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

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V.L. Davidson, unpublished results).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].

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

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

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.

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

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-

)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

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

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

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

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

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-

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

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

)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

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

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

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

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

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

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

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

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