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 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 concentration differed between ‘fast’ (tryptamine) and ‘slow’ (benzylamine) substrates, 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 reductive 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 potential origin of these kinetically influential ionizations is discussed Aromatic amine dehydrogenase (AADH) is a tryptophan tryptophylquinone (TTQ)-dependent quinoprotein that catalyses the oxidative deamination of a wide range of amines to their corresponding aldehydes and ammonia [1] Electrons released upon substrate oxidation are transferred to the TTQ cofactor (Fig 1) and then to the physiological electron acceptor, azurin, which mediates electron transfer from the dehydro- 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 P Hothi et al Fig (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 aminoquinol intermediate is converted back to the oxidized enzyme by electron transfer to azurin and elimination of ammonium genase to a c-type cytochrome [2,3] Oxidation of substrate 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 a2b2 structure (a, 40 kDa; b, 12 kDa) [1,4], highly similar to the related TTQ-dependent methylamine dehydrogenase (MADH) [5] Each b subunit contains a covalently bound TTQ prosthetic group (Fig 1), which is formed by post-translational modification 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 products are required for protein export to the periplasm, synthesis of the TTQ prosthetic group, and formation FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS Cofactor biogenesis in TTQ-dependent AADH 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 biogenesis [8] The genes were placed under the regulatory control of the coxII promoter which, unlike the native mau promoter, was not controlled by methylamine levels [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 nature 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 number 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 promoter of P denitrificans and introduced into P denitrificans 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 identified ionizable groups in the enzyme–substrate complex that control the rate of TTQ reduction One of these 5895 Cofactor biogenesis in TTQ-dependent AADH P Hothi et al 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 control 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 generally produced by A faecalis grown on b-phenylethylamine Given that P denitrificans is known to express MADH when grown on methylamine [18], the periplasmic 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 Experimental 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 tryptamine When P denitrificans lacking plasmid pRKAADH was grown on methylamine, no AADH was detected MADH expression was similar in wildtype P denitrificans and pRKAADH containing P denitrificans Characterization of recombinant AADH During the purification of recombinant AADH, the elution conditions during ion-exchange chromatography, hydrophobic interaction chromatography and gel filtration were identical to those observed for the native enzyme The purification of recombinant AADH is illustrated in Fig and summarized in Table 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 5896 Fig Purification of recombinant AADH from P denitrificans monitored 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, following DE-52 ion exchange chromatography; lane 4, phenyl Sepharose chromatography; lane 5, pure AADH following gel-filtration chromatography Table Purification of recombinant AADH from Paracoccus denitrificans Purification step Total Total Specific protein activity activity (mg) (units) (unitsỈmg)1) Cell extract 3668 Ammonium sulphate 2580 fractionation DE52 chromatography 128 Phenyl Sepharose 58 Sephacryl S-200 gel filtration 17 1427 1172 1012 884 524 0.38 0.45 7.9 15.2 30.8 Yield (%) 100 82 71 61 37 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 corrected sequence presented in Supplementary Material.) N-Terminal sequence analysis of both native and recombinant b subunits indicated that the first six residues are Ala-Gly-Gly-Gly-Gly-Ser The mature protein product is therefore truncated by 47 amino acids compared 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, suggesting 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 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS P Hothi et al predicted site for removal of the periplasmic localization 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 Cofactor biogenesis in TTQ-dependent AADH 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 reductive half-reaction (Table 2) 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 comparable 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 Fig Spectral and kinetic properties of recombinant AADH (A) Spectral changes accompanying the titration of oxidized enzyme with substrate AADHox (6.5 lM), in 10 mM BisTris propane buffer (pH 7.5), was reduced by the addition of benzylamine under anaerobic 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 activity; open circles, deuterated benzylamine-dependent activity Reactions were performed using lM enzyme (reaction cell concentration) in 10 mM 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) Photodiode array studies of enzyme reduction AADHox (4 lM) contained in 10 mM BisTris propane buffer, pH 7.5, was rapidly mixed with 200 lM protiated or deuterated benzylamine (reaction cell concentrations) at 25 °C Spectral changes accompanying enzyme reduction 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) FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS 5897 Cofactor biogenesis in TTQ-dependent AADH P Hothi et al Table 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 klim (s)1) Deuterated benzylamine klim (s)1) Protiated benzylamine Kd (lM) Deuterated benzylamine Kd (lM) KIE Native Recombinant 1.47 ± 0.01 1.54 ± 0.02 0.32 ± 0.01 0.36 ± 0.01 10.38 ± 0.25 10.8 ± 0.6 10.4 ± 0.29 12.7 ± 0.6 4.6 ± 0.2 4.3 ± 0.2 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 previously, 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 therefore performed as described in Experimental procedures Given that single buffers were used to determine pH profiles, points were confirmed by overlapping the pH ranges of the different buffers Owing to limitations 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 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 kinetically influential macroscopic ionization in the free enzyme, and most likely the presence of two ionizations (Fig 4D) The relatively large increase in 5898 klim ⁄ Kd at pH 10 (Fig 4D, inset) needs to be interpreted with caution owing to the poor buffering capacity of BisTris propane at this pH Analysis of the data (omitting the pH 10 data point) using the equation for a double ionization yielded a pKa1 value of 7.1 ± 0.2 and pKa2 value of 9.3 ± 0.2 for protiated benzylamine With deuterated benzylamine the corresponding 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 reported 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 significantly 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 Apparent 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 reactions with benzylamine [% 2.5 in the presence of mm phenazine ethosulfate (PES) and % 2.0 with 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 subFEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS P Hothi et al Cofactor biogenesis in TTQ-dependent AADH Fig The pH dependence of TTQ reduction in AADH with protiated and deuterated benzylamine Individual parameters determined from curve fitting to plots of observed rate (kobs) against substrate concentration are shown in Table (A) Data set collected at pH 8.0 (B) Plot of Kd vs 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 (pKa 5.65 ± 0.15) The errors associated with the pKa values are those determined from curve fitting (D) Plot of klim ⁄ Kd 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, deuterated benzylamine dependent activity Analysis of the data (omitting the pH 10 data point) using the equation for a double ionization yielded a pKa1 value of 7.1 ± 0.2 and pKa2 value of 9.3 ± 0.2 for protiated benzylamine With deuterated benzylamine the corresponding values were pKa1 7.0 ± 0.2 and pKa2 11.1 ± 0.4 Inset, plot of klim ⁄ Kd including data collected at pH 10 and fits to the equation for a double ionization Conditions: lM native AADH, various buffers as described in Experimental procedures, at 25 °C strate for AADH in steady-state reactions is unclear However, we have observed that detection of activity with the ‘slow’ substrate benzylamine requires a substantially higher enzyme concentration (% 50 nm) than with those assays performed with the ‘fast’ substrate tryptamine (% 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), FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS suggesting competition between PES and benzylamine at a common binding site This might account for, or contribute to, the apparent lack of benzylaminedependent activity reported by Hyun and Davidson [21] Effects of substrate concentration on initial velocity profiles Previous studies have established that substrate inhibition occurs during steady-state reactions of AADH 5899 Cofactor biogenesis in TTQ-dependent AADH P Hothi et al Table 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 klim and Kd were determined by fitting data to the standard hyperbolic expression pH k lim H (s)1) K d H (lM) k lim D (s)1) K d D (lM) KIE 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 0.36 0.56 0.92 1.2 1.45 1.53 1.56 1.57 1.54 1.52 1.7 381.9 228.2 118.1 58.4 29.87 17.45 14.69 12.0 9.97 8.08 3.91 0.13 0.18 0.26 0.3 0.34 0.35 0.34 0.34 0.32 0.31 0.33 418.2 263.9 118.3 48.8 31.83 15.62 11.92 12.58 10.33 6.68 4.11 2.8 3.1 3.5 4.0 4.3 4.4 4.6 4.6 4.8 4.9 5.15 ± ± ± ± ± ± ± ± ± ± ± 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.02 0.02 0.02 0.02 ± ± ± ± ± ± ± ± ± ± ± 24 8.5 4.0 2.4 2.0 0.9 0.8 0.8 0.6 0.6 0.3 with aromatic amines such as tyramine, b-phenylethylamine and tryptamine [1,22] In a previous report, data collected with tyramine [1] were fit to the following equation: m¼ Vmax ẵS Km ỵ ẵS ỵ ẵS2 =Ki 1ị 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)   bẵS Vmax 1ỵ Ki 2ị mẳ Ks Ks ẵS 1ỵ ỵ ỵ ẵS Ki Ki where KS and Ki are the Michaelis and inhibition constants for substrate, respectively Vmax is 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 breakage 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 5900 ± ± ± ± ± ± ± ± ± ± ± 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 ± ± ± ± ± ± ± ± ± ± ± 17 14 3.6 1.4 1.0 0.9 0.9 0.8 0.3 0.3 0.3 ± ± ± ± ± ± ± ± ± ± ± 0.29 0.23 0.17 0.16 0.19 0.18 0.16 0.19 0.20 0.19 0.26 Stopped-flow studies of the oxidative half-reaction with PES To investigate the kinetics of the oxidative halfreaction, AADH was reduced stoichiometrically with benzylamine and rapidly mixed with different concentrations of PES under anaerobic conditions (Fig 7) Transients were followed at 483 nm, which is an isosbestic point for PES but also a wavelength at which there is reasonable absorbance from the TTQ cofactor At mm PES the rate of enzyme oxidation is % 35 s)1 at 25 °C At 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 and 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 steadystate 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, FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS P Hothi et al Cofactor biogenesis in TTQ-dependent AADH were obtained by fitting the Eyring equation to the data and are summarized in Table Analysis of the temperature dependence of reaction rates with protiated and deuterated benzylamine provides a sensitive test for the functional equivalence of native and recombinant AADH We infer, therefore, that both enzymes are identical in their functional properties and that the TTQ cofactor is assembled correctly in the recombinant enzyme Discussion Fig 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 described in Experimental procedures with 50 nM AADH and mM PES in 10 mM BisTris propane buffer, pH 7.5, at 25 °C Filled circles, protiated benzylamine-dependent activity; open circles, deuterated benzylamine-dependent activity Similar plots were collected in the presence of mM PES (data not shown) Apparent Michaelis constants were determined by fitting initial velocity data to the Michaelis–Menten equation Similar data were also collected for native AADH (not shown) (B) Initial velocity data as a function of tryptamine concentration Conditions: nM native AADH, mM PES in 10 mM BisTris propane buffer, pH 7.5, at 25 °C Filled circles, protiated tryptamine-dependent activity; open circles, deuterated tryptamine-dependent activity Fits to Eqn (1) (solid line) and Eqn (2) (dashed line) are shown Kinetic parameters determined from fitting to Eqn (1) are: kcat (s)1), 54.4 ± 2.5; Ks (lM), 1.3 ± 0.13; Ki (lM), 26 ± 3.3 Similar plots were collected in the presence of mM PES (data not shown) and for recombinant AADH (data not shown) FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS 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 similar to mauD and mauE, respectively, from the methylamine 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 probably not involved in the oxidation of aromatic amines in A faecalis [7] Of the remaining ORFs, sequence similarity 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 uroporphyrinogen decarboxylase Here, we have described the heterologous expression of functional TTQ-dependent 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 denitrificans using a broad-host-range vector The successful production of active enzyme suggests that orf-1, orf-3, orf-4 and hemE are not required for the biosynthesis of AADH, consistent with there being no inferred biological function in TTQ biogenesis for the polypeptides 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, 5901 Cofactor biogenesis in TTQ-dependent AADH P Hothi et al Table 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] (mM) Protiated benzylamine kcat (s)1) Deuterated benzylamine kcat (s)1) Protiated benzylamine Km (lM) Deuterated benzylamine Km (lM) KIE Native Recombinant Native Recombinant 1 5 1.14 1.02 1.23 1.03 0.46 0.35 0.55 0.49 6.7 9.9 14.4 13.1 12.7 13.2 12.6 14.3 2.5 2.9 2.2 2.1 ± ± ± ± 0.01 0.02 0.01 0.02 ± ± ± ± 0.01 0.01 0.01 0.02 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 denitrificans 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 similar to those of the native enzyme purified from A faecalis Our studies have shown that benzylamine is a substrate in multiple turnover assays and stopped-flow mixing reactions Unlike with fast substrates (e.g tryptamine 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 mechanistic reasons for the smaller KIEs seen with benzylamine 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 measured 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 consistent with TTQ reduction being partially rate limiting in steady-state turnover Our studies of the pH dependence of TTQ reduction by benzylamine have indicated that a single kinetically influential ionization of pKa 6.0 controls the rate of TTQ reduction The crystal structure of AADH indicates the presence of only two ionizable 5902 ± ± ± ± 0.1 0.7 0.3 0.5 ± ± ± ± 0.5 0.7 0.8 0.7 ± ± ± ± 0.2 0.3 0.1 0.2 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 deprotonated in the reactive iminiquinone enzyme–substrate complex We suggest that the ionizable group of pKa 6.0 represents the deprotonation of this aspartate residue 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 ionization observed in pH-dependent studies with benzylamine 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 deprotonation 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 pKa of 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 pKa 7.1 has a substantial affect on the affinity of the enzyme for substrate In the protonated form, the Kd for the enzyme substrate complex increases substantially over that seen in the deprotonated form of the enzyme (% 20-fold on moving from pH 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 protonated 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 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS P Hothi et al Cofactor biogenesis in TTQ-dependent AADH Fig 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 concentrations of PES under anaerobic conditions Conditions: AADHred (2 lM), 10 mM BisTris propane buffer, pH 7.5, 25 °C The monophasic 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 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 benzylamine were obtained from Sigma (St Louis, MO) Deuterated benzylamine HCl (C6D5CD2NH2 HCl, 99.6%) and deuterated 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 Fig Apparent inhibition of AADH as a function of PES concentration (A) Initial velocity data as a function of PES concentration Conditions: 50 nM native AADH and 500 lM benzylamine (or deuterated benzylamine) in 10 mM 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 kcat for benzylamine vs PES concentration (C) Plot of apparent Km for benzylamine vs PES concentration FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS Growth of cells and media The bacterial strains and plasmids used in this study are listed in Table 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- 5903 Cofactor biogenesis in TTQ-dependent AADH P Hothi et al Fig Eyring plots for reactions of AADH with benzylamine and deuterated benzylamine (A) Plot of ln kobs ⁄ T vs ⁄ T for the reductive half-reaction of native AADH with benzylamine and deuterated benzylamine Filled circles, protiated benzylamine; open circles, deuterated benzylamine Inset, plot of ln KIE vs ⁄ T Conditions: lM AADH, 10 mM BisTris propane buffer, pH 7.5 Rate constants are observed rate constants measured at 200 lM 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 exponential expression (C) Plot of ln vi ⁄ T vs ⁄ T for steady-state reactions of native AADH with benzylamine and deuterated benzylamine Filled circles, protiated benzylamine; open circles, deuterated benzylamine Inset, plot of ln KIE vs ⁄ T Conditions: 100 nM AADH, 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 sion of AADH, A faecalis and P denitrificans were cultured 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 h prior to harvesting When appropriate, antibiotics were added to the following final concentrations: ampicillin, 100 lgỈmL)1; kanamycin, 25 lgỈmL)1; tetracycline, 10 lgỈmL)1 for E coli and tetracycline, lgỈmL)1; rifampicin 20–50 lgỈmL)1 for P denitrificans 5904 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 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 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS P Hothi et al Cofactor biogenesis in TTQ-dependent AADH Table Kinetic and thermodynamic parameters determined from stopped-flow and steady-state reactions of AADH with protiated and deuterated benzylamine Parameters were obtained from fitting the data shown in Fig to the Eyring equation Enzyme DHàH (kJỈmol)1) DHàD (kJỈmol)1) DDHà (kJỈmol)1) A’H: A’D KIE 63.6 ± 0.6 62.0 ± 1.1 65.0 ± 0.7 61.4 ± 1.1 1.4 ± 0.03 0.6 ± 0.02 2.7 ± 0.07 5.3 ± 0.26 4.7 ± 0.1 4.3 ± 0.2 DHàH (kJỈmol)1) DHàD (kJỈmol)1) DDHà (kJỈmol)1) A’H: A’D KIE at 24 °C 49.6 ± 0.6 48.2 ± 1.1 55.4 ± 0.7 55.2 ± 0.9 5.8 ± 0.1 7.0 ± 0.3 0.24 ± 0.01 0.15 ± 0.01 2.5 ± 0.2 2.5 ± 0.3 Temp range (°C) Reductive half-reaction Native 4–40 Recombinant 4–40 Enzyme Temp range (°C) Steady-state reactions Native 4–40 Recombinant 4–40 Fig Strategy for the construction of plasmid, pRKAADH, used in the heterologous expression of AADH The aau gene region of A faecalis consists of nine genes (orf-1, aauBEDA, orf-2, orf-3, orf-4 and hemE) [7] Genes are represented by their corresponding letter, numbers denote orfs 1–4, and hE is an abbreviation for hemE The mauF promoter and mauR gene from P denitrificans are represented by mFp and mR, respectively Open boxes, periplasmic proteins; shaded boxes, cytoplasmic proteins; diagonally hatched boxes, membrane proteins Arrows indicate the direction of transcription genomic DNA using forward (5¢-GGAGGGATCCCATATG AAGTCTAAATTTAAATTAACG-3¢) and reverse (5¢-GC primers GTGCTCGAGCGATCCATGGAGCCGTA-3¢) 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 (Invitrogen) as a BamHI–XhoI fragment, generating plasmid pKAK01 The P denitrificans mauF promoter and a neigh- FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS bouring ORF (mauR) coding for a transcriptional activator of the mauF promoter were amplified from genomic DNA using forward (5¢-TTGGTAAGCTTGGGCATTTCTGAT CGGGTCGC-3¢) and reverse (5¢-AAACCATATGACGCC 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, 5905 Cofactor biogenesis in TTQ-dependent AADH P Hothi et al Table Bacterial strains and plasmids used in the heterologous expression of AADH in Paracoccus denitrificans Source or reference Strain or plasmid Relevant features Bacteria A faecalis P denitrificans E coli strain DH5 E coli S17-1 Wild-type Wild-type, Rifr General cloning strain Conjugative donor ATCC OF1144 ATCC13543 Invitrogen Laboratory strain TA cloning vector General cloning vector pCDNAII, aauBEDAorf-2 pCDNA II, mauFR::aauBEDAorf-2 Broad-host-range vector pRK415-1, mauFR::aauBEDAorf-2 Invitrogen Invitrogen This study This study Plasmids pCR 4-TOPO pCDNA II pKAK01 pKAK02 pRK415-1 pRKAADH [24] This study 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 rifampicinresistant P denitrificans cells in Luria–Bertani media for h at 30 °C, plated on Luria–Bertani media without antibiotic selection and then incubated for a further h Cells were scraped off the Luria–Bertani plates, washed once with Sistroms’ medium [25] and then plated on Luria–Bertani plates containing lgỈmL)1 tetracycline and 20 lgỈmL)1 rifampicin to select for exconjugates tained in 10 mm 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 containing 35% ammonium sulfate, pH 7.5 Enzyme was eluted using a 35 to 0% ammonium sulfate gradient Fractions containing AADH were pooled, concentrated by ultrafiltration, 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 potassium 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 °C in 10 mm potassium phosphate 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 BisTris propane buffer, pH 7.5, by gel exclusion chromatography 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 % 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 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 produce the mass spectrum 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-phenylethylamine 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 harvested 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., °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 equilibrated with the same buffer AADH was eluted with 200 mm NaCl using a salt gradient (0–250 mm NaCl con- 5906 Anaerobic titrations of oxidized AADH The following procedure was performed in a Belle Technology (Portesham, UK) glove box (< p.p.m oxygen) and buffer was made anaerobic by bubbling with argon for h and left to equilibrate overnight in the glove box Anaerobic solutions of substrate were prepared by dissolving preweighed solid in anaerobic buffer Enzymes were reoxidized with potassium ferricyanide and exchanged into 10 mm BisTris propane buffer, pH 7.5, by gel-exclusion chromatography Enzymes were reduced by the addition of benzylamine 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 cm light path in 10 mm BisTris propane buffer, pH 7.5, FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS P Hothi et al at 25 °C in a total volume of 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 UV-visible spectrophotometer The reaction mixture typically contained 50 nm AADH (for benzylamine-dependent reactions) or nm (for tryptamine-dependent reactions), 0.04 mm DCPIP and 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 velocity data collected as a function of tryptamine concentration [S] were fitted to Eqn (2) Equation has previously been applied in the determination of steady-state kinetic parameters 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 (reaction cell concentration lm) in 10 mm BisTris propane buffer, 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 collected 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), absorbance 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 analysed using the single exponential expression For multiple wavelength studies of the reductive halfreaction, AADHox (4 lm) contained in 10 mm BisTris propane buffer, pH 7.5, was mixed with 200 lm protiated or deuterated benzylamine (reaction cell concentrations) at FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS Cofactor biogenesis in TTQ-dependent AADH 25 °C Multiple-wavelength absorption studies were carried out using a photodiode array detector and x-scan software (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 breakage, 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 thermometer in the UV ⁄ visible spectrophotometer) Control studies of the concentration dependence of bond cleavage at and 40 °C indicated that the Kd (or Km for steady-state reactions) was not substantially perturbed on changing temperature (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 °C in 50 mm 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 stimulate 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 klim =Kd ẳ Lim1 ỵ Lim2 tempị=temp þ 1Þ ð3Þ In Eqn (3), Lim1 is the lower limit and Lim2 is the upper limit of the curve, klim =Kd ẳ Lim1 ỵ Lim2 temp1ị=temp1 ỵ 1ị Lim2 Lim3ị temp2ị=temp2 ỵ 1ịịị 4ị In Eqn (4), temp1 ¼ alog(pH ) pKa1), temp2 ¼ alog (pH ) pKa2), Lim1 is the lower limit of the curve, Lim is the middle limit of the curve and Lim 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- 5907 Cofactor biogenesis in TTQ-dependent AADH photometer housed in a Belle Technology anaerobic glove box (< p.p.m oxygen) Solutions used were made anaerobic by bubbling with argon for h and left to equilibrate overnight in the glove box Studies of the oxidative half-reaction of the enzyme were performed by rapid mixing of lm AADHred (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 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 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 References Govindaraj S, Eisenstein E, Jones LH, Sanders-Loehr J, Chistoserdov AY, Davidson VL & Edwards SL (1994) Aromatic amine dehydrogenase, a second tryptophan tryptophylquinone enzyme J Bacteriol 176, 2922–2929 Edwards SL, Davidson VL, Hyun YL & Wingfield PT (1995) Spectroscopic evidence for a common electron transfer pathway for two tryptophan tryptophylquinone enzymes J Biol Chem 270, 4293–4298 Hyun YL & Davidson VL (1995) Electron transfer reactions between aromatic amine dehydrogenase and azurin Biochemistry 34, 12249–12254 Masgrau L, Roujeinikova A, Johannissen L, Basran J, Ranaghan K, Hothi P, Mulholland A, Sutcliffe M, Scrutton N & Leys D (2005) Atomic description of an enzyme reaction dominated by hydrogen tunnelling, submitted Vellieux FM, Huitema F, Groendijk H, Kalk KH, Jzn JF, Jongejan JA, Duine JA, Petratos K, Drenth J & Hol WG (1989) Structure of quinoprotein methylamine dehy˚ drogenase at 2.25 A resolution EMBO J 8, 2171–2178 McIntire WS, Wemmer DE, Chistoserdov A & Lidstrom ME (1991) A new cofactor in a prokaryotic enzyme: tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase Science 252, 817–824 Chistoserdov AY (2001) Cloning, sequencing and mutagenesis of the genes for aromatic amine dehydrogenase from Alcaligenes faecalis and evolution of amine dehydrogenases Microbiology 147, 2195–2202 Graichen ME, Jones LH, Sharma BV, van Spanning RJ, Hosler JP & Davidson VL (1999) Heterologous expression of correctly assembled methylamine dehydrogenase in Rhodobacter sphaeroides J Bacteriol 181, 4216–4222 5908 P Hothi et al Jones L, Pearson AR, Tang Y, Wilmot C & Davidson V (2005) J Biol Chem 280, 17392–17396 10 Wang Y, Graichen ME, Liu A, Pearson AR, Wilmot CM & Davidson VL (2003) MauG, a novel diheme protein required for tryptophan tryptophylquinone biogenesis Biochemistry 42, 7318–7325 11 Pearson AR, 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indophenol Biochim Biophys Acta 86, 194–197 27 Falzon L & Davidson VL (1996) Kinetic model for the regulation by substrate of intramolecular electron transfer in trimethylamine dehydrogenase Biochemistry 35, 2445–2452 28 Basran J, Chohan KK, Sutcliffe MJ & Scrutton NS (2000) Differential coupling through Val-344 and Tyr-442 of trimethylamine dehydrogenase in electron transfer reactions with ferricenium ions and electron transferring flavoprotein Biochemistry 39, 9188–9200 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS Cofactor biogenesis in TTQ-dependent AADH 29 Harris TK & Davidson VL (1993) A new kinetic model for the steady-state reactions of the quinoprotein methanol dehydrogenase from Paracoccus denitrificans Biochemistry 32, 4362–4368 30 Hothi P, Basran J, Sutcliffe MJ & Scrutton NS (2003) Effects of multiple ligand binding on kinetic isotope effects in PQQ-dependent methanol dehydrogenase Biochemistry 42, 3966–3978 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 5909 ... 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... tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase Science 252, 817–824 Chistoserdov AY (2001) Cloning, sequencing and mutagenesis of the genes for aromatic amine dehydrogenase. .. Davidson VL & Wilmot CM (2004) Further insights into quinone cofactor biogenesis: probing the role of mauG in methylamine dehydrogenase tryptophan tryptophylquinone formation Biochemistry 43,