Evidence of a sequestered imine intermediate during reduction of nitrile to amine by the nitrile reductase QueF from Escherichia coli

Article abstract of DOI:10.1074/jbc.M117.804583

In the biosynthesis of the tRNA-inserted nucleoside queuosine, the nitrile reductase QueF catalyzes conversion of 7-cyano-7-deazaguanine (preQ₀) to 7-aminomethyl-7-deaza-guanine (preQ₁), a biologically unique four-electron reduction

Full text of DOI:10.1074/jbc.M117.804583

JBC Papers in Press. Published on January 16, 2018 as Manuscript M117.804583
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.804583
Imine intermediate during nitrile reduction by QueF
Evidence of a sequestered imine intermediate during reduction of nitrile to amine by the nitrile reductase
QueF from Escherichia coli
Jihye Jung1,2 and Bernd Nidetzky1,2,
*
From the 1Austrian Centre of Industrial Biotechnology, Petersgasse 14, and the 2Institute of Biotechnology
and Biochemical Engineering, NAWI Graz, Graz University of Technology, Petersgasse 12/1, A-8010
Graz, Austria
Running title: Imine intermediate during nitrile reduction by QueF
* To whom correspondence should be addressed. Tel.: +43-316-873-8400; Fax: +43-316-873-8434; E-
mail: bernd.nidetzky@tugraz.at
Keywords: nitrile reductase, QueF, catalytic mechanism, thioimide, imine, aldehyde, intermediate
sequestration
Abstract
the imine intermediate. Collectively, these results
provide direct evidence for the intermediacy of an
imine in the QueF-catalyzed reaction. They reveal
determinants of QueF structure required for imine
sequestration and hence for a complete nitrile-to-
amine conversion by this class of enzymes.
In the biosynthesis of the tRNA-inserted
nucleoside queuosine, the nitrile reductase QueF
catalyzes conversion of 7-cyano-7-deazaguanine
(preQ0) to 7-aminomethyl-7-deazaguanine (preQ1),
a biologically unique four-electron reduction of a
nitrile to an amine. The QueF mechanism involves
a covalent thioimide adduct between the enzyme
and preQ0 which undergoes reduction to preQ1 in
two NADPH-dependent steps, presumably via an
imine intermediate. Protecting a labile imine from
interception by water is fundamental to QueF
catalysis for proper enzyme function. In the QueF
from E. coli, the conserved Glu89 and Phe228
residues together with a mobile structural element
comprising the catalytic Cys190 form a substrate-
binding pocket that secludes the bound preQ0
completely from solvent. We show here that
residue substitutions (E89A, E89L, F228A)
targeted at opening up the binding pocket
weakened preQ0 binding at the preadduct stage, by
up to +10 kJ/mol, and profoundly affected on
catalysis. Unlike wildtype enzyme, the QueF
variants, including L191A and I192A, were no
longer selective for preQ1 formation. The E89A,
E89L and F228A variants performed primarily (≥
90%) a two-electron reduction of preQ0, releasing
hydrolyzed imine (7-formyl-7-deazaguanine) as
the product. The preQ0 reduction by L191A and
I192A gave preQ1 and 7-formyl-7-deazaguanine at
a 4:1 and 1:1 ratio, respectively. The proportion of
7-formyl-7-deazaguanine in total product increased
with increasing substrate concentration, suggesting
a role for preQ0 in a competitor-induced release of
Introduction
Sequestration of reactive intermediates, to avoid
their release into solvent where they might
decompose, is a hallmark of enzyme catalysis (1,
2). Mobile elements of the protein structure are
often used to close down over the substrate while it
undergoes the catalytic transformation into
product. The nitrile reductase QueF deals with a
notably difficult problem of intermediate
sequestration (3–7). QueF catalyzes a four-electron
reduction of a nitrile to an amine in two NADPH-
dependent steps, presumably via an imine
intermediate. However, imines are extremely
unstable molecular groups in water and will
hydrolyze to aldehyde immediately. Therefore, to
ensure that every substrate "makes it through" to
the amine, QueF must do both, keep a firm grip on
the labile imine during the catalytic act and
exclude water from the binding site. This study
was performed to identify elements of the QueF
catalytic apparatus that make nitrile reductases the
high-fidelity enzymes they are by nature,
converting nitrile to amine without detectable loss
of the supposed imine to off-pathway reactions (3–
7).
QueF was described from the biosynthetic
pathway of the nucleoside queuosine (Q) where
1
Copyright 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Imine intermediate during nitrile reduction by QueF
the enzyme functions in turning 7-cyano-7-
deazaguanine (preQ0) into 7-aminomethyl-7-
deazaguanine (preQ1) (8, 9). The preQ1 is inserted
into tRNA and subsequently converted to Q.
Bound at the wobble position of tRNAs decoding
NAC/U codons, Q plays a role in modulating
codon-anticodon binding efficiency (8, 10, 11). De
novo biosynthesis of Q is exclusively found in
bacteria (11–13). Although Q is not essential for
bacterial life (14), knock out of its biosynthesis can
reduce, or eliminate, pathogenicity (15, 16). QueF
among all other enzymes of the Q pathway,
therefore, presents a potential target for anti-
infective strategies directed selectively against
bacteria. QueF also has attracted considerable
mechanistic interest (3, 5–7, 17), for the reaction
catalyzed by it is apparently unique to biology.
Two-fold enzymatic reduction of preQ0 is
proposed to proceed in three catalytic steps, as
shown in Scheme 1 (3–7). A covalent thioimide
adduct is formed between the enzyme's catalytic
nucleophile (cysteine) and preQ0 (3, 5, 7). Its
subsequent reduction by NADPH yields a covalent
thiohemiaminal intermediate. After exchange of
coenzyme and breakdown of the covalent
thiohemiaminal to imine, a second reduction by
NADPH gives the amine product. The QueF
mechanism necessitates that the different chemical
steps of the enzymatic reaction are coordinated
exactly with the intermediate physical steps of
coenzyme binding and release, and these in turn
must be precisely timed with the disengagement of
free imine from the covalent thiohemiaminal. The
way of how QueF orchestrates these multiple steps
(4–6) immediately suggests strategies possibly
utilized for intermediate sequestration.
QueF enzymes are oligomeric proteins built
from subunits that possess a small, so-called
tunneling-fold (T-fold) domain (18). The core T-
fold domain consists of a ββααββ arrangement of
secondary structure (4, 5, 9). The known QueF
enzymes are divided into unimodular (e.g., QueF
from Bacillus subtilis; bsQueF) (3, 5, 19, 20) and
bimodular groups (e.g., QueF from Escherichia
coli; ecQueF) (4, 7, 21–24), depending on whether
they comprise subunits containing only a single T-
fold domain or two T-fold domains in tandem
repeat, respectively. Both types of QueF have their
active sites located at the structural interface of
two T-fold domains. The active site residues, and
their structural arrangement into a functional
catalytic center, are highly conserved in both QueF
types (4, 5, 21). Besides the catalytic cysteine
(Cys190 in ecQueF), there is an invariant pair of
acidic residues of which the aspartic acid (Asp197
in ecQueF) plays a role in proton transfer, as
shown in Scheme 1, and the glutamic acid (Glu89
in ecQueF) is involved in binding recognition of
preQ0.
Crystallographic studies of bsQueF (5, 20) and
the bimodular QueF from Vibrio cholerae
(vcQueF; (4)) show that preQ0 binding involves an
induced-fit conformational change in protein
structure, with the consequence that the substrate
binding site, which is quite open in the apo-
enzyme, is closed up completely in the holo-
enzyme. The bound preQ0 becomes sequestered
completely from the solvent as a result (Fig 1).
In vcQueF, for which crystal structures of apo-
enzyme (PDB: 3RJ4), noncovalent complex of a
C194A variant with preQ0 (PDB: 3RZP, 3UXV)
and covalent complexes of H233A (PDB: 4GHM)
and R262L (PDB: 3S19, 3UXJ) variants with
preQ0 were determined, the induced-fit binding of
preQ0 comprises three main elements in particular.
A characteristic device, generally referred to as the
"QueF motif", is embedded structurally in a α-
helix flanking the active site (5, 9). At its N-
terminal end, this helix has a glutamic acid (Glu89
in ecQueF), which forms a hydrogen bond with
preQ0 (2.8 Å) in the enzyme-substrate complex, as
shown in Fig. 1. The glutamic acid is positioned
opposite to an adaptable element of the QueF
structure (Fig. 1 B,C) contributing the catalytic
cysteine to the active site. This "Cys element",
which is completely conserved within bimodular
QueF enzymes, additionally comprises Leu191 and
Ile192 (ecQueF numbering). The Cys element
rearranges to cover the active site in the preQ0-
enzyme complex structure (PDB: 4GHM; Fig. 1C).
Finally, a conserved phenylalanine (Phe228 in
ecQueF), which adopts a position to open up the
preQ0 binding pocket in the apo-enzyme, closes
down over the bound substrate, by forming pi-
stacking interactions with it (3.7 Å), in the holo-
enzyme (Fig. 1C). Two key residues of preQ0
binding, Glu89 and Phe228, and the catalytic Cys190
thus get “locked” onto the bound substrate at the
end of the structural rearrangement. These features
of vcQueF structure important for enzymatic
function appear to be completely conserved in
2

Imine intermediate during nitrile reduction by QueF
ecQueF. Overall, the sequence identity between
the two enzymes is 65%.
corresponding thermodynamic characteristics were
determined, in ITC experiments. Binding of preQ0
by ecQueF is a two-step process in which an
initially formed noncovalent enzyme-preQ0
complex is converted to a covalent thioimide
adduct (Scheme 2; ref. 7). Note that with ITC, it
was not possible to distinguish between the two
steps of preQ0 binding and the data report on the
binding process as a whole. The results are shown
in Fig. 2 and the parameters calculated from the
data are summarized in Table 1.
Strong heat release on titrating enzyme solution
with preQ0 solution (Fig. 2), similarly as it
occurred with the wildtype enzyme (7), was good
evidence that ecQueF variants had retained the
ability to bind preQ0. Substitution of Glu89 caused
substantial weakening of the preQ0 binding as
compared to wildtype enzyme, reflected in a
Kinetic studies of ecQueF indicate that
NADPH binds to QueF already at the non-covalent
complex with preQ0 (7). The presence of NADPH
enhances about 4-fold the thioimide formation rate
compared to reaction of free enzyme and preQ0.
The substrate-binding pocket has
a narrow
aperture, made up from Glu89 and the Cys element,
Ile192 in particular (Fig. 1), that appears to be used
for threading the nicotinamide moiety of NADPH
into the active site (21). Structurally, therefore,
substrate cannot be released before the coenzyme
has dissociated. Accordingly, an important feature
of QueF efficiency in intermediate sequestration,
suggested from kinetic analysis, is the enzyme's
extremely tight binding of NADPH at the stage of
the thiohemiaminal/imine (7).
The evidence just summarized suggests that the
residues Glu89 and Phe228 and also the Cys element
are significant for intermediate sequestration in
ecQueF. Anchoring the proposed imine through
interactions from these QueF structural elements
might be crucially important. In this study,
therefore, we performed mutagenesis of ecQueF,
replacing the "anchoring" Glu89 and Phe228 by
residues (Ala or Leu) unable to fulfill the
analogous function. We additionally replaced
Leu191 and Ile192 by Ala with the aim of perturbing
the active site-closing movement of the Cys
element. Overall, we considered that due to a
substrate-binding site not properly closable any
more, the ecQueF variants might no longer behave
as high-fidelity nitrile reductases. The putative
imine intermediate might become intercepted by
water able to enter an opened-up active site in the
variant enzymes. Detailed characterization of these
ecQueF variants reveals two-electron reduction of
nitrile to imine for the first time in a nitrile
reductase, thus providing direct evidence of the
intermediacy of an elusive imine in the native
catalytic reaction; and provides a structural and
mechanistic explanation for complete nitrile-to-
amine conversion by QueF enzymes, emphasizing
smaller negative value of ΔGbinding (ΔΔGbinding =
+14 kJ/mol) and increased overall apparent
dissociation constant Kd (292-fold). The degree of
disruptive effect was similar in both enzyme
variants. The F228A variant also showed
weakened preQ0 binding, reflected in a smaller
negative value of ΔGbinding (ΔΔGbinding = +10.8
kJ/mol) and 90-fold increased Kd. In terms of
ΔGbinding and Kd, preQ0 binding was unaffected by
the replacement of Leu191 or Ile192 with Ala.
Interestingly, the ΔGbinding of the E89A, E89L, and
F228A variants was comparable to the ΔGbinding of
the previously reported C190A variant which can
bind preQ0 just noncovalently (7). However,
contrasting their similarity overall, the ΔGbinding
values of these ecQueF variants involved distinct
relative contributions from enthalpy (ΔHbinding) and
entropy (-TΔSbinding). The ΔHbinding became more
negative (favorable of binding) and the -TΔSbinding
more positive (nonfavorable of binding) in going
from C190A via the variants. A covalent thioimide
formation more pronounced in E89L compared to
the E89A and F228A variants and lacking
completely in C190A variant could explain these
trends in ΔHbinding and -TΔSbinding
.
In comparison to the wildtype enzyme, the
ΔΔGbinding of the E89A variant was primarily due to
effect on ΔHbinding. In the E89L and F228A
variants, however, the ΔΔGbinding involved effects,
of a similar degree, on ΔHbinding and -TΔSbinding. The
ΔGbinding of L191A variant involved a ΔHbinding
the
critical
importance
of
intermediate
sequestration.
Results
Isothermal titration calorimetry study of preQ0
binding to ecQueF variants - Purified ecQueF
variants were shown to bind preQ0, and the
lower and
a
-TΔSbinding higher than the
corresponding preQ0 binding parameters of the
3

Imine intermediate during nitrile reduction by QueF
wildtype enzyme. However, the ΔHbinding and -
TΔSbinding terms compensated each other in an
overall unchanged ΔGbinding. A -TΔSbinding term
substantially higher in the ecQueF variants (E89L,
L191A) is therefore worth noting. In the I192A
variant, the ΔHbinding and -TΔSbinding terms of preQ0
binding were comparable to as they were in the
wildtype enzyme.
Kinetic analysis of covalent thioimide adduct
formation in ecQueF variants - Absorbance at
380 nm (ε = 10.02 ± 0.14 mM-1cm-1) indicates the
covalent thioimide intermediate of wildtype
ecQueF (7). Titration of the enzyme variants with
preQ0 also gave rise to a "thioimide" absorbance
band, as shown in Fig. 3. Maximum absorption
as the wildtype enzyme. The k3 was decreased in
all enzyme variants, most strongly so in the F228A
variant (10-fold). The ratio k3/k4 describes the
tightening of preQ0 binding in consequence of
covalent thioimide formation. The effect was more
pronounced in the L191A variant (k3/k4 = 440)
than in the E89A (k3/k4 = 53), I192A (k3/k4 = 26),
and F228A variant (k3/k4 = 40). Including the
wildtype enzyme in this comparison is difficult
because only a rough lower limit of 53 could be
given for k3/k4. Since in kinetic experiments the k4
was not different from zero (7), the relationship
Kd_overall = Kd_kinetic/(1+k3/k4) was used to calculate
the upper bound of k4 from the wildtype enzyme's
apparent Kd_overall determined in ITC experiments.
The values of Kd_overall determined kinetically and
obtained from ITC data agreed for the L191A and
F228A variant. They however differed by as much
as 5-fold for the E89A and I192A variant. We
explain this difference by the fact that in the ITC
measurement, both the covalent and the
noncovalent complex contribute to the recorded
heat signal whereas absorbance is completely
specific for detecting the thioimide adduct.
Therefore, only absorbance measurements allow
was however shifted to
a
slightly higher
wavelength (≤ 5 nm) in the Glu89 variants. In
wildtype ecQueF based on half-of-the-sites
reactivity of the enzyme dimer, the molar
equivalent of preQ0 was sufficient to convert all
enzyme present into the covalent adduct (7). In
ecQueF variants, especially the ones having Glu89
replaced, excess preQ0 (∼10-fold) was required for
full conversion of the enzyme (Fig. 3A).
In wildtype ecQueF as shown recently (7), the
for a clear-cut determination of Kd_overall
.
thioimide formation involves
a non-covalent
Reduction of preQ0 by the ecQueF variants –
Direct analysis of the reaction mixtures by H
enzyme-preQ0 complex in rapid equilibrium,
which reacts only relatively slowly to the covalent
adduct (Scheme 2). Absorbance time courses from
preQ0 binding experiments with the E89A, L191A,
I192A and F228A variant were best fit with single
exponentials. The overall fit was not improved
when double exponentials were used (data not
shown). The corresponding rate constants and
amplitudes (data not shown) were hyperbolically
dependent on the preQ0 concentration, as expected
if the ecQueF variants reacted in two distinct steps,
as shown in Scheme 2, analogous to the wildtype
enzyme (7). A global fit of the reaction time
courses was therefore performed using the kinetic
mechanism in Scheme 2. Experimental data are
superimposed in Fig. 3 with the fitting results.
Parameters of preQ0 noncovalent binding (Kd_kinetic
= k2/k1) and thioimide formation (k3, k4) by the
ecQueF variants are summarized in Table 2 along
with the corresponding parameters of the wildtype
enzyme.
1
NMR (Fig. 4) and HPLC (Fig. 5) was used to
examine preQ0 reduction by the ecQueF variants.
Initial evidence was that while F228A and both
Glu89 variants showed conversion of preQ0 at a
slow rate, they hardly produced preQ1. We
considered that any imine intermediate exposed to
water after the first reduction would be detected as
7-formyl-7-deazaguanine.
Using
authentic
reference material from chemical synthesis (7), we
identified 7-formyl-7-deazaguanine as the main
product of preQ0 reduction by E89A, E89L and
F228A variants, as shown in Fig. 4 and 5. Proton
signals characteristic of preQ1 were below
detection in these reactions. Conversely, 7-formyl-
7-deazaguanine was completely absent from, and
preQ1 the only detectable product in, reactions of
the wildtype enzyme. Reaction of the L191A
variant gave a mixture of products comprising
mainly preQ1 (80 - 90%), but also 7-formyl-7-
deazaguanine that accounted for the rest of preQ0
converted. The preQ0 reduction by the I192A
variant also proceeded to form both preQ1 and 7-
formyl-7-deazaguanine, roughly at a ratio of 1:1.
Compared to the wildtype enzyme, the Kd_kinetic
was increased in all ecQueF variants (F228A: 59-
fold; E89A: 41-fold; L191A: 7.1-fold), except for
the I192A variant that exhibited the same Kd_kinetic
4

Imine intermediate during nitrile reduction by QueF
LC and LC-MS analysis confirmed the ecQueF
variants to exhibit the particular change in product
formation from preQ0 (Fig. 5). When the preQ0
reduction with the variants were done in Tris
buffer, LC traces revealed a putative product peak
of unknown identity, appearing always together
with the expected product peak for 7-formyl-7-
deazaguanine (Fig. 5B). The unknown peak
exhibited absorbance at 262 nm and 340 nm, just
like 7-formyl-7-deazaguanine but contrary to
preQ0 and preQ1, which both show absorbance
only at 262 nm. When the same enzymatic
reactions were done in phosphate buffer, the
second peak was missing and only the peak for 7-
formyl-7-deazaguanine was found (Fig. 5C, D).
This together with the characteristic mass of the
unknown peak (H+, 282) suggested an imine
adduct between 7-formyl-7-deazaguanine and Tris,
as shown in Fig. 5, that may have formed in
solution already during the enzymatic reaction or
during sample preparation.
Ability of the LC and LC-MS analytic method
to quantify even small amounts of the secondary
reaction product (e.g., 5% preQ1 in preQ0
conversion by the E89A variant) made it possible
to determine whether the NADPH or the preQ0
concentration had an influence on the product
distribution in the enzymatic conversions. Whereas
high concentrations of NADPH might prevent the
loss of imine intermediate from the enzyme and so
pull the enzymatic reaction towards preQ1
formation, the opposite effect, namely preference
for formation of aldehyde, could be expected at
high preQ0 concentration (Scheme 3). In each
enzyme including wildtype ecQueF, there was no
effect from variation in [NADPH] between 1 and
10 mM. Note that relatively high NADPH
concentrations were used in the experiment to
maximize the possible "pull" towards preQ1. The
case of the L191A and I192A variants, in which on
average 1 imine was lost in every 5 (L191A) and 2
(I192A) preQ0 conversion events, clearly shows
that exchange between NADP+ and NADPH for
complete two-fold reduction of preQ0 was still
somewhat functional after the replacements of
Leu191 and Ile192. This result immediately suggests
the possibility that imine was intercepted by water
in, or escaped into solution from, the L191A and
I192A variants before the new NADPH had the
chance to bind, implying in turn an early
disengagement of the imine from the covalent
thiohemiaminal. The alternative possibility is that
imine was intercepted by water in, or was released
from, the ternary complexes of L191A and I192A
variants with NADPH.
Contrary to NADPH, the preQ0 substrate, on
variation of its concentration, affected strongly the
product distribution from nitrile reduction by the
different ecQueF variants. In each enzyme as
shown in Fig. 6, the percent imine in the total
reaction product released increased upon increase
in the molar ratio [preQ0]/[enzyme dimer] used in
the experiment. The effect was most pronounced in
the E89A and F228A variants, which switched
from forming primarily preQ1 (80%) when preQ0
was used at the same concentration as the enzyme
dimer to being almost completely selective for
imine
formation
(≥
95%)
at
higher
[preQ0]/[enzyme dimer] ratios. The E89L variant
gave predominantly imine at all conditions used. In
the I192A variant, the percent imine in total
product increased steadily dependent on the
[preQ0]/[enzyme] ratio and reached a limiting
value of about 50% when preQ0 was added in
excess. The L191A variant formed imine as the
minor product (≤ 20%). In all enzyme variants
except for F228A, the percent imine in total
product typically exhibited a roughly hyperbolic
dependence on the [preQ0]/[enzyme] ratio used
(Fig. 6A-D), from which the maximum imine
formation at saturating preQ0 was determined. In
the F228A variant, maximum imine formation was
established from the data, but the underlying
dependence
of
product
selectivity
on
[preQ0]/[enzyme] ratio appeared complex (Fig.
6E).
Kinetic analysis and KIE studies – To analyze the
enzyme kinetics in more detail, and determine
primary isotope effects (KIE) from the use of (4R)-
[2H]-NADPH as reductant, we monitored the
preQ0 conversion by wildtype and variant enzymes
by in situ proton NMR and also by LC. Kinetic
data obtained from time-course analysis are
summarized in Table 3. Material balances were
fully consistent with the chemical reaction, preQ0
+ NADPH + H3O+ → 7-formyl-7-deazaguanine +
NADP+ + NH3. At long reaction times (≥ 24 h),
however, decomposition of NADPH became
significant as a spontaneous side reaction. The
reaction of wildtype ecQueF was confirmed as
preQ0 + 2 NADPH + 2H+ → preQ1 + 2 NADP+.
Utilization of NADPH for preQ0 reduction by the
5

Imine intermediate during nitrile reduction by QueF
L191A and I192A variants was fully consistent
with the preQ1 and 7-formyl-7-deazaguanine
obtained. Product selectivity of the enzyme
variants was confirmed and shown to be constant
over the whole time course prevailing steady state
conditions ([preQ0] > 15 [enzyme dimer]). The
specific rate of preQ0 conversion was slowed in the
enzyme variants compared to wildtype ecQueF,
most strongly in E89L (700-fold), then E89A (160-
fold), F228A (140-fold), L191A (24-fold) and
I192A (2.6-fold).
was higher than the KIE of 1.5 (±0.3) on formation
of 7-formyl-7-deazaguanine, but lower than the
KIE of 8.9 (±0.3) on preQ1 formation. The KIE on
conversion of NADPH to NADP+ was 5.0 (±0.6).
There is only one isotope-sensitive step
involved in 7-formyl-7-deazaguanine formation,
that of reduction of preQ0 to imine. The KIEs for
the E89A, E89L and F228A variants therefore
reflect the slowing down of the enzymatic
reactions at just this step. The individual KIEs
associated with reductions of the preQ0 substrate
and the imine intermediate by wildtype ecQueF
were determined previously as 3.3 and 1.8,
respectively (7). Global fitting of reaction time
courses of preQ0 conversion were used in our
earlier study (7) to obtain these KIEs. The KIEs on
conversion of preQ0 to 7-formyl-7-deazaguanine
by the Glu89 variants and the F228A variant were
of a similar magnitude as the first-step KIE, i.e.,
preQ0 reduction to imine, for the reaction of
wildtype ecQueF.
In the L191A and the I192A variant,
partitioning of the imine intermediate between
reaction to preQ1 and reaction to 7-formyl-7-
deazaguanine constitutes the point of departure for
analyzing the different KIEs in these two enzymes.
The molar ratio (R) of preQ1 and 7-formyl-7-
deazaguanine reflects the rate constant ratio of
imine reduction/imine hydrolysis (k6/k7), as shown
in the kinetic mechanism depicted in Scheme 3.
Because only the imine reduction is sensitive to
isotopic substitution in coenzyme, the isotope
effect on the product ratio (4/1.8 = 2.22 for L191A;
2.6/0.48 = 5.4 for I192A) is also the KIE on k6. A
KIE on preQ0 conversion smaller than on preQ1
formation arises because partitioning of the imine
intermediate is also subject to an isotope effect.
The fraction of imine intermediate reacting to 7-
formyl-7-deazaguanine is defined from Scheme 3
as F = 1/(1 + R). F has a value of 0.2 and 0.36 in
L191A reactions with NADPH (FH) and NADPD
(FD), respectively. The corresponding F values for
the I192A variant are 0.28 (FH) and 0.67 (FD). The
relative flux from preQ0 to 7-formyl-7-
deazaguanine, therefore, increases 1.8-fold
(L191A variant) and 2.4-fold (I192A variant) due
to deuteration of the coenzyme. Considerations of
flux balance reveal the set of KIE data for the
L191A and the I192A variant to be internally
consistent.
Using (4R)-[2H]-NADPH instead of NADPH,
we showed that the deuterium was transferred
from coenzyme to form 7-formyl-7-deazaguanine
(all enzyme variants) and preQ1 (L191A and
I192A variants), indicating the same pro-R
stereoselectivity for NADPH conversion in the
variants as in wildtype enzyme (7).
The KIE determined from a direct comparison
of reaction rates with NADPH and (4R)-[2H]-
NADPH was 2.9 (±0.7) for the E89A variant, 3.0
(±0.6) for the E89L variant and 2.8 (±0.4) for the
F228A variant. The "reference KIE" for the
reaction of the wildtype enzyme was 1.8 (±0.5).
The shown KIEs are means (± S.D.) from 5
independent determinations and represent the
averaged isotope effects on the rates of substrate
consumption (preQ0, NADPH) and product
formation (preQ1 or 7-formyl-7-deazaguanine,
NADP+). The L191A and I192A variants differed
from the wildtype enzyme and the Glu89 and Phe228
variants in that their reaction rates based on
substrate consumption and product consumption
were all affected by a different KIE, as shown in
Table 3.
In the reaction of the L191A variant, the KIE
on preQ0 consumption was 3.7 (±0.2), higher than
the KIE of 2.3 (±0.2) on the release of 7-formyl-7-
deazaguanine, but lower than the KIE of 5.1 (±0.2)
on the enzyme's main reaction path to preQ1. The
KIE on conversion of NADPH to NADP+ was 4.4
(±0.2). Using (4R)-[2H]-NADPH as coenzyme, the
molar ratio between preQ1 and 7-formyl-7-
deazaguanine was just 1.8, significantly smaller
than the ratio of 4 received from preQ0 reduction
by NADPH with the L191A variant.
Using the I192A variant, the ratio of preQ1 to 7-
formyl-7-deazaguanine formed in the reaction was
5.4-fold lower when (4R)-[2H]-NADPH (ratio:
0.48) instead of NADPH was used (ratio: 2.6). The
KIE on preQ0 consumption was 4.1 (±0.3), which
6

Imine intermediate during nitrile reduction by QueF
With FH and FD known, the KIE on preQ0
consumption obtains, according to Scheme 3, from
sequestration (e.g., Glu89, Phe228) were also
strongly disruptive on the hydride transfer revealed
that the major physical steps of the enzymatic
process proceeded tightly interconnected with the
catalytic act(s). It furthermore indicated that
ecQueF made parsimonious use of its active-site
structural devices to accomplish the different tasks
in the overall multistep catalytic reaction. The
thermodynamic signatures of preQ0 binding by the
different ecQueF variants appear consistent with
the enzyme-substrate interactions revealed in the
crystallographic evidence on vcQueF (Fig. 1).
the KIE on preQ1 formation as KIE_preQ0
=
KIE_preQ1 (1 - FD)/(1 - FH). The calculated value
of KIE_preQ0 is therefore 4.08 for the L191A
variant. The same value is obtained for the I192A
variant. The KIE on formation of 7-formyl-7-
deazaguanine obtains from the relationship,
KIE_preQ0 FH/FD, as 2.27 and 1.70 for the L191A
and the I192A variant, respectively. Finally, the
KIE on NADPH consumption, which is the same
as the KIE on NADP+ formation, is calculated with
the relationship, KIE_preQ0 (2 - FH)/(2 - FD), as
The hydride transfers from NADPH are the
4.48 and 5.28 for the L191A and the I192A variant, slowest steps in the overall conversion of preQ0 to
respectively. These calculated KIE data agree well
the experimentally obtained values.
preQ1 by ecQueF (7). Evidence that catalytic rates
were lowered by up to ∼700-fold in the ecQueF
variants, while their associated KIEs due to
deuteration of the coenzyme were not decreased as
compared to the wildtype KIEs, strongly supports
the suggestion that catalysis was also rate limiting
in the enzyme variants. The crucial importance of
substrate positioning at the stage of imine
reduction was most vividly evident from the fact
that substitutions of Leu191 and Ile192, and even
more so those of Glu89 and Phe228, caused loss of
imine from the enzyme during the reaction. The
fraction of preQ0, which on conversion by the
ecQueF variants didn't make it through to the fully
reduced amine preQ1, increased dependent on the
substrate amount present. A simple model, having
preQ0 compete with imine intermediate for binding
to the enzyme in rapid equilibrium, fails to explain
the experimental product distributions, for it
predicts that at "saturating" [preQ0] no preQ1 will
be formed. This is most evidently refuted from the
behavior of the L191A and the I192A variants.
Evidence summarized in Table 3, that in preQ1
formation the complete reaction of the L191A or
the I192A variant was affected by a substantially
larger KIE than the partial reaction of imine
reduction (k6), identifies nitrile reduction to imine
as the rate-determining step of the overall preQ0
conversion by these two ecQueF variants. Linear
time courses of NADPH consumption by the
enzyme, with no sign of a presteady state burst,
were consistent with this notion (data not shown).
Discussion
Conversion of preQ0 to 7-formyl-7-deazaguanine
represents the first reaction of this kind catalyzed
by an enzyme. Chemically, it consists in a single
hydride transfer reduction of a nitrile group from
NADPH followed by addition of water to the
imine thus obtained. Catalysis from the enzyme is
required only for nitrile reduction. Imine
hydrolysis is
a
spontaneous reaction, well
established from other enzymatic transformations,
like those of amino acid dehydrogenases for
Formally,
a general model of molecular
complex dissociation, induced by a competing
ligand as shown in literature (28), would be
consistent with the effect of [preQ0] on the product
distribution in the ecQueF variants (Scheme 4).
The model involves ligand dissociation effectively
from a pre-associated complex, which enables
rapid rebinding of the ligand to give the actual
biological form of the complex. The model
predicts that competitor molecules will accelerate
the breakdown of enzyme-ligand complexes by
occluding the rapid rebinding of enzyme and
ligand from the level of their pre-associated
complex. It is therefore worth noting that the
model does not imply the formation of a ternary
instance
(25–27).
Mechanistically,
the
"interrupted" (two-electron) reduction of preQ0 is
significant for it provides first-time direct evidence
on the occurrence of an imine intermediate in the
reaction pathway of QueF nitrile reductases.
Besides reinforcing the current mechanistic
proposal for QueF (Scheme 1) (3, 5–7), these
results advance comprehension of the enzyme's
mode of action, demonstrating how critically
important is imine intermediate sequestration for
complete two-fold reduction of preQ0 to preQ1.
The finding that site-directed substitutions
interferring most significantly with intermediate
7

Imine intermediate during nitrile reduction by QueF
complex between enzyme, ligand and competitor.
The ligand dissociation rate (koff), enhanced in the
presence of competitor (C), is thus given by the
relationship, derived from Scheme 4, koff (C) =
kd × (kesc + konC) ÷ (ka + kesc + konC), where ka and
kd are first-order association and dissociation rate
constants for (rapid) formation of the fully
associated complex from the pre-associated
complex, kesc is the first-order rate constant of
ligand release into solution, and kon is the rate
constant for competitor binding (28). Note that the
kesc step involves hydrolysis of imine. Scheme 4
does not distinguish mechanistically between
whether the hydrolysis occurs in solution or, more
likely, with imine still bound to the enzyme.
Results in Fig. 6 suggest that the ecQueF
variants differed in the relative contribution of kesc
and kon to koff. Whereas in the E89L variant the
escape of imine appeared to be largely controlled
by kesc (since koff was hardly dependent on the
preQ0 concentration), the product formation by the
L191A and I192A variants reflected a prominent
effect from kon. The E89A variant adopted an
intermediate position between the two extremes,
with both kesc and kon affecting the product
distribution dependent on the competitor preQ0. A
molecular account of the proposed kinetic scenario
for ecQueF is not easy to conceive, and the model
is essentially independent of such details. However,
a preassociated enzyme-imine complex in a
somewhat open conformation might make possible
the kinetically implied displacement of the imine
by preQ0.
In conclusion, identification of residues in the
catalytic apparatus of ecQueF essential (Glu89,
Phe228) or auxiliary (Leu191, Ile192) for complete
twofold reduction of nitrile to amine has led to
uncover the imine intermediate, and the necessity
of its efficient sequestration in the active site, as
central features of the enzymatic mechanism.
Structure-based interpretation of the effect of site-
directed substitutions on the reaction selectivity of
ecQueF is that due to a substrate-binding site
(Figure 1 B-C) not fully closable any longer, the
enzyme variants lose the imine intermediate to
hydrolysis, completely in E89A, E89L and F228A
and partly in L191A and I192A. Although not
proven by the evidence shown, a likely scenario is
that the imine is captured by water now able to
enter the partly opened-up active site in the variant
enzymes. The hydrolyzed imine, 7-formyl-7-
deazaguanine, is not recognized by ecQueF as a
substrate of NADPH-dependent reduction, as
demonstrated previously (7). It would therefore be
released into solvent.
While in terms of reaction catalyzed, QueF
nitrile reductases share commonalities with other
four-electron reductases, such as 3-hydroxy-3-
methyl-glutaryl-coenzyme A reductase (29–31)
and the non-ribosomal peptide synthetase module
myxalamid reductase (32, 33), that use NAD(P)H
to convert thioester-activated forms of carboxylic
acids into primary alcohols via an aldehyde
intermediate, they are individuated strongly from
these other enzymes in having to deal with a
highly decomposition-prone intermediate. The
central role of intermediate sequestration in the
QueF mechanism seems to be reflected in the fact
that structural devices used to keep a firm grip on
the imine intermediate are likewise involved
directly in the catalytic events of the enzymatic
reaction.
Experimental Procedures
Chemicals and materials used ― NADPH (purity
>98%) and NADP+ (purity >97%) were from Carl
Roth (Karlsruhe, Germany). 2-Propanol-d8 (99.5
atom % D) was from Sigma (St. Louis, Missouri,
USA). All other materials were of the highest
purity available from Carl Roth and Sigma. The
preQ0
synthesized as described previously (7, 19). A
pET-28a(+) expression vector encoding
and
7-formyl-7-deazaguanine
were
Thermoanaerobium brockii alcohol dehydrogenase
as an N-terminally His-tagged protein was ordered
from GenScript (Piscataway, New Jersey, USA).
Standard expression in Escherichia coli BL21-
DE3 and His-tag purification were used to prepare
the enzyme.
Site-directed mutagenesis―Mutagenesis leading
to site-directed substitution of Leu191 by Ala
(L191A) was performed according to a standard
two-stage PCR protocol (34, 35). A pEHISTEV
expression vector comprising the ecQueF gene was
used as the template. The oligonucleotide primers
used are shown with the mismatched bases
underlined.
L191A forward:
5’-GCTGAAATCAAACTGCGCGATCACCCATCAACC-3’
L191A reverse:
5’-GGTTGATGGGTGATCGCGCAGTTTGATTTCAGC-3’
Mutagenesis to substitute Glu89 by Ala or Leu was
reported in an earlier study (21). Replacement of
8

Imine intermediate during nitrile reduction by QueF
at different protein and preQ0 concentrations were
Ile192 or Phe228 by Ala was ordered from Genscript.
All mutations were verified by gene sequencing.
Enzyme preparation―The ecQueF wildtype and
site-directed variants thereof (E89A, E89L,
L191A, I192A and F228A) were obtained as N-
terminally His-tagged proteins using expression in
E. coli BL21-DE3 as described previously (7, 21).
All enzymes were purified by a reported two-step
procedure, consisting of immobilized metal ion
affinity chromatography and gel filtration (7).
Enzyme purity was confirmed by SDS-PAGE. The
background of the E. coli expression host did not
contain QueF activity in amounts detectable with
the assays used. Contamination of ecQueF variants
with endogenous wildtype activity could thus be
ruled out firmly. The protein concentration was
measured with a Pierce BCA protein assay kit
(Thermo Fisher Scientific, Germering, Germany).
Enzyme stock solutions (0.8 - 1.2 mM) were stored
at -20 °C and used up within 3 weeks due to
limited stability.
analyzed by
a global fitting procedure, as
previously described (7), that employed the
program COPASI (version 4.11_build 65) and
used the mechanism in Scheme 2. A least squares
fitting routine implemented in COPASI was used
to determine the kinetic rate constants and their
standard deviations. In the fitting, k1 and k2 were
not determined individually, but only their ratio
Kd_kinetic (= k2/k1) was obtained. We showed that the
fitting always converged to a unique and well-
defined solution that was independent of the
parameter start values used. All fitted parameters
(Kd_kinetic, k3, k4) were well determined as regards
their associated standard deviation. An overall
binding constant (Kd_overall) was calculated from
the results, using the relationship Kd_overall
=
Kd_kinetic/(1+k3/k4). Note that Kd_overall thus includes
the covalent thioimide formation.
Binding constants were transformed into
binding energies using the relationship ΔG = -RT
lnKd. A 1 M standard state for all reactants was
assumed. R is the gas constant (8.314 J/mol·K) and
T is the temperature (298.15 K).
ITC and spectrophotometric analysis of preQ0
binding in ecQueF variants―A VP-ITC Micro
Calorimeter from Microcal (Malvern Instruments
Ltd., Malvern, UK) was used at 25 °C. The
enzyme was gel-filtered twice to phosphate buffer
(100 mM Na2HPO4-NaH2PO4, pH 7.5, 50 mM
Enzymatic reduction of preQ0 – Experiments were
performed to characterize the preQ0 conversion by
the
different
ecQueF
enzymes.
Besides
KCl) using illustra NAP
5
columns (GE
determining the specific rate of substrate
consumption, determination of the product(s)
formed by preQ0 reduction was of main interest.
Reactions were carried out by incubating buffered
solutions of enzyme, preQ0 and NADPH on a
Thermomixer Comfort (Eppendorf, Hamburg,
Germany) at 25 °C with agitation at 700 rpm for
about 24 h. The DMSO concentration used was
below 2.5%. The concentrations of preQ0 and
NADPH were varied as indicated. The enzyme
concentration was 50 µM (E89A, E89L, I192A
and F228A variants), 10 µM (L191A variant) and
1.8 µM (wildtype enzyme). Reactions were
stopped by precipitating the enzyme with methanol
(10%, by volume) at 70 °C for 10 min (1000 rpm
in a Thermomixer Comfort) (7). Samples were
Healthcare, Buckinghamshire, UK). A DMSO
concentration of maximally 2.0% (v/v) was used in
both the enzyme and the preQ0 solution.
Experiments were conducted and data evaluation
done as described previously (7). The enzyme
molar concentration was based on the protein
concentration assuming
a functional ecQueF
homodimer with a molecular mass of 71,772 Da
(wildtype enzyme), 71,656 Da (E89A variant),
71,740 Da (E89L variant), 71,688 Da (L191A or
I192A variant), or 71,620 Da (F228A variant).
The covalent thioimide adduct between preQ0
and ecQueF in wildtype or variant form was
detected from its characteristic absorbance with
maximum absorption at around 380 nm.
Absorbance titrations were carried out with a
Beckman DU 800 spectrophotometer (Beckman
Coulter, Pasadena, CA, USA) as described
previously (7). Kinetic study of the thioimide
formation was done with a SX.18 MV Stopped
flow spectrophotometer (Applied Photophysics;
Leatherhead, UK). The experimental progress
curves of absorbance (380 nm) increase recorded
1
applied to H NMR, HPLC, and LC-MS from
comprehensive analysis.
HPLC analytics―The samples were analyzed
using an Agilent 1200 HPLC system (Santa Clara,
CA, USA) equipped with a 5 µm SeQuant ZIC-
HILIC column (200 Å, 250 × 2.1 mm; Merck,
Billerica, MA, USA), the corresponding guard
column (20 × 2.1 mm; Merck) and a UV detector
9

Imine intermediate during nitrile reduction by QueF
(λ = 262 and 340 nm). A 20 mM ammonium
acetate buffer (pH 6.67) was used. A decreasing
gradient in acetonitrile was applied for compound
separation over a 20-min run time. Specifically,
the acetonitrile concentration was decreased only
slightly from 80% to 78% over 5 min, and a
steeper ramp from 78% to 60% was then used
from 5 to 20 min. The column was washed with
60% and 80% acetonitrile for 5 min after each
analysis. The flow rate was 0.5 ml/min. The
column temperature was 30 °C.
Optionally, a mass detector (Agilent 6200
Quadrupole) was coupled to the HPLC system,
which in this case was equipped with a 2.7 µm
Poroshell 120 EC-C18 column (120 Å, 100 × 3
mm; Agilent) and a UV detector (λ = 210, 262, and
340 nm). The masses of the products from the
enzymatic reactions were scanned in a mass range
of 100-850 using a positive mode. The masses of
preQ0 (1H+, 176), preQ1 (1H+, 180), and 7-formyl-
7-deazaguanine (1H+, 179) were also analyzed in
SIM mode. A linear gradient of acetonitrile was
used in water containing 0.01% formic acid. The
gradient was from 0% to 25% acetonitrile over 5
min. The column was washed with 90%
acetonitrile for 1 min and water for 0.5 min. The
post time was 1 min. The flow rate was 0.7
ml/min. The column temperature was 30 °C.
through addition of the enzyme. Spectra of the
reaction with wildtype ecQueF or the L191A
variant were collected at every 5 - 20 min for 5 -
20 h in the magnet. As the reactions with the Glu89
variants were relatively slow, the spectra were
collected for 48 h in the magnet and the solutions
were temporarily kept at room temperature. Data
were analyzed in the software program ACD/NMR
Processor Academic Edition (version 12.01_build
39104). The total amount of 7-formyl-7-
deazaguanine formed in the reaction was
determined from the free aldehyde product present
and the corresponding imine adduct between 7-
1
formyl-7-deazaguanine and Tris (3.82 ppm in H
NMR spectra, not shown).
KIE studies―NADPH and (4R)-[2H]-NADPH
were prepared by reducing NADP+ in the oxidation
of 2-propanol and 2-propanol-d8 with T. brockii
alcohol dehydrogenase. A reported protocol was
used for their synthesis (7, 38). Primary KIEs on
preQ0 reduction were obtained by comparing
initial rates measured with NADPH and (4R)-[2H]-
NADPH, both synthesized as described above.
Enzymatic reactions were monitored by HPLC and
for certain enzymes (wildtype, E89A, E89L,
1
L191A) additionally by in situ H-NMR. Initial
rates were obtained from linear fits to HPLC or
spectral data. Note: since all substrates and
products of the reaction were measured in the
analysis, a total of four or five reaction rates was
obtained: preQ0 and NADPH consumption and
preQ1, 7-formyl-7-deazaguanine and NADP+
formation. The enzyme concentrations used in the
reactions were: wildtype, 1.5 µM; E89A and
E89L, 25 µM; L191A, 8 µM; I192A, 10 µM; and
F228A, 20 µM. The preQ0 concentration was
saturating, that is, 500 µM using the wildtype
enzyme, the L191A, and the I192A variant and
800 µM using the E89A, the E89L and the F228A
variant. The NADPH concentration was 1.5 mM.
The temperature was 30 °C. The DMSO
concentration was always at or below 2 %.
Phosphate buffer (100 mM Na2HPO4-NaH2PO4,
pH 7.5) containing 50 mM KCl was used.
1H NMR measurements―NMR spectra were
recorded at 499.98 MHz and 30 °C using a Varian
INOVA 500 MHz spectrometer (Agilent
Technologies) with VNMRJ 2.2D software. The
spectra of coenzymes, preQ0 and preQ1 in
D2O/H2O solution were reported previously (7, 36,
37). The reduction of preQ0 by the different
1
ecQueF enzymes was monitored by in situ H
NMR measurements that involved presaturation of
the residual water signal. Tris buffer (30 mM, pH
7.5) in 120 mM KCl was used which also
contained an internal standard solution of 30 µL
(trimethylamine hydrochloride, 1.82 × 10-5 mol/L;
trimethylsilylpropanoic acid, 0.05% in 99.5%
D2O) to obtain data for correct integration. The
D2O content was 14% (v/v) and the total reaction
volume was 0.6 ml. The reactions were started
ACKNOWLEDGMENTS
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
10

Imine intermediate during nitrile reduction by QueF
Program managed by the Austrian Research Promotion Agency FFG. We thank Prof. Norbert Klempier
and Dr. Birgit Wilding (Institute of Organic Chemistry, Graz University of Technology) for providing
preQ0 and 7-formyl-7-deazaguanine; Dr. Tibor Czabany (Institute of Biotechnology and Biochemical
Engineering, Graz University of Technology) for HPLC analysis; Jan Braun (Institute of Biotechnology
and Biochemical Engineering, Graz University of Technology) for site-directed mutagenesis; Dr.
Hansjörg Weber (Institute of Organic Chemistry, Graz University of Technology) for 1H NMR
measurements; and Dr. Gernot Strohmeier (Austrian Centre of Industrial Biotechnology) for HPLC-MS
analysis.
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this
article.
Author’s Contributions - J.J. and B.N. designed the research and wrote the paper. J.J. conducted
experiments and analyzed data together with B.N. All authors agreed on the final version of the paper.
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31. Oliveira, E. F., Cerqueira, N. M. F. S. A., Ramos, M. J., and Fernandes, P. A. (2016) QM/MM study
of the mechanism of reduction of 3-hydroxy-3-methylglutaryl coenzyme A catalyzed by human
HMG-CoA reductase. Catal. Sci. Technol. 6, 7172–7185
32. Chhabra, A., Haque, A. S., Pal, R. K., Goyal, A., Rai, R., Joshi, S., Panjikar, S., Pasha, S.,
Sankaranarayanan, R., and Gokhale, R. S. (2012) Nonprocessive [2 + 2]e- off-loading reductase
domains from mycobacterial nonribosomal peptide synthetases. Proc. Natl. Acad. Sci. U. S. A. 109,
5681–5686
33. Barajas, J. F., Phelan, R. M., Schaub, A. J., Kliewer, J. T., Kelly, P. J., Jackson, D. R., Luo, R.,
Keasling, J. D., and Tsai, S. C. (2015) Comprehensive structural and biochemical analysis of the
terminal myxalamid reductase domain for the engineered production of primary alcohols. Chem. Biol.
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Imine intermediate during nitrile reduction by QueF
34. Wang, W., and Malcolm, B. A. (1999) Two-stage PCR protocol allowing introduction of multiple
mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. Biotechniques 26,
680–682
35. Schmölzer, K., Luley-Goedl, C., Czabany, T., Ribitsch, D., Schwab, H., Weber, H., and Nidetzky, B.
(2014) Mechanistic study of CMP-Neu5Ac hydrolysis by α2,3-sialyltransferase from Pasteurella
dagmatis. FEBS Lett. 588, 2978–2984
1
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electrochemical reduction of NADP+. Biochim. Biophys. Acta. 1076, 49–60
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Acta. 88, 2610–2616
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reduced nicotinamide adenine dinucleotide phosphate. Anal. Biochem. 221, 273–277
FOOTNOTES
preQ0, 7-cyano-7-deazaguanine; preQ1, 7-aminomethyl-7-deazaguanine; Q, queuosine; T-fold, tunneling-
fold; bsQueF, Bacillus subtilis QueF; ecQueF, E.coli QueF; ITC, isothermal titration calorimetry; KIE,
kinetic isotope effect.
13

Imine intermediate during nitrile reduction by QueF
Table 1. Thermodynamic parameters of preQ0 binding by ecQueF wildtype and variants are shown.
Apparent Kd
(µM)a
∆Hbinding
(kJ/mol)
-T∆Sbinding
(kJ/mol)
∆Gbinding
(kJ/mol)
Ref.
wildtype
C190A
E89A
0.039 ± 0.004
5.5 ± 0.1
-80.3 ± 0.5
-44.7 ± 0.2
-63.3 ± 0.7
-73.3 ± 3.7
-97.5 ± 0.6
-82.9 ± 4.0
-61.7 ± 1.4
37.9
14.6
34.9
44.7
55.1
39.0
30.1
-42.4 ± 0.5
-30.1 ± 0.2
-28.4 ± 0.7
-28.6 ± 3.3
-42.4 ± 0.6
-43.8 ± 1.1
-31.6 ± 0.5
(7)
(7)
11.4 ± 0.4
This study
This study
This study
This study
This study
E89L
10.3 ± 1.1
L191A
I192A
F228A
0.040 ± 0.004
0.023 ± 0.011
3.3 ± 0.5
a The Kd is denoted apparent because the binding process analyzed involves noncovalent binding of preQ0
and covalent thioimide formation with the enzyme.
Table 2. Kinetic constants of preQ0 binding by ecQueF wildtype and variants are shown.
Kd_kinetic (k2/k1)
k3
k4
Kd_overall
≤ 0.039b µM
2.0 µM
Ref.
wildtype
E89A
(2.63 ± 0.01) × 10-6 M 1.63 ± 0.01 s-1
≤ 0.024a s-1
0.006 ± 0.001 s-1
(7)
(109 ± 10) × 10-6 M
(18.7 ± 7.0) × 10-6 M
(2.8 ± 1.0) × 10-6 M
(155 ± 30) × 10-6 M
0.32 ± 0.01 s-1
0.79 ± 0.02 s-1
0.36 ± 0.03 s-1
0.16 ± 0.02 s-1
This study
This study
This study
This study
L191A
I192A
F228A
0.0018 ± 0.0003 s-1 0.042 µM
0.014 ± 0.001 s-1
0.004 ± 0.001 s-1
0.10 µM
3.8 µM
a k4 was obtained from the relationship Kd_overall = Kd_kinetic/(1+k3/k4) as described in Ref. (7).
b
As an upper bound of Kd_overall for preQ0 binding to wildtype ecQueF, the apparent Kd from ITC
measurement (Table 1) was taken.
Table 3. Catalytic reaction ratesa and their associated KIEsb are shown for ecQueF wildtype and variants
thereof.
wildtype
enzyme
E89A
E89L
L191A
I192A
F228A
-0.17 ± 0.03
(3.7 ± 0.2)c
-1.5 ± 0.3
preQ0
preQ1
-4.1 ± 0.1
-0.025 ± 0.001
-0.006 ± 0.001
-0.030 ± 0.006
(4.1 ± 0.3)c
0.16 ± 0.02
(5.1 ± 0.2)c
[2.2]d
0.8 ± 0.1
(8.9 ± 0.3)c
[5.4]d
3.9 ± 0.3
-8.4 ± 0.4
0.03 ± 0.01
(2.3 ± 0.2)c
0.3 ± 0.1
7-formyl-7-deazaguanine
NADPH
0.022 ± 0.001
-0.018 ± 0.007
0.006 ± 0.001
-0.005 ± 0.002
0.035 ± 0.006
-0.022 ± 0.005
0.024 ± 0.005
(1.5 ± 0.3)c
-0.27 ± 0.02
(4.4 ± 0.2)c
-1.1 ± 0.1
(5.0 ± 0.6)c
0.29 ± 0.02
(4.4 ± 0.2)c
1.8 ± 0.3
NADP+
7.6 ± 0.3
1.8 ± 0.5
0.022 ± 0.001
2.9 ± 0.7
0.009 ± 0.001
3.0 ± 0.6
(5.3 ± 0.5)c
DVb
not applicablee not applicablee 2.8 ± 0.4
a The catalytic reaction rates are given as µmol product released (or substrate converted)/(µmol enzyme ×
min). They were determined individually for each compound participating in the reaction. Reaction rates
14

Imine intermediate during nitrile reduction by QueF
of substrate (preQ0, NADPH) consumption are negative, those of product (preQ1, NADP+) formation are
positive.
b DV is the primary KIE, due to deuteration of NADPH, on the catalytic rate. It is the average of the five
individual KIEs determined from preQ0, preQ1, 7-formyl-7-deazaguanine, NADH and NADP+. Each
individual KIE is the average of 5 independent reactions. Note that in reactions of the E89A, E89L and
F228A variants, which perform a single-step reduction of preQ0 to produce 7-formyl-7-deazaguanine
from, the experimental DV equals Dk5 according to Scheme 3. In the wildtype enzyme, DV involves
contribution from the KIEs on k5 and k6.
c In parenthesis is shown the primary KIE on the consumption rate or the formation rate of the compound
indicated.
d The KIE on imine reduction (k6 in Scheme 3) is shown in square brackets. As explained in text, this KIE
is the same as the KIE on the experimental product ratio R, preQ1/7-formyl-7-deazaguanine, formed in the
reaction.
e
Contrary to the wildtype enzyme and the E89A, E89L and F228A variants in which DV was not
dependent on whether substrate consumption or product formation was analyzed and so an average value
could be given, the L191A and I192A variants necessitated KIE evaluation separately for each compound
involved in the reaction. The concept of an average KIE was therefore not applicable.
15

Imine intermediate during nitrile reduction by QueF
Figure Legends
Scheme 1. The proposed mechanism of ecQueF reducing preQ0 to preQ1 is shown. Residues involved
in catalysis are indicated. Also shown are residues involved in closing up the binding site of preQ0 and
targeted by mutational analysis in this study. The amino acid residues are arranged to indicate their
relative positions in the enzyme structure. Amino acid numbering of ecQueF is used. The dashed line
indicates an electrostatic stabilization.
Scheme 2. The two-step kinetic mechanism of preQ0 binding is shown. E is free ecQueF. E•preQ0
indicates the noncovalent complex. E-preQ0 indicates the covalent thioimide adduct.
Scheme 3. The proposed kinetic mechanism of preQ0 conversion by L191A and I192A variants of
ecQueF via partitioning of the imine intermediate between reduction to preQ1 and hydrolysis to 7-
formyl-7-deazaguanine is shown. Covalent (-) and noncovalent (•) binding of substrates/products and
coenzymes to the enzyme are indicated. Rate constant numbering starts from Scheme 2 with the
assumption that enzyme was saturated with NADPH. Nitrile reduction to imine (k5) and imine reduction to
preQ1 (k6) are isotope-sensitive steps. Coenzyme exchange in imine intermediate is assumed to be in rapid
equilibrium.
Scheme 4. A hypothetical kinetic mechanism of ecQueF-imine complex dissociation induced by
preQ0 is shown. E is the free enzyme. The enzyme-bound preQ0 and imine are indicated by subscript.
E~imine indicates a preassociated complex of ecQueF with imine intermediate. The model is adapted
from literature (28).
Figure 1. Sequestration of preQ0 is revealed in crystal structures of vcQueF in apoform (C194A
variant, slate, PDB: 3RJ4) and holoenzyme complex with preQ0 (H233A variant, green, PDB:
4GHM). (A) A superimposition of apoenzyme and holoenzyme structures of the vcQueF homodimer is
shown. (B-C) A close-up view of the binding site for preQ0 in apoenzyme (B) and holoenzyme (C) is
shown. The orientation of NADPH binding is from the crystal structure of the ternary complex of enzyme,
preQ0 and NADPH (vcQueF R262L variant, PDB: 3UXJ). Despite missing density for the nicotinamide
ring in the protein-bound NADPH (dark gray), the likely orientation of the nicotinamide moiety can be
inferred from the binding positions of preQ0 and the structurally resolved portion of NADPH. Binding of
NADPH in ecQueF was also examined with structure modeling (21). The ecQueF residues corresponding
to the vcQueF residues shown in panels A - C are: Glu89, Ser90, Cys190, Leu191, Ile192, Asp197, Phe228, His229
and Glu230. The preQ0, NADPH, and amino acids in the binding pocket are indicated by element-based
colors.
Figure 2. ITC analysis of preQ0 binding to ecQueF variants at 25 °C is shown. E89A (A, 58 µM),
E89L (B, 60 µM), L191A (C, 9.4 µM), I192A (D, 10 µM), and F228A (E, 37 µM) were used. The preQ0
solution (1 mM for E89A and E89L, 0.25 mM for L191A and I192A, 0.8 mM for F228A) was titrated into
the enzyme solution. The obtained thermodynamic parameters are summarized in Table 1. The c values (c
=[dimeric protein]/Kd) obtained from the experiments were 5 for the E89A, 6 for the E89L, 232 for the
L191A, 525 for the I192A and 14 for the F228A variant.
Figure 3. Stopped-flow progress curves of thioimide formation by different ecQueF variants are
shown together with fitting results. The thioimide formation by the ecQueF variants was recorded by
absorbance at 380 nm. E89A (A, 33 µM), L191A (B, 35 µM), I192A (C, 57 µM) or F228A (D, 53 µM)
variant was mixed with varied concentration of preQ0 (bottom to top; 15, 30, 45, 60, 120, and 300 µM).
The DMSO concentration did not exceed 2%. Black dashed lines are the averaged data from triplicate
measurements. Global fitting was performed using the two-step binding mechanism in Scheme 2. The fits
are shown with orange lines. The obtained kinetic constants are summarized in Table 2.
16

Imine intermediate during nitrile reduction by QueF
1
Figure 4. H NMR analysis of the products formed on preQ0 conversion by ecQueF wildtype and
variants thereof is shown. (A) Enzymatic routes of conversion of preQ0: two-fold reduction gives preQ1
while single reduction results in an imine which on release to water decomposes to 7-formyl-7-
1
deazaguanine. (B) H NMR spectra of product mixtures obtained with the different enzymes are shown
together with spectra of authentic reference material. Phosphate buffer (100 mM, Na2HPO4-NaH2PO4, pD
7.5, 99.8% D2O; pD = pH meter reading + 0.4), additionally containing 50 mM KCl and 1.15 mM tris(2-
carboxyethyl)phosphine, was used for the reactions of ecQueF wildtype and Glu89 variants. NADPH (1.5
mM) and preQ0 (0.5 mM) were added to the enzyme solution. The total reaction volume was 0.6 mL. The
spectrum of 7-formyl-7-deazaguanine (3 mM) was obtained in the phosphate buffer. The preQ0 reduction
with L191A variant (6 µM) was conducted in Tris buffer (30 mM, pD 7.5) containing 50 mM KCl and
1.15 mM tris(2-carboxyethyl)phosphine). All peaks in the spectra of the ecQueF L191A reaction were
slightly shifted by 0.03-0.06 ppm due to the presence of enzyme. The spectra of preQ0, preQ1, and
coenzyme were compared with the previously reported spectra of these components (7, 36, 37).
Figure 5. HPLC-MS analysis of the products formed on preQ0 conversion by ecQueF wildtype and
variants thereof is shown. The enzyme concentration was varied: 1 µM for wildtype (A), 40 µM for
E89A (B), and 20 µM for each I192A (C) and F228A (D). The preQ0 concentration was 200 µM and the
NADPH concentration was 500 µM. Incubation at 25 °C proceeded for 3-12 h. The total reaction volume
was 1.5 (A, B) and 0.1 mL (C, D). (A-B) Tris buffer (100 mM, pH 7.5) containing 50 mM KCl and 1.15
mM tris(2-carboxyethyl)phosphine was used for wildtype enzyme and E89A variant. The Tris-imine
complex (1H+, 282) formed in solution between 7-formyl-7-deazaguanine and Tris was separated at 9.2
min. (C-D) Phosphate buffer (100 mM Na2HPO4-NaH2PO4, pH 7.5) containing 50 mM KCl was used for
I192A and F228A variant. The preQ1 was separated at 12.8 min (C) but it was not detectable in the
reaction of the F228A variant (D).
Figure 6. Product distribution analysis for preQ0 reduction by the ecQueF variants is shown. The
relative proportion of preQ1 and 7-formyl-7-deazaguanine in total product is shown in dependence of the
molar ratio of preQ0 and enzyme ([preQ0]/[E]) used in the reaction. The panels show A, E89A; B, E89L;
C, L191A; D, I192A; E, F228A. Phosphate buffer (100 mM Na2HPO4·NaH2PO4, pH 7.5), additionally
containing 50 mM KCl and 1.15 mM tris(2-carboxyethyl)phosphine, was used. The enzyme solution (10,
50, and 100 µM) was mixed with preQ0 (25 - 500 µM) and NADPH (0.6 or 1.5 mM). After incubation (30
°C, 2-24 hours), the samples were analyzed by LC-MS and HPLC. Open circles show preQ1 and closed
circles show 7-formyl-7-deazaguanine. The dotted line is a hyperbolic fit to the relative product proportion
of 7-formyl-7-deazaguanine, and its maximum relative content is shown.
17

Imine intermediate during nitrile reduction by QueF
Scheme 1
18

Imine intermediate during nitrile reduction by QueF
Scheme 2
19

Imine intermediate during nitrile reduction by QueF
Scheme 3
20

Imine intermediate during nitrile reduction by QueF
Scheme 4
21

Imine intermediate during nitrile reduction by QueF
Figure 1
22

Imine intermediate during nitrile reduction by QueF
Figure 2
23

Imine intermediate during nitrile reduction by QueF
Figure 3
24

Imine intermediate during nitrile reduction by QueF
Figure 4
25

Imine intermediate during nitrile reduction by QueF
Figure 5
26

Imine intermediate during nitrile reduction by QueF
Figure 6
27

Evidence of a sequestered imine intermediate during reduction of nitrile to amine by
the nitrile reductase QueF from Escherichia coli
Jihye Jung and Bernd Nidetzky
J. Biol. Chem. published online January 16, 2018
Access the most updated version of this article at doi: 10.1074/jbc.M117.804583
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