueF from E. coli

Figure 7

30

Article Doi

Kinetic analysis and probing with substrate analogues of the reaction pathway of the nitrile reductase QueF from Escherichia coli

DOI: 10.1074/jbc.M116.747014

Source and publish data:

Authors:

Article abstract of DOI:10.1074/jbc.M116.747014

Full text of DOI:10.1074/jbc.M116.747014

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