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

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 inh

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: nigel.scrutton@manchester.ac.uk

(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 (klim) for TTQ reduction is controlled by a single ionization with pKa value of 6.0, with maximum activity realized in the alkaline region Two kinetically influential ionizations were identified in plots of klim⁄ Kd of pKa values 7.1 and 9.3, again with the maximum value realized in the alkaline region The poten-tial origin of these kinetically influenpoten-tial 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.

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 a2b2structure (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 maupromoter, 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.

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 (%)

Ammonium sulphate fractionation

Sephacryl S-200 gel filtration 17 524 30.8 37

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

ais 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; KIE¼ 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 (KIE¼ 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 AADHox(6.5 l M ), in 10 m M 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 l M ) 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).

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 Kd vs pH (Fig 4B),

klim vs pH (Fig 4C) and the plot of klim⁄ Kd vs pH

(Fig 4D) Limiting rate constants for TTQ reduction

and Kd 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 pKa

values of 6.0 ± 0.1 (protiated benzylamine) and

5.65 ± 0.15 (deuterated benzylamine) A plot of

klim⁄ Kd 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

klim⁄ Kd 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 ionizaequa-tion yielded a pKa1 value of 7.1 ± 0.2 and pKa2 value of 9.3 ± 0.2 for protiated benzylamine With deuterated benzylamine the corres-ponding values were pKa1 7.0 ± 0.2 and pKa2 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 (kcat

< 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

1 mm phenazine ethosulfate (PES) and
2.0 with

5 mm 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 (l M )

Deuterated benzylamine K d (l M ) KIE

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 (
3 nm) Moreover, we have

observed that AADH enzyme activity is inhibited at

high concentrations of PES (Ki is 1.8 ± 0.14 mm;

Fig 6A) Increasing the PES concentration leads to

an increase in the apparent Km for benzylamine

(Fig 6C) and decrease in apparent kcat (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 reacinhibi-tions 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

Kdvs pH (C) Plot of klim vs pH Inset, plot of KIE vs pH (pKa 6.3 ± 0.2) Filled circles, protiated benzylamine-dependent activity (pKa 6.0 ± 0.07); open circles, deuterated benzylamine-dependent activity (pKa5.65 ± 0.15) The errors associated with the pKavalues 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 yielddeuterat-ed 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 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.

with aromatic amines such as tyramine,

b-phenylethyl-amine and tryptb-phenylethyl-amine [1,22] In a previous report, data

collected with tyramine [1] were fit to the following

equation:

m¼ Vmax½S

Kmþ ½S þ ½S2=Ki

ð1Þ

where v is the initial velocity, Vmax is the maximum

initial velocity, [S] is the substrate concentration and

Ki 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)

m¼

1þb½S

Ki

Vmax

1þKs

½Sþ

Ks

Ki

þ½S

Ki

ð2Þ

where KS and Kiare the Michaelis and inhibition

con-stants for substrate, respectively Vmaxis the theoretical

maximum initial velocity and b is a factor by which

the Vmax 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 DH
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 klimand Kdwere determined by fitting data to the standard hyperbolic expres-sion.

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 faecaliscomprises 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 mauFpromoter and introducing these genes into P den-itrificansusing 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 m M 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 m M PES in

10 m M BisTris propane buffer, pH 7.5, at 25
C Filled circles,

proti-ated tryptamine-dependent activity; open circles, deuterproti-ated

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 (l M ), 1.3 ± 0.13; K i (l M ),

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).

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

maugene 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 pKa 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 pKa 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

klim⁄ Kd vs pH, which reports on kinetically influential ionizations in the free enzyme and free substrate forms The more alkaline ionization has a pKa value identical, within error, to that of free benzylamine (pKa 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 (pKa 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 pKaof this group to 6.0 (i.e the value measured in the plot of klim

vs pH; Fig 4C) The more acidic ionization in the free enzyme of pKa7.1 has a substantial affect on the affin-ity of the enzyme for substrate In the protonated form, the Kdfor 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 (l M )

Deuterated benzylamine K m (l M ) KIE

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 obtabenzylam-ined from Sigma (St Louis, MO) Deuterated benzylamine HCl (C6D5CD2NH2HCl, 99.6%) and deuter-ated tryptamine HCl (tryptamine-b,b-d2 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 concentraconcentra-tion

Condi-tions: 50 n M native AADH and 500 l M 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 kcatfor 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: AADHred (2 l M ), 10 m M 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.