Tài liệu Báo cáo khoa học: Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis Cofactor assembly and catalytic properties of recombinant enzyme expressed in Paracoccus denitrificans pptx

Tryptophan tryptophylquinone cofactor biogenesis in the
aromatic amine dehydrogenase of Alcaligenes faecalis
Cofactor assembly and catalytic properties of recombinant enzyme
expressed in Paracoccus denitrificans
Parvinder Hothi, Khalid Abu Khadra, Jonathan P. Combe, David Leys and Nigel S. Scrutton
Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, UK
Aromatic amine dehydrogenase (AADH) is a trypto-
phan tryptophylquinone (TTQ)-dependent quinopro-
tein that catalyses the oxidative deamination of a wide
range of amines to their corresponding aldehydes and
ammonia [1]. Electrons released upon substrate oxida-
tion are transferred to the TTQ cofactor (Fig. 1) and
then to the physiological electron acceptor, azurin,
which mediates electron transfer from the dehydro-
Keywords
amine oxidation; aromatic amine
dehydrogenase; cofactor biogenesis;
stopped-flow spectroscopy; tryptophan
tryptophyl quinone
Correspondence
N. S. Scrutton, Manchester Interdisciplinary
Biocentre and Faculty of Life Sciences,
University of Manchester, Stopford Building,
Oxford Road, Manchester, M13 9PT, UK
Fax: +44 161275 5586
Tel: +44 161275 5632
E-mail:
(Received 15 August 2005, revised 19
September 2005, accepted 22 September
2005)
doi:10.1111/j.1742-4658.2005.04990.x

The heterologous expression of tryptophan trytophylquinone (TTQ)-
dependent aromatic amine dehydrogenase (AADH) has been achieved in
Paracoccus denitrificans. The aauBEDA genes and orf-2 from the aromatic
amine utilization (aau) gene cluster of Alcaligenes faecalis were placed
under the regulatory control of the mauF promoter from P. denitrificans
and introduced into P. denitrificans using a broad-host-range vector. The
physical, spectroscopic and kinetic properties of the recombinant AADH
were indistinguishable from those of the native enzyme isolated from
A. faecalis. TTQ biogenesis in recombinant AADH is functional despite
the lack of analogues in the cloned aau gene cluster for mauF, mauG,
mauL, mauM and mauN that are found in the methylamine utilization
(mau) gene cluster of a number of methylotrophic organisms. Steady-state
reaction profiles for recombinant AADH as a function of substrate concen-
tration differed between ‘fast’ (tryptamine) and ‘slow’ (benzylamine) sub-
strates, owing to a lack of inhibition by benzylamine at high substrate
concentrations. A deflated and temperature-dependent kinetic isotope effect
indicated that C-H ⁄ C-D bond breakage is only partially rate-limiting in
steady-state reactions with benzylamine. Stopped-flow studies of the reduc-
tive half-reaction of recombinant AADH with benzylamine demonstrated
that the KIE is elevated over the value observed in steady-state turnover
and is independent of temperature, consistent with (a) previously reported
studies with native AADH and (b) breakage of the substrate C-H bond by
quantum mechanical tunnelling. The limiting rate constant (k
lim
) for TTQ
reduction is controlled by a single ionization with pK
a
value of 6.0, with
maximum activity realized in the alkaline region. Two kinetically influential
ionizations were identified in plots of k

lim
⁄ K
d
of pK
a
values 7.1 and 9.3,
again with the maximum value realized in the alkaline region. The poten-
tial origin of these kinetically influential ionizations is discussed.
Abbreviations
AADH, aromatic amine dehydrogenase; aau, aromatic amine utilization; DCPIP, dichlorophenol indophenol; KIE, kinetic isotope effect;
MADH, methylamine dehydrogenase; mau, methylamine utilization; ORF, open reading frame; PES, phenazine ethosulfate; TTQ, tryptophan
tryptophylquinone.
5894 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS
genase to a c-type cytochrome [2,3]. Oxidation of sub-
strate proceeds via a pathway that involves the release
of two electrons. Time-resolved crystallographic studies
have provided structures for a number of intermediates
along the reaction pathway (M.E. Graichen, L.H.
Jones, B.V. Sharma, R.J. van Spanning, J.P. Hosler &
V.L. Davidson, unpublished results). AADH is known
to adopt a a
2
b
2
structure (a, 40 kDa; b, 12 kDa) [1,4],
highly similar to the related TTQ-dependent methyl-
amine dehydrogenase (MADH) [5]. Each b subunit
contains a covalently bound TTQ prosthetic group
(Fig. 1), which is formed by post-translational modifi-
cation of two gene-encoded tryptophan residues [6].

The mechanism by which TTQ biosynthesis occurs
is not well known. The biosynthesis of AADH from
Alcaligenes faecalis requires a number of additional
genes, not present in Escherichia coli , as well as those
that encode the large and small protein subunits (aauB
and aauA, respectively) [7]. The accessory gene prod-
ucts are required for protein export to the periplasm,
synthesis of the TTQ prosthetic group, and formation
of structural disulfide bonds [7]. Thus, functional
AADH cannot be obtained by cloning and expressing
the two structural genes in a heterologous host in the
absence of the TTQ biosynthesis genes. Heterologous
expression of Paracoccus denitrificans TTQ-dependent
MADH has been achieved in Rhodobacter sphaeroides
by using a broad-host-range vector incorporating the
MADH structural genes (mauA and mauB) and the
additional genes (mauFEDCJG) required for TTQ bio-
genesis [8]. The genes were placed under the regulatory
control of the coxII promoter which, unlike the native
mau promoter, was not controlled by methylamine lev-
els [8]. Aspartate residues in the active site of MADH
have been identified for their role in TTQ biogenesis
[9]. Also, the dihaem c-type cytochrome mauG [10] is
known to (a) initiate the TTQ crosslink in MADH, (b)
convert a single hydroxyl group on Trp57 of the small
subunit to a carbonyl group, and (c) insert a second
oxygen atom into the TTQ ring [11]. The essential nat-
ure of some of the genes in the mau gene cluster of
P. denitrificans (mauF, mauE, mauD and mauG) has
been shown [12,13]; other genes in the cluster (mauR,

mauC, mauJ, mauM and mauN) are not essential for
TTQ biogenesis [12,14,15].
TTQ-dependent quinoproteins are important model
systems for studies of enzymatic hydrogen tunnelling
[4,16,17]. An understanding of the factors that drive
tunnelling reactions in TTQ-dependent enzymes requires
detailed structural, kinetic and mutagenesis studies.
High-resolution crystal structures of AADH and a num-
ber of reaction intermediates have been reported (M.E.
Graichen, L.H. Jones, B.V. Sharma, R.J. van Spanning,
J.P. Hosler & V.L. Davidson, unpublished results), but
a source of recombinant enzyme for mutagenesis studies
has not been made available. With this in mind, we have
developed a system for the heterologous expression of
recombinant AADH exploiting P. denitrificans as host.
The aauBEDA genes and orf-2 from the aromatic
amine utilization (aau) gene cluster of A. faecalis were
placed under the regulatory control of the mauF promo-
ter of P. denitrificans and introduced into P. denitrifi-
cans by using a broad-host-range vector. This leads to
the synthesis of active recombinant AADH that requires
the cooperation of TTQ biogenesis genes from the mau
gene cluster. By performing detailed kinetic studies of
both AADH enzymes, we show that the recombinant
enzyme is indistinguishable from the native AADH of
A. faecalis and benzylamine is a substrate during
steady-state reactions of AADH, contrary to previous
reports using native AADH. In stopped-flow kinetic
studies of TTQ reduction with benzylamine, we identi-
fied ionizable groups in the enzyme–substrate complex

that control the rate of TTQ reduction. One of these
Fig. 1. (A) Structure of tryptophan tryptophylquinone (TTQ) for
AADH isolated from A. faecalis. (B) The reductive half-reaction of
AADH. I, oxidized enzyme; II, substrate carbinolamine intermediate;
III, iminoquinone intermediate; IV, product Schiff base intermediate;
V, aminoquinol intermediate. In the oxidative half-reaction the ami-
noquinol intermediate is converted back to the oxidized enzyme by
electron transfer to azurin and elimination of ammonium.
P. Hothi et al. Cofactor biogenesis in TTQ-dependent AADH
FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS 5895
groups is tentatively assigned to the active site aspartate
residue that accepts a proton from the iminoquinone
intermediate formed in the reductive half-reaction of
the catalytic cycle.
Results
Expression of recombinant AADH
Plasmid pRKAADH was introduced via conjugation
into P. denitrificans to test for AADH expression. The
level of recombinant AADH produced under the con-
trol of the mauF promoter and subsequently purified,
when grown on methylamine as a sole carbon source,
was 52 mg of pure enzyme per 100 g of cells. This is
approximately twice the level of native AADH gener-
ally produced by A. faecalis grown on b-phenylethyl-
amine. Given that P. denitrificans is known to express
MADH when grown on methylamine [18], the peri-
plasmic extract of P. denitrificans transformed with
pRKAADH contained MADH as well as recombinant
AADH. The TTQ-dependent enzymes were easily
separated by ion-exchange chromatography (second

step of the purification procedure described in Experi-
mental procedures). Fractions containing AADH were
eluted from the DE-52 cellulose column with 200 mm
NaCl, whereas MADH fractions were eluted with
400 mm NaCl. AADH was assayed with tryptamine as
described in Experimental procedures and MADH was
assayed with methylamine. AADH fractions were
highly active with tryptamine but methylamine was a
poor substrate. MADH fractions were highly active
with methylamine and completely inactive with trypt-
amine. When P. denitrificans lacking plasmid
pRKAADH was grown on methylamine, no AADH
was detected. MADH expression was similar in wild-
type P. denitrificans and pRKAADH containing
P. denitrificans.
Characterization of recombinant AADH
During the purification of recombinant AADH, the
elution conditions during ion-exchange chromatogra-
phy, hydrophobic interaction chromatography and gel
filtration were identical to those observed for the
native enzyme. The purification of recombinant
AADH is illustrated in Fig. 2 and summarized in
Table 1. The recombinant enzyme migrates as two
subunits (corresponding to a and b subunits) in
SDS ⁄ PAGE and migration is identical to that
observed for the native enzyme. The migration of both
subunits is consistent with the predicted masses of the
mature form of the subunits (14 472 and 40 421 Da
for the small and large subunits, respectively). (The
published nucleotide sequence for the aau B gene is

incorrect [7]. The predicted mass is based on the cor-
rected sequence presented in Supplementary Material.)
N-Terminal sequence analysis of both native and
recombinant b subunits indicated that the first six resi-
dues are Ala-Gly-Gly-Gly-Gly-Ser. The mature protein
product is therefore truncated by 47 amino acids com-
pared with the conceptual protein sequence inferred
from the gene sequence, consistent with removal of the
periplasmic localization sequence (see Supplementary
material). We were unable to obtain N-terminal
sequence for recombinant and native a subunit, sug-
gesting that the sequence is N-blocked. However,
unlike the b subunit (which we infer lacks sufficient
surface protonatable residues for analysis by ESMS in
the scanned mass range), we were able to obtain a
mass for the a subunit by electrospray ionization mass
spectrometry. The mass obtained for both native
and recombinant a subunits were 40 421 Da. This
mass correlates with cleavage of the a subunit at the
Fig. 2. Purification of recombinant AADH from P. denitrificans mon-
itored by SDS ⁄ PAGE analysis. Lane 1, molecular mass markers as
follows: phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin
(45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa)
and a-lactalbumin (14.4 kDa); lane 2, crude cell extract; lane 3, fol-
lowing DE-52 ion exchange chromatography; lane 4, phenyl Seph-
arose chromatography; lane 5, pure AADH following gel-filtration
chromatography.
Table 1. Purification of recombinant AADH from Paracoccus deni-
trificans.
Purification step

Total
protein
(mg)
Total
activity
(units)
Specific
activity
(unitsÆmg
)1
)
Yield
(%)
Cell extract 3668 1427 0.38 100
Ammonium sulphate
fractionation
2580 1172 0.45 82
DE52 chromatography 128 1012 7.9 71
Phenyl Sepharose 58 884 15.2 61
Sephacryl S-200 gel filtration 17 524 30.8 37
Cofactor biogenesis in TTQ-dependent AADH P. Hothi et al.
5896 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS
predicted site for removal of the periplasmic localiza-
tion sequence (i.e. cleavage prior to residue Gln26 with
expected mass of cleaved subunit is 40 438 Da; see
Supplementary material). As the large subunit is
N-blocked, we infer the N-terminal glutamine residue
has cyclized to form the pyrollidone. This brings the
expected mass of the a subunit to 40 421 Da, which is
within error of the measured mass of 40 422 Da.

Stopped-flow studies of the reductive
half-reaction of AADH
Studies of the reductive half-reaction were performed
to allow comparison of kinetic parameters for native
and recombinant AADH. Reduction of the TTQ
cofactor by benzylamine (or deuterated benzylamine)
was followed at 456 nm on rapid mixing of enzyme
with substrate. The plot of observed rate constant
against benzylamine concentration for recombinant
AADH is shown in Fig. 3B. Fitting of the standard
hyperbolic expression to the data revealed that kinetic
parameters for the recombinant enzyme are compar-
able with parameters obtained for the native enzyme
(Table 2).
Photodiode array detection revealed that spectral
changes accompanying enzyme reduction (for both
enzymes) were best described by a one-step kinetic
model A fi B by global analysis (Fig. 3C). Spectrum
a is the oxidized enzyme and spectrum b is the reduced
enzyme. Rate constants for native AADH were
1.64 ± 0.01 and 0.37 ± 0.01 s
)1
, for protiated and
deuterated benzylamine, respectively (kinetic isotope
effect; KIE ¼ 4.4). Rate constants for recombinant
AADH were 1.52 ± 0.01 and 0.36 ± 0.01 s
)1
, for
protiated and deuterated benzylamine, respectively
(KIE ¼ 4.2). These parameters are similar to those

obtained during single wavelength studies of the reduc-
tive half-reaction (Table 2).
Fig. 3. Spectral and kinetic properties of recombinant AADH. (A)
Spectral changes accompanying the titration of oxidized enzyme
with substrate. AADH
ox
(6.5 lM), in 10 mM BisTris propane buffer
(pH 7.5), was reduced by the addition of benzylamine under anaer-
obic conditions at 25 °C. (B) Stopped-flow kinetic data for the
reaction of recombinant AADH with benzylamine and deuterated
benzylamine. Filled circles, protiated benzylamine-dependent activ-
ity; open circles, deuterated benzylamine-dependent activity. Reac-
tions were performed using 1 l
M enzyme (reaction cell
concentration) in 10 m
M BisTris propane buffer, pH 7.5, at 25 °C.
Transients were measured at 456 nm. Observed rates were
obtained by fitting to a standard single exponential expression. The
fits shown are to the standard hyperbolic expression. (C) Photo-
diode array studies of enzyme reduction. AADH
ox
(4 lM) contained
in 10 m
M BisTris propane buffer, pH 7.5, was rapidly mixed with
200 l
M protiated or deuterated benzylamine (reaction cell concen-
trations) at 25 °C. Spectral changes accompanying enzyme reduc-
tion are as in Fig. 3A. Spectral intermediates were identified by
fitting to a one step kinetic model. Spectrum a is the oxidized
enzyme and spectrum b is the reduced enzyme. Similar data to

those in (A–C) were obtained for the native enzyme (not shown).
P. Hothi et al. Cofactor biogenesis in TTQ-dependent AADH
FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS 5897
pH dependence of TTQ reduction with
benzylamine and deuterated benzylamine
Initially, we attempted pH dependence studies of TTQ
reduction by substrate using a three buffer system (e.g.
Mes, TAPSO and diethanolamine) of constant ionic
strength [19]. However, we demonstrated that this was
not possible owing to rapid reduction of the TTQ
cofactor by the buffer components. Also, as noted pre-
viously, univalent cations stimulate spectral changes in
AADH, particularly at higher pH values [20] and our
own studies revealed a similar trend at higher pH
values with various univalent cations (data not shown).
Thus, owing to a rapid loss in absorbance at 456 nm
(attributed to chemical modification of the TTQ to
form an hydroxide adduct) [20], it was not feasible to
perform pH-dependent studies of TTQ reduction in
the presence of added salt (although studies performed
in the presence of 100 mm NaCl at lower pH values
yielded results comparable with those obtained in the
absence of NaCl). pH-dependence studies were there-
fore performed as described in Experimental proce-
dures. Given that single buffers were used to determine
pH profiles, points were confirmed by overlapping the
pH ranges of the different buffers. Owing to limita-
tions in the buffering range of BisTris propane, studies
in the alkaline region were not extended beyond
pH 10. Alternative buffers that might be employed in

the alkaline region (e.g. sodium borate) were avoided
to reduce complications from cation induced adduct
formation.
A typical example of data collected is presented in
Fig. 4A, as well as the plot of K
d
vs. pH (Fig. 4B),
k
lim
vs. pH (Fig. 4C) and the plot of k
lim
⁄ K
d
vs. pH
(Fig. 4D). Limiting rate constants for TTQ reduction
and K
d
values at different pH values are summarized
in Table 3. Fitting of the equation describing a single
ionization to the data shown in Fig. 4C yielded pK
a
values of 6.0 ± 0.1 (protiated benzylamine) and
5.65 ± 0.15 (deuterated benzylamine). A plot of
k
lim
⁄ K
d
indicates the presence of at least one kinetic-
ally influential macroscopic ionization in the free
enzyme, and most likely the presence of two ioniza-

tions (Fig. 4D). The relatively large increase in
k
lim
⁄ K
d
at pH 10 (Fig. 4D, inset) needs to be inter-
preted with caution owing to the poor buffering capa-
city of BisTris propane at this pH. Analysis of the
data (omitting the pH 10 data point) using the equa-
tion for a double ionization yielded a pK
a1
value of
7.1 ± 0.2 and pK
a2
value of 9.3 ± 0.2 for protiated
benzylamine. With deuterated benzylamine the corres-
ponding values were pK
a1
7.0 ± 0.2 and pK
a2
11.1 ± 0.4.
Plots of initial velocity vs. benzylamine
concentration for steady-state reactions of AADH
Benzylamine can reduce AADH and function as an
effective substrate in the reductive half-reaction
[17,21]. Hyun and Davidson, however, have re-
ported that benzylamine-dependent activity is barely
detected during steady-state reactions of AADH (k
cat
< 0.01 s

)1
) [21]. Our steady-state analyses, performed
with native and recombinant enzyme, revealed that
benzylamine and deuterated benzylamine are signifi-
cantly better substrates during steady-state turnover
reactions than suggested by previous studies. A plot
of initial velocity against benzylamine concentration
for recombinant AADH is shown in Fig. 5A. Appar-
ent Michaelis constants were determined by fitting the
Michaelis–Menten equation to initial velocity
data and apparent Michaelis constants were found
to be similar for native and recombinant enzymes
(Table 4). Also, steady-state kinetic parameters are
comparable with kinetic parameters determined from
stopped-flow studies of the reductive half-reaction
(Table 2). The KIE observed during steady-state reac-
tions with benzylamine [% 2.5 in the presence of
1mm phenazine ethosulfate (PES) and % 2.0 with
5mm PES] is deflated compared with the KIE
observed during stopped-flow studies (% 4.5 in the
absence of PES). An observed KIE of % 2.0 suggests
that C-H ⁄ C-D bond breakage is partially rate limiting
during steady-state reactions employing benzylamine
as substrate.
The origin of the apparent discrepancy between
our work and that reported by Hyun and Davidson
concerning the effectiveness of benzylamine as a sub-
Table 2. Kinetic parameters determined from stopped-flow reactions of native and recombinant AADH. Parameters were obtained by least
squares fitting of data to the standard hyperbolic expression.
Enzyme

Protiated
benzylamine k
lim
(s
)1
)
Deuterated
benzylamine k
lim
(s
)1
)
Protiated
benzylamine K
d
(lM)
Deuterated
benzylamine K
d
(lM)KIE
Native 1.47 ± 0.01 0.32 ± 0.01 10.38 ± 0.25 10.4 ± 0.29 4.6 ± 0.2
Recombinant 1.54 ± 0.02 0.36 ± 0.01 10.8 ± 0.6 12.7 ± 0.6 4.3 ± 0.2
Cofactor biogenesis in TTQ-dependent AADH P. Hothi et al.
5898 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS
strate for AADH in steady-state reactions is unclear.
However, we have observed that detection of activity
with the ‘slow’ substrate benzylamine requires a sub-
stantially higher enzyme concentration (% 50 nm)
than with those assays performed with the ‘fast’ sub-
strate tryptamine (% 3nm). Moreover, we have

observed that AADH enzyme activity is inhibited at
high concentrations of PES (K
i
is 1.8 ± 0.14 mm;
Fig. 6A). Increasing the PES concentration leads to
an increase in the apparent K
m
for benzylamine
(Fig. 6C) and decrease in apparent k
cat
(Fig. 6B),
suggesting competition between PES and benzylamine
at a common binding site. This might account for,
or contribute to, the apparent lack of benzylamine-
dependent activity reported by Hyun and Davidson
[21].
Effects of substrate concentration on initial
velocity profiles
Previous studies have established that substrate inhibi-
tion occurs during steady-state reactions of AADH
Fig. 4. The pH dependence of TTQ reduction in AADH with protiated and deuterated benzylamine. Individual parameters determined from
curve fitting to plots of observed rate (k
obs
) against substrate concentration are shown in Table 3. (A) Data set collected at pH 8.0. (B) Plot of
K
d
vs. pH. (C) Plot of k
lim
vs. pH. Inset, plot of KIE vs. pH (pK
a

6.3 ± 0.2). Filled circles, protiated benzylamine-dependent activity (pK
a
6.0 ± 0.07); open circles, deuterated benzylamine-dependent activity (pK
a
5.65 ± 0.15). The errors associated with the pK
a
values are those
determined from curve fitting. (D) Plot of k
lim
⁄ K
d
vs. pH in the pH range 5–9.5. Hatched lines indicate fits to the equation for a single ionization;
solid lines represent fits to the equation for a double ionization. Filled circles, protiated benzylamine dependent activity; open circles, deuterat-
ed benzylamine dependent activity. Analysis of the data (omitting the pH 10 data point) using the equation for a double ionization yielded a
pK
a1
value of 7.1 ± 0.2 and p K
a2
value of 9.3 ± 0.2 for protiated benzylamine. With deuterated benzylamine the corresponding values were
pK
a1
7.0 ± 0.2 and pK
a2
11.1 ± 0.4. Inset, plot of k
lim
⁄ K
d
including data collected at pH 10 and fits to the equation for a double ionization. Con-
ditions: 1 l
M native AADH, various buffers as described in Experimental procedures, at 25 °C.

P. Hothi et al. Cofactor biogenesis in TTQ-dependent AADH
FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS 5899
with aromatic amines such as tyramine, b-phenylethyl-
amine and tryptamine [1,22]. In a previous report, data
collected with tyramine [1] were fit to the following
equation:
m ¼
V
max
½S
K
m
þ½Sþ½S
2
=K
i
ð1Þ
where v is the initial velocity, V
max
is the maximum
initial velocity, [S] is the substrate concentration and
K
i
is the inhibition constant for substrate. We also
observed substrate inhibition with the ‘fast’ substrate
tryptamine (Fig. 5B) and b-phenylethylamine (data not
shown). Fitting of Eqn (1) generated poor fits to the
data (Fig. 5B) and thus data collected with tryptamine
as substrate were analysed using Eqn (2).
m ¼

1 þ
b½S
K
i

V
max
1 þ
K
s
½S
þ
K
s
K
i
þ
½S
K
i
ð2Þ
where K
S
and K
i
are the Michaelis and inhibition con-
stants for substrate, respectively. V
max
is the theoretical
maximum initial velocity and b is a factor by which

the V
max
is adjusted owing to inhibition. The initial
velocity profile for deuterated tryptamine was similar
to the profile obtained with protiated tryptamine with
a KIE close to unity indicating that C-H bond break-
age is not rate limiting with ‘fast’ substrates. The lack
of inhibition observed with benzylamine (Fig. 5A) in
comparison to the inhibition observed with tryptamine
(Fig. 5B) suggests differences in binding of the two
substrates within the active site of the enzyme and ⁄ or
indicates that different steps are rate limiting during
steady-state reactions of AADH with ‘fast’ and ‘slow’
substrates.
Stopped-flow studies of the oxidative
half-reaction with PES
To investigate the kinetics of the oxidative half-
reaction, AADH was reduced stoichiometrically with
benzylamine and rapidly mixed with different concen-
trations of PES under anaerobic conditions (Fig. 7).
Transients were followed at 483 nm, which is an isos-
bestic point for PES but also a wavelength at which
there is reasonable absorbance from the TTQ cofactor.
At 1 mm PES the rate of enzyme oxidation is % 35 s
)1
at 25 °C. At 5 mm PES the extrapolated rate of
enzyme oxidation is %53 s
)1
at 25 °C. This is much
faster than the corresponding turnover number of

% 1.2 s
)1
(with 1 and 5 mm PES), suggesting that the
chemistry of the oxidative half-reaction and binding
of PES to enzyme is not rate limiting in steady-state
turnover.
Temperature dependence studies and kinetic
isotope effects with benzylamine as reducing
substrate
The temperature dependence of the observed KIE was
investigated for reductive half-reactions and steady-
state reactions of native and recombinant AADH. As
shown previously [17], Eyring plots of the reductive
half-reaction indicate that the KIE is independent of
temperature (Fig. 8A,B), although reaction rates are
strongly dependent on temperature. In contrast, Eyring
plots of steady-state reactions indicate that the KIE is
dependent on temperature, suggesting that C-H ⁄ C-D
bond breakage is not fully rate-limiting (Fig. 8C,D).
The parameters DHà and A’
H
: A’
D
, which were found
to be similar for native and recombinant enzymes,
Table 3. Limiting rate constants for TTQ reduction and enzyme–substrate dissociation constants for the reaction of AADH with benzylamine
and deuterated benzylamine at different pH values. Values of k
lim
and K
d

were determined by fitting data to the standard hyperbolic expres-
sion.
pH k
lim
H
(s
)1
) K
d
H
(lM) k
lim
D
(s
)1
) K
d
D
(lM)KIE
5.0 0.36 ± 0.01 381.9 ± 24 0.13 ± 0.01 418.2 ± 17 2.8 ± 0.29
5.5 0.56 ± 0.01 228.2 ± 8.5 0.18 ± 0.01 263.9 ± 14 3.1 ± 0.23
6.0 0.92 ± 0.01 118.1 ± 4.0 0.26 ± 0.01 118.3 ± 3.6 3.5 ± 0.17
6.5 1.2 ± 0.01 58.4 ± 2.4 0.3 ± 0.01 48.8 ± 1.4 4.0 ± 0.16
7.0 1.45 ± 0.02 29.87 ± 2.0 0.34 ± 0.01 31.83 ± 1.0 4.3 ± 0.19
7.5 1.53 ± 0.02 17.45 ± 0.9 0.35 ± 0.01 15.62 ± 0.9 4.4 ± 0.18
8.0 1.56 ± 0.01 14.69 ± 0.8 0.34 ± 0.01 11.92 ± 0.9 4.6 ± 0.16
8.5 1.57 ± 0.02 12.0 ± 0.8 0.34 ± 0.01 12.58 ± 0.8 4.6 ± 0.19
9.0 1.54 ± 0.02 9.97 ± 0.6 0.32 ± 0.01 10.33 ± 0.3 4.8 ± 0.20
9.5 1.52 ± 0.02 8.08 ± 0.6 0.31 ± 0.01 6.68 ± 0.3 4.9 ± 0.19
10.0 1.7 ± 0.02 3.91 ± 0.3 0.33 ± 0.01 4.11 ± 0.3 5.15 ± 0.26

Cofactor biogenesis in TTQ-dependent AADH P. Hothi et al.
5900 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS
were obtained by fitting the Eyring equation to the
data and are summarized in Table 5. Analysis of the
temperature dependence of reaction rates with protiated
and deuterated benzylamine provides a sensitive test
for the functional equivalence of native and recombin-
ant AADH. We infer, therefore, that both enzymes are
identical in their functional properties and that the
TTQ cofactor is assembled correctly in the recombin-
ant enzyme.
Discussion
The aromatic amine utilization (aau) gene cluster of
A. faecalis comprises nine genes (orf-1 , aauBEDA, orf-2,
orf-3, orf-4 and hemE) all putatively transcribed in the
same direction [7]. The second and fifth genes (aauA
and aauB) encode the large and small subunits of
AADH, respectively. The genes aauD and aauE are sim-
ilar to mauD and mauE, respectively, from the methyl-
amine utilization (mau) gene cluster, and the latter two
genes are essential for MADH biosynthesis [13]. Like
mauE, aauE is predicted to be a membrane-spanning
protein and both aauD and mauD contain a C-X-X-C
motif similar to that found in disulfide isomerases. The
identity of the first open reading frame (ORF) (orf-1)in
the aau gene cluster is not certain and it is not related to
mauF, which is found at the corresponding position in
the mau gene cluster [7]. The gene orf-2 in the aau gene
cluster is predicted to be a small periplasmic monohaem
c-type cytochrome. One might suppose that orf-2 is the

functional counterpart of mauG a novel dihaem protein
[10,11] required for TTQ biogenesis in MADH [12] even
though orf-2 (aau cluster) and mauG (mau cluster) lack
substantial similarity in sequence. However, insertion
mutagenesis studies have indicated that orf-2 is prob-
ably not involved in the oxidation of aromatic amines in
A. faecalis [7]. Of the remaining ORFs, sequence simi-
larity searches have failed to establish roles for orf-3
and orf-4, whereas the final gene in the cloned aau gene
cluster, hemE, has 59% identity with E. coli uro-
porphyrinogen decarboxylase. Here, we have described
the heterologous expression of functional TTQ-depend-
ent AADH by placing aauBEDA and orf-2 (directly
downstream of aauA; Fig. 9) under the control of the
mauF promoter and introducing these genes into P. den-
itrificans using a broad-host-range vector. The success-
ful production of active enzyme suggests that orf-1,
orf-3, orf-4 and hemE are not required for the biosyn-
thesis of AADH, consistent with there being no inferred
biological function in TTQ biogenesis for the polypep-
tides encoded by orf-1, orf-3 and orf-4 by comparison
with gene sequences in the mau cluster [7]. The mau gene
cluster for MADH contains the mauF, mauG, mauL,
Fig. 5. Effects of substrate concentration on initial velocity profiles.
(A) Initial velocity vs. benzylamine concentration for steady-state
reactions of recombinant AADH. Assays were performed as des-
cribed in Experimental procedures with 50 n
M AADH and 5 mM PES
in 10 m
M BisTris propane buffer, pH 7.5, at 25 °C. Filled circles, pro-

tiated benzylamine-dependent activity; open circles, deuterated ben-
zylamine-dependent activity. Similar plots were collected in the
presence of 1 m
M PES (data not shown). Apparent Michaelis con-
stants were determined by fitting initial velocity data to the Michael-
is–Menten equation. Similar data were also collected for native
AADH (not shown). (B) Initial velocity data as a function of trypta-
mine concentration. Conditions: 3 n
M native AADH, 5 mM PES in
10 m
M BisTris propane buffer, pH 7.5, at 25 °C. Filled circles, proti-
ated tryptamine-dependent activity; open circles, deuterated trypta-
mine-dependent activity. Fits to Eqn (1) (solid line) and Eqn (2)
(dashed line) are shown. Kinetic parameters determined from fitting
to Eqn (1) are: k
cat
(s
)1
), 54.4 ± 2.5; K
s
(lM), 1.3 ± 0.13; K
i
(lM),
26 ± 3.3. Similar plots were collected in the presence of 1 m
M PES
(data not shown) and for recombinant AADH (data not shown).
P. Hothi et al. Cofactor biogenesis in TTQ-dependent AADH
FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS 5901
mauM and mauN genes, but analogues for these genes
are not found around the aauBEDA gene cluster [7]. We

have shown that TTQ biogenesis in recombinant
AADH is functional despite the lack of equivalent genes
for mauFGLM in the cloned aau gene cluster. Studies
have shown that mauL and mauM are not required for
TTQ biogenesis, but mauG and mauF are essential [12].
The expression of active recombinant AADH in P. deni-
trificans might therefore require the cooperation of
some TTQ biogenesis genes (mauF and mauG ) from the
mau gene cluster.
We have shown that the physical, spectroscopic and
kinetic properties of the recombinant AADH are sim-
ilar to those of the native enzyme purified from A. fae-
calis. Our studies have shown that benzylamine is a
substrate in multiple turnover assays and stopped-flow
mixing reactions. Unlike with fast substrates (e.g. tryp-
tamine and tyramine) substrate inhibition is not
observed with the ‘slow’ substrate benzylamine, which
likely reflects a different and less optimal mode of
binding in the active site for benzylamine. The mech-
anistic reasons for the smaller KIEs seen with benzyl-
amine compared with fast substrates such as
tryptamine are not known at this stage, but barrier
shape and inductive effects (e.g. through the use of
per-C-deuterated benzylamine) should be considered.
That TTQ reduction is partially, but not fully, rate
limiting in steady-state reactions with benzylamine is
consistent with (a) the suppressed KIE observed in
steady-state turnover assays compared with that meas-
ured by stopped-flow methods, and (b) the similarity
of the limiting rate constant for TTQ reduction and

the steady-state turnover value. Also, the temperature
dependence of the KIE observed in steady-state assays
contrasts with the essentially temperature-independent
KIE observed in stopped-flow studies, which is consis-
tent with TTQ reduction being partially rate limiting
in steady-state turnover.
Our studies of the pH dependence of TTQ reduc-
tion by benzylamine have indicated that a single kin-
etically influential ionization of pK
a
6.0 controls the
rate of TTQ reduction. The crystal structure of
AADH indicates the presence of only two ionizable
groups in the immediate vicinity of the active site
(M.E. Graichen, L.H. Jones, B.V. Sharma, R.J. van
Spanning, J.P. Hosler & V.L. Davidson, unpublished
results). Asp128b accepts a proton from substrate
during breakage of the substrate C–H bond (i.e. the
tunnelling reaction) [17] and thus needs to be deproto-
nated in the reactive iminiquinone enzyme–substrate
complex. We suggest that the ionizable group of pK
a
6.0 represents the deprotonation of this aspartate resi-
due. Given that benzylamine is oxidized to the
corresponding aldehyde product in a reaction that
requires only a single proton abstraction by Asp128b,
it seems probable that the kinetically influential ioniza-
tion observed in pH-dependent studies with benzylam-
ine is attributable to the ionization of Asp128b.
Two ionizations were also identified in the plot of

k
lim
⁄ K
d
vs. pH, which reports on kinetically influential
ionizations in the free enzyme and free substrate
forms. The more alkaline ionization has a pK
a
value
identical, within error, to that of free benzylamine
(pK
a
9.3), and we suggest that this represents deproto-
nation of the substrate benzylamine to generate the
reactive, free amine form of the substrate. The more
acidic ionization (pK
a
7.1) is attributed to a group in
the free enzyme, and we speculate this represents the
ionization of Asp128b. This being the case, the effect
of substrate binding would be to lower the pK
a
of this
group to 6.0 (i.e. the value measured in the plot of k
lim
vs. pH; Fig. 4C). The more acidic ionization in the free
enzyme of pK
a
7.1 has a substantial affect on the affin-
ity of the enzyme for substrate. In the protonated

form, the K
d
for the enzyme substrate complex increa-
ses substantially over that seen in the deprotonated
form of the enzyme (% 20-fold on moving from pH 5
to 7.5; Fig. 4B and Table 3). A further increase in
affinity (approximately fivefold) is seen on moving
from pH 7.5 to 10 (Table 3), over which pH range the
substrate benzylamine is converted from the protonat-
ed to free base form. Formal assignment of the
observed kinetically influential ionizations must await
studies with mutant enzymes and different substrates.
These studies can now proceed given the availability of
recombinant AADH containing a correctly assembled
Table 4. Kinetic parameters determined from steady-state reactions of native and recombinant AADH. Parameters were obtained by least
squares fitting of data to the standard Michaelis–Menten expression.
E Enzyme
[PES]
(m
M)
Protiated
benzylamine k
cat
(s
)1
)
Deuterated
benzylamine k
cat
(s

)1
)
Protiated
benzylamine K
m
(lM)
Deuterated
benzylamine K
m
(lM)KIE
Native 1 1.14 ± 0.01 0.46 ± 0.01 6.7 ± 0.1 12.7 ± 0.5 2.5 ± 0.2
Recombinant 1 1.02 ± 0.02 0.35 ± 0.01 9.9 ± 0.7 13.2 ± 0.7 2.9 ± 0.3
Native 5 1.23 ± 0.01 0.55 ± 0.01 14.4 ± 0.3 12.6 ± 0 .8 2.2 ± 0.1
Recombinant 5 1.03 ± 0.02 0.49 ± 0.02 13.1 ± 0.5 14.3 ± 0 .7 2.1 ± 0.2
Cofactor biogenesis in TTQ-dependent AADH P. Hothi et al.
5902 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS
TTQ reaction centre. Analysis of wild-type and mutant
forms of AADH with a range of substrates is now in
progress in an attempt to identify those residues that
are responsible for the observed kinetically influential
ionizations in AADH.
Experimental procedures
Materials
BisTris propane buffer, 2,6-dichlorophenol indophenol;
sodium salt (DCPIP), PES (N-ethyldibenzopyrazine ethyl
sulfate salt), b-phenylethylamine, tryptamine and benzylam-
ine were obtained from Sigma (St. Louis, MO). Deuterated
benzylamine HCl (C
6
D

5
CD
2
NH
2
HCl, 99.6%) and deuter-
ated tryptamine HCl (tryptamine-b,b-d
2
HCl, 98%) were
from CDN Isotopes (Quebec, Canada). The chemical purity
of the deuterated reagents was determined to be > 98% by
high performance liquid chromatography, NMR, and gas
chromatography, by the suppliers.
Growth of cells and media
The bacterial strains and plasmids used in this study are lis-
ted in Table 6. For strain stocks and DNA isolation, E. coli
and A. faecalis were cultured with Luria–Bertani media
at 37 and 30 °C, respectively. P. denitrificans was grown in
nutrient broth or on nutrient agar at 30 °C. For the expres-
Fig. 6. Apparent inhibition of AADH as a function of PES concentra-
tion. (A) Initial velocity data as a function of PES concentration. Condi-
tions: 50 n
M native AADH and 500 lM benzylamine (or deuterated
benzylamine) in 10 m
M BisTris propane buffer, pH 7.5, at 25 °C.
Filled circles, protiated benzylamine-dependent activity; open circles,
deuterated benzylamine-dependent activity. The fits shown are to
Eqn (1). (B) Plot of k
cat
for benzylamine vs. PES concentration. (C)

Plot of apparent K
m
for benzylamine vs. PES concentration.
Fig. 7. Plot of observed rate for the oxidative half-reaction of AADH
as a function of PES concentration. AADH was stoichiometrically
reduced with benzylamine and rapidly mixed with different concen-
trations of PES under anaerobic conditions. Conditions: AADH
red
(2 lM), 10 mM BisTris propane buffer, pH 7.5, 25 °C. The mono-
phasic increase in absorbance, representing oxidation of reduced
enzyme, was followed at 483 nm (isosbestic point of PES).
Observed rates were obtained by fitting to the standard single
exponential expression.
P. Hothi et al. Cofactor biogenesis in TTQ-dependent AADH
FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS 5903
sion of AADH, A. faecalis and P. denitrificans were cul-
tured in minimal salts media according to Iwaki et al. [23]
and Davidson [18], respectively. In P. denitrificans, AADH
was expressed from plasmid pRKAADH, by inducing the
mauF promoter cassette with 1% methylamine 8 h prior to
harvesting. When appropriate, antibiotics were added to
the following final concentrations: ampicillin, 100 lgÆmL
)1
;
kanamycin, 25 lgÆmL
)1
; tetracycline, 10 lgÆmL
)1
for E. coli
and tetracycline, 1 lgÆmL

)1
; rifampicin 20–50 lgÆmL
)1
for
P. denitrificans.
Construction of plasmids for the expression
of AADH
The strategy employed for constructing plasmid
pRKAADH for the expression of AADH in P. denitrificans
is summarized in Fig. 9. pRKAADH consisted of a 3.5 kb
region of the A. faecalis aau gene cluster (containing aauB,
aauE, aauD, aauA and orf-2) fused to the P. denitrificans
methylamine utilizing (mau) gene F promoter. First, the
3.5 kb aau fragment was amplified by PCR from A. faecalis
Fig. 8. Eyring plots for reactions of AADH with benzylamine and deuterated benzylamine. (A) Plot of ln k
obs
⁄ T vs. 1 ⁄ T for the reductive half-reac-
tion of native AADH with benzylamine and deuterated benzylamine. Filled circles, protiated benzylamine; open circles, deuterated benzylamine.
Inset, plot of ln KIE vs. 1 ⁄ T. Conditions: 1 l
M AADH, 10 mM BisTris propane buffer, pH 7.5. Rate constants are observed rate constants meas-
ured at 200 l
M benzylamine. Observed rates were obtained by fitting to a standard single exponential expression. For each reaction at least four
replicate measurements were collected and averaged, each containing 1000 data points. (B) As (A) but with recombinant AADH. Above 25 °C,
absorbance changes observed for the recombinant enzyme were biphasic and thus observed rates were obtained by fitting to a double expo-
nential expression. (C) Plot of ln v
i
⁄ T vs. 1 ⁄ T for steady-state reactions of native AADH with benzylamine and deuterated benzylamine. Filled cir-
cles, protiated benzylamine; open circles, deuterated benzylamine. Inset, plot of ln KIE vs. 1 ⁄ T. Conditions: 100 n
M AADH, 5 mM PES, 500 lM
benzylamine, 10 mM BisTris propane buffer, pH 7.5. (D) As (C) but with recombinant AADH. In these assays, one standard deviation in each

activity measurement (n ¼ 5) at a defined temperature is < 6% of the determined value. Kinetic and thermodynamic parameters were obtained
from fitting data to the Eyring equation.
Cofactor biogenesis in TTQ-dependent AADH P. Hothi et al.
5904 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS
genomic DNA using forward (5¢-GGAGGGATCCCATATG
AAGTCTAAATTTAAATTAACG-3¢) and reverse (5¢-GC
GTG
CTCGAGCGATCCATGGAGCCGTA-3¢) primers
that incorporated BamHI and NdeI restriction sites in the
5¢-end of the amplification product, and an XhoI site in the
3¢-end. The amplification product was cloned into the TA
cloning vector PCR4-TOPO (Invitrogen, Carlsbad, CA) for
sequencing and then subcloned into vector pCDNA II (In-
vitrogen) as a BamHI–XhoI fragment, generating plasmid
pKAK01. The P. denitrificans mauF promoter and a neigh-
bouring ORF (mauR) coding for a transcriptional activator
of the mauF promoter were amplified from genomic DNA
using forward (5¢ -TTGGT
AAGCTTGGGCATTTCTGAT
CGGGTCGC-3¢) and reverse (5¢-AAAC
CATATGACGCC
TCCTCTCGCT-3¢) primers that incorporated HindIII and
NdeI restriction sites into the 5¢- and 3¢-ends, respectively.
The amplification product was cloned into pCR4-TOPO for
sequencing and subsequently subcloned into pKAK02 as an
HindIII–NdeI fragment, generating a transcriptional fusion
between the mauF promoter and aauBEDAorf-2. Finally,
Fig. 9. Strategy for the construction of plas-
mid, pRKAADH, used in the heterologous
expression of AADH. The aau gene region of

A. faecalis consists of nine genes (orf-1, aau-
BEDA, orf-2, orf-3, orf-4 and hemE) [7].
Genes are represented by their correspond-
ing letter, numbers denote orfs 1–4, and hE
is an abbreviation for hemE.ThemauF pro-
moter and mauR gene from P. denitrificans
are represented by mFp and mR, respect-
ively. Open boxes, periplasmic proteins; sha-
ded boxes, cytoplasmic proteins; diagonally
hatched boxes, membrane proteins. Arrows
indicate the direction of transcription.
Table 5. Kinetic and thermodynamic parameters determined from stopped-flow and steady-state reactions of AADH with protiated and deu-
terated benzylamine. Parameters were obtained from fitting the data shown in Fig. 8 to the Eyring equation.
Enzyme Temp. range (°C) DHà
H
(kJÆmol
)1
) DHà
D
(kJÆmol
)1
) DDHà (kJÆmol
)1
) A’
H
: A’
D
KIE
Reductive half-reaction
Native 4–40 63.6 ± 0.6 65.0 ± 0.7 1.4 ± 0.03 2.7 ± 0.07 4.7 ± 0.1

Recombinant 4–40 62.0 ± 1.1 61.4 ± 1.1 0.6 ± 0.02 5.3 ± 0.26 4.3 ± 0.2
Enzyme Temp. range (°C) DHà
H
(kJÆmol
)1
) DHà
D
(kJÆmol
)1
) DDHà (kJÆmol
)1
) A’
H
: A’
D
KIE at 24 °C
Steady-state reactions
Native 4–40 49.6 ± 0.6 55.4 ± 0.7 5.8 ± 0.1 0.24 ± 0.01 2.5 ± 0.2
Recombinant 4–40 48.2 ± 1.1 55.2 ± 0.9 7.0 ± 0.3 0.15 ± 0.01 2.5 ± 0.3
P. Hothi et al. Cofactor biogenesis in TTQ-dependent AADH
FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS 5905
the mauF::aauBEDAorf-2 fusion was cloned as an HindIII–
XbaI fragment into broad-host range vector pRK415 [24],
creating pRKAADH.
Plasmid pRKAADH was transformed into E. coli
strain S17-1 and conjugated into P. denitrificans using a
method adapted from Graichen et al. [8]. E. coli S17-1
cells containing pRKAADH were mixed with rifampicin-
resistant P. denitrificans cells in Luria–Bertani media for
2 h at 30 °C, plated on Luria–Bertani media without

antibiotic selection and then incubated for a further 6 h.
Cells were scraped off the Luria–Bertani plates, washed
once with Sistroms’ medium [25] and then plated on
Luria–Bertani plates containing 1 l g ÆmL
)1
tetracycline
and 20 lgÆmL
)1
rifampicin to select for exconjugates.
Purification of native and recombinant AADH
For the isolation of native AADH, A. faecalis IFO 14479
was grown aerobically at 30 °C on 0.15% (w ⁄ v) b-phenyl-
ethylamine as described previously [1,23]. For the isolation
of recombinant AADH, P. denitrificans transformed with
pRKAADH was grown aerobically at 30 °C on 0.3% (w ⁄ v)
methylamine. The following purification procedure applies
to both native and recombinant enzymes. Cells were harves-
ted in the late exponential phase and resuspended in 10 mm
potassium phosphate buffer, pH 7.5. Cells were broken by
passage through a French pressure cell (14 000 p.s.i., 4 °C)
or by sonication. DNA was hydrolysed (DNase I) and cell
debris removed by centrifugation. The cell extract was
fractionated between 35 and 85% ammonium sulfate. The
precipitate from the ammonium sulfate fractionation was
dialysed exhaustively against 10 mm potassium phosphate,
pH 7.5, and applied to a DE-52 cellulose column equili-
brated with the same buffer. AADH was eluted with
200 mm NaCl using a salt gradient (0–250 mm NaCl con-
tained in 10 m m potassium phosphate buffer, pH 7.5).
Fractions containing AADH were pooled, concentrated by

ultrafiltration, and applied to a phenyl–Sepharose column
equilibrated with 10 mm potassium phosphate buffer con-
taining 35% ammonium sulfate, pH 7.5. Enzyme was elut-
ed using a 35 to 0% ammonium sulfate gradient. Fractions
containing AADH were pooled, concentrated by ultrafiltra-
tion, and dialysed against 10 mm potassium phosphate
buffer, pH 7.5. The enzyme was then applied to a Sephacryl
S-200 gel filtration column equilibrated with 10 mm potas-
sium phosphate buffer containing 100 mm KCl, pH 7.5.
AADH fractions were concentrated by ultrafiltration and
dialysed against 10 mm potassium phosphate, pH 7.5.
Enzyme was judged to be pure by SDS ⁄ PAGE. Purified
enzyme was stored at )80 °Cin10mm potassium phos-
phate buffer, pH 7.5, with 10% ethylene glycol.
Prior to use in kinetic studies, AADH was reoxidized
with potassium ferricyanide and exchanged into 10 mm Bis-
Tris propane buffer, pH 7.5, by gel exclusion chromato-
graphy. Enzyme concentration was determined using an
extinction coefficient of 27 600 m
)1
cm
)1
at 433 nm [1].
Mass spectrometry
ESMS was performed on a Micromass (Milford, MA) LCT
time of flight mass spectrometer operating in positive ion
mode. A mobile phase of 50% acetonitrile ⁄ 50% formic acid
(1% in deionized water) was pumped through the spraying
capillary, which was maintained at % 3 kV. Samples were
dissolved in deionized water and were introduced into the

mobile phase via a Rheodyne injector. Scans were taken at
the rate of 1 s per scan in the mass range 600–2000 a.m.u.
Several scans were averaged to give raw data, which was
further processed using maximum entropy software to pro-
duce the mass spectrum.
Anaerobic titrations of oxidized AADH
The following procedure was performed in a Belle Technol-
ogy (Portesham, UK) glove box (< 5 p.p.m. oxygen) and
buffer was made anaerobic by bubbling with argon for 2 h
and left to equilibrate overnight in the glove box. Anaero-
bic solutions of substrate were prepared by dissolving pre-
weighed solid in anaerobic buffer. Enzymes were reoxidized
with potassium ferricyanide and exchanged into 10 mm
BisTris propane buffer, pH 7.5, by gel-exclusion chromato-
graphy. Enzymes were reduced by the addition of benzyl-
amine and followed spectroscopically using a Jasco (Great
Dunmow, UK) V-550 UV ⁄ Vis spectrophotometer housed
in the glove box.
Steady-state kinetic analysis
Steady-state kinetic measurements were performed with a
1 cm light path in 10 mm BisTris propane buffer, pH 7.5,
Table 6. Bacterial strains and plasmids used in the heterologous
expression of AADH in Paracoccus denitrificans.
Strain or plasmid Relevant features
Source or
reference
Bacteria
A. faecalis Wild-type ATCC OF1144
P. denitrificans Wild-type, Rif
r

ATCC13543
E. coli strain DH5 General cloning strain Invitrogen
E. coli S17-1 Conjugative donor Laboratory
strain
Plasmids
pCR 4-TOPO TA cloning vector Invitrogen
pCDNA II General cloning vector Invitrogen
pKAK01 pCDNAII, aauBEDAorf-2 This study
pKAK02 pCDNA II,
mauFR::aauBEDAorf-2
This study
pRK415-1 Broad-host-range vector [24]
pRKAADH pRK415-1,
mauFR::aauBEDAorf-2
This study
Cofactor biogenesis in TTQ-dependent AADH P. Hothi et al.
5906 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS
at 25 °C in a total volume of 1 mL. AADH activity was
measured using a dye-linked assay in which the reduction
of PES is followed by coupling its oxidation to the
reduction of DCPIP. Reduction of DCPIP was monitored
at 600 nm using a Perkin-Elmer (Boston, MA) Lambda 5
UV-visible spectrophotometer. The reaction mixture typic-
ally contained 50 nm AADH (for benzylamine-dependent
reactions) or 3 nm (for tryptamine-dependent reactions),
0.04 mm DCPIP and 5 mm PES (unless stated otherwise).
Substrates were added to the reaction mix at the appropriate
concentration (see Results). Initial velocity was expressed as
lmole product formed per lmole enzyme per s using a
molar absorption coefficient at 600 nm of 22 000 m

)1
cm
)1
for DCPIP [26]. Initial velocity data collected as a function
of benzylamine concentration were analysed by fitting using
the standard Michaelis–Menten rate equation. Initial velo-
city data collected as a function of tryptamine concentration
[S] were fitted to Eqn (2). Equation 2 has previously been
applied in the determination of steady-state kinetic parame-
ters for trimethylamine dehydrogenase [27,28] and methanol
dehydrogenase [29,30].
Stopped-flow kinetic studies of the reductive
half-reaction
Rapid kinetic experiments were performed using an Applied
Photophysics (Leatherhead, UK) SX.18MV stopped-flow
spectrophotometer. Studies of the reductive half-reaction
were performed by rapid mixing of oxidized AADH (reac-
tion cell concentration 1 lm)in10mm BisTris propane buf-
fer, pH 7.5, with various concentrations of protiated or
deuterated substrate (Results), at 25 °C. The absorbance
change, representing reduction of the TTQ cofactor, was
followed at 456 nm. Data were analysed by nonlinear least
squares regression analysis on an Acorn RISC PC using
spectrakinetics software (Applied Photophysics). For each
reaction, at least three replicate measurements were collec-
ted and averaged, each containing 1000 data points. The
absorbance changes accompanying enzyme reduction with
benzylamine were monophasic, and observed rates were
obtained by fitting using the standard single exponential
expression. Under some conditions (see Results), absorb-

ance changes for the recombinant enzyme were biphasic and
were analysed using a double exponential expression. The
fast phase of these transients (> 90% of the total amplitude
change for reactions at or below 32 °C, and > 80% for
reactions above 32 °C) exhibits a KIE and thus reflects C-H
bond breakage. The slow phase (the origin of which remains
uncertain) was not observed in reactions of AADH with
deuterated benzylamine and therefore transients were ana-
lysed using the single exponential expression.
For multiple wavelength studies of the reductive half-
reaction, AADH
ox
(4 lm) contained in 10 mm BisTris pro-
pane buffer, pH 7.5, was mixed with 200 lm protiated or
deuterated benzylamine (reaction cell concentrations) at
25 °C. Multiple-wavelength absorption studies were carried
out using a photodiode array detector and x-scan soft-
ware (Applied Photophysics). Spectra were analysed and
intermediates of the reaction identified by global analysis
and numerical integration methods using prokin software
(Applied Photophysics).
In studies of the temperature dependence of bond break-
age, enzyme was equilibrated in the stopped-flow apparatus
(or in the UV-visible spectrophotometer for steady-state
reactions) at the appropriate temperature prior to the
acquisition of kinetic data. Temperature control was
achieved using a thermostatic circulating water bath, and
the temperature was monitored directly in the stopped-flow
apparatus using a semiconductor sensor (or using a ther-
mometer in the UV ⁄ visible spectrophotometer). Control

studies of the concentration dependence of bond cleavage
at 4 and 40 °C indicated that the K
d
(or K
m
for steady-state
reactions) was not substantially perturbed on changing tem-
perature (ensuring that substrate was saturating at all the
temperatures investigated). Kinetic and thermodynamic
parameters were obtained by fitting the Eyring equation to
the data as described previously [16,17].
Studies of the pH dependence of TTQ reduction
pH studies were performed at 25 °Cin50mm potassium
acetate (pH 5.0–5.5), 50 mm potassium phosphate (pH 6.0–
7.0) or 50 mm BisTris propane buffer (pH 7.5–10.0).
AADH was exchanged into the required buffer by
gel-exclusion chromatography and substrates were also
dissolved in the required buffer (see Results). Given that
previous studies have established that univalent cations sti-
mulate spectral changes in AADH, particularly at higher
pH values [20], experiments were conducted in the absence
of added salt. pH profiles for the kinetic parameters were
constructed and the data fitted to the equation describing a
single (Eqn 3) or double (Eqn 4) ionization as appropriate.
k
lim
=K
d
¼ððLim1 þ Lim2  tempÞ=ðtemp þ 1Þð3Þ
In Eqn (3), Lim1 is the lower limit and Lim2 is the upper

limit of the curve,
k
lim
=K
d
¼ððLim1 þ Lim2  temp1Þ=ðtemp1 þ 1Þ
À ðððLim2 À Lim3ÞÂtemp2Þ=ðtemp2 þ 1ÞÞÞ ð4Þ
In Eqn (4), temp1 ¼ alog(pH ) pK
a1
), temp2 ¼ alog (pH )
pK
a2
), Lim1 is the lower limit of the curve, Lim 2 is the
middle limit of the curve and Lim 3 is the upper limit of the
curve.
Stopped-flow kinetic studies of the oxidative
half-reaction with PES
Anaerobic rapid kinetic experiments were performed using
an Applied Photophysics SX.18MV stopped-flow spectro-
P. Hothi et al. Cofactor biogenesis in TTQ-dependent AADH
FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS 5907
photometer housed in a Belle Technology anaerobic glove
box (< 5 p.p.m. oxygen). Solutions used were made anaero-
bic by bubbling with argon for 2 h and left to equilibrate
overnight in the glove box. Studies of the oxidative half-reac-
tion of the enzyme were performed by rapid mixing of 2 lm
AADH
red
(stoichiometrically reduced with benzylamine)
with various concentrations of PES (see Results) in 10 mm

BisTris propane buffer, pH 7.5, at 25 °C. The absorbance
change, representing oxidation of the TTQ cofactor, was
followed at 483 nm (isosbestic point of PES). Data were ana-
lysed by nonlinear least squares regression analysis on an
Acorn RISC PC using spectrakinetics software (Applied
Photophysics). For each reaction at least three replicate
measurements were collected and averaged, each containing
1000 data points. The absorbance change monitored was
monophasic, and observed rates were obtained by fitting
using the standard single exponential expression.
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Supplementary material
The following supplementary material is available for
this article online:
Figure S1. Corrected nucleotide sequence of aauB.
Errors in the published nucleotide sequence (accession
AF302652), shown in emboldened and underlined text,
and are as follows: T835 is now C835, C836 is now
T836 and an extra T between A1376 and T1377.
Underlined amino acid residues denote the periplasmic
signal peptide predicted by SignalP.
Figure S2. Nucleotide and predicted polypeptide

sequence of aauA. Underlined amino acid residues
denote the periplasmic signal peptide predicted by
SignalP.
P. Hothi et al. Cofactor biogenesis in TTQ-dependent AADH
FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS 5909