Mechanistic aspects and redox properties of hyperthermophilic L-proline dehydrogenase from Pyrococcus furiosus related to dimethylglycine dehydrogenase⁄oxidase Phillip J Monaghan, David Leys and Nigel S Scrutton Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, UK Keywords amine oxidation; flavoprotein; hyperthermophile; mechanism; proline dehydrogenase Correspondence N S Scrutton, Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK Fax: +44 161 275 5586 Tel: +44 161 275 5632 E-mail: nigel.scrutton@manchester.ac.uk (Received January 2007, revised February 2007, accepted 19 February 2007) doi:10.1111/j.1742-4658.2007.05750.x Two ORFs encoding a protein related to bacterial dimethylglycine oxidase were cloned from Pyrococcus furiosus DSM 3638 The protein was expressed in Escherichia coli, purified, and shown to be a flavoprotein amine dehydrogenase The enzyme oxidizes the secondary amines l-proline, l-pipecolic acid and sarcosine, with optimal catalytic activity towards l-proline The holoenzyme contains one FAD, FMN and ATP per ab complex, is not reduced by sulfite, and reoxidizes slowly following reduction, which is typical of flavoprotein dehydrogenases Isolation of the enzyme in a form containing only FAD cofactor allowed detailed pH dependence studies of the reaction with l-proline, for which a bell-shaped dependence (pKa values 7.0 ± 0.2 and 7.6 ± 0.2) for kcat ⁄ Km as a function of pH was observed The pH dependence of kcat is sigmoidal, described by a single macroscopic pKa of 7.7 ± 0.1, tentatively attributed to ionization of l-proline in the Michaelis complex The preliminary crystal structure of the enzyme revealed active site residues conserved in related amine dehydrogenases and potentially implicated in catalysis Studies with H225A, H225Q and Y251F mutants ruled out participation of these residues in a carbanion-type mechanism The midpoint potential of enzyme-bound FAD has a linear temperature dependence () 3.1 ± 0.05 mVỈC°)1), and extrapolation to physiologic growth temperature for P furiosus (100 °C) yields a value of ) 407 ± mV for the two-electron reduction of enzyme-bound FAD These studies provide the first detailed account of the kinetic ⁄ redox properties of this hyperthermophilic l-proline dehydrogenase Implications for its mechanism of action are discussed The membrane-associated flavoprotein PutA in enteric bacteria is a proline catabolic enzyme that catalyzes the oxidation of proline to glutamate in a two-step reaction to form glutamate (Fig 1) The protein is also a transcriptional repressor of the proline utilization (put) genes [1–3] Cytoplasmic PutA represses transcription from its own gene and also from the Na+ ⁄ proline transporter PutP [4–6] Proline catabolism enables enteric bacteria to use l-proline as a source of carbon, nitrogen and electrons, and the reaction is initiated in the FAD-binding domain by two-electron oxidation of l-proline to form D1-pyrroline-5-carboxylate (P5C) [5,6] Following oxidation of l-proline, the two-electron reduced FAD cofactor passes electrons to an acceptor in the electron transfer chain The intermediate P5C is hydrolyzed to glutamate 5-semialdehyde, which is then oxidized to glutamate by the P5C dehydrogenase domain, with NAD+ acting as electron Abbreviations DMGO, dimethylglycine oxidase; P5C, D1-pyrroline-5-carboxylate; PRODH, L-proline dehydrogenase; TAPSO, 3-{[tris(hydroxymethyl)methyl]amino}-2-hydroxypropane sulfonic acid; TMADH, trimethylamine dehydrogenase 2070 FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS L-proline P J Monaghan et al dehydrogenase from P furiosus Fig Reactions catalyzed by the bifunctional PutA protein of enteric bacteria Hydrolysis of P5C is nonenzymatic In eukaryotes, distinct enzymes encoded by separate genes catalyze these reactions acceptor (Fig 1) The structure of a truncated form of PutA comprising the FAD-containing proline dehydrogenase domain has been elucidated, and reveals a domain-swapped dimer, with each subunit containing three domains [7] These domains comprise a helical dimerization arm, a three-helix bundle, and a b ⁄ a barrel l-proline dehydrogenase (PRODH) domain A model of the enzyme–substrate complex has been constructed from which a mechanism for the oxidation of l-proline has been proposed [7] In eukaryotes, distinct enzymes encoded by separate genes catalyze the oxidation of l-proline to glutamate A mitochondrial l-proline oxidase is the human homolog of the PutA protein of enteric bacteria, and it has roles in p53mediated apoptosis, the production of reactive oxygen species, and also schizophrenia [8,9] A new type of dye-linked PRODH was recently identified in crude extracts of the hyperthermophile Thermococcus profundus [10] The enzyme is a heterotetramer, contains moles of FAD per mole of enzyme, and is bifunctional, having proline dehydrogenase and dye-linked NADH dehydrogenase activities [10,11] The enzyme is a complex iron–sulfur flavoprotein, with the a subunit containing a 2Fe)2S center Additionally, the c subunit contains a broad absorption peak around 420 nm typical of an 8Fe)8S ferredoxin The a and b subunits show sequence similarity with other putative amine-oxidizing proteins, including the putative sarcosine oxidase a and b subunits of P furiosus Recent studies have also identified a dyelinked d-proline dehydrogenase from Pyrobaculum islandicum [12] In searching for other new classes of PRODH, we have identified two ORFs [annotated gi_18977617 (a subunit) and gi_18977618 (b subunit), in the protein extraction, description and analysis tool (pedant) database] in the genome of P furiosus DSM 3638 that show sequence homology to the a and b subunits of the flavoprotein amine oxidoreductase, tetrameric sarcosine oxidase [13] The translated amino acid sequence of gi_18977618 (b subunit) also aligns with another member of the amine oxidoreductase family, dimethylglycine oxidase (DMGO) from Arthrobacter globiformis [13], for which the crystal ˚ structure has been determined to 1.6 A resolution [14] This alignment indicates the conservation of three residues (His225, Tyr251 and Gly262 in the gi_18977618 translation), known to reside in the active site of DMGO, that have been implicated in the catalytic mechanism of dimethylglycine oxidation [14] The crystal structure of a related enzyme from Pyrococcus horikoshii OT-3 has been elucidated [15], and its basic solution properties have been analyzed [16], but, to date, detailed mechanistic studies of the activity of this or related enzymes have not been reported With this aim in mind, we present an analysis of recombinant protein expressed from the two ORFs found in P furiosus DSM 3638 that share sequence similarity with the gene encoding DMGO We show that these genes encode a new member of the hyperthermophilic class of PRODHs that couples the oxidation of l-proline to the reduction of a noncovalently bound FAD and the production of P5C Oxidation of enzyme-bound FADH2 is accomplished using the artificial electron acceptor ferricenium hexafluorophosphate, but not molecular oxygen, NAD+, or pyrococcal 4Fe)4S ferredoxin This article represents the first detailed report of the kinetic and spectroscopic properties of this novel type of PRODH Results Analysis of a and b subunit sequences Sequence analysis of both subunits suggests the presence of an ADP-binding motif in the N-terminal region of the a subunit, with the 11 participating FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS 2071 L-proline dehydrogenase from P furiosus P J Monaghan et al residues satisfying the physicochemical parameters of the consensus [17] The a subunit also contains a conserved GG doublet five nucleotides downstream of the dinucleotide-binding domain An ATG motif is also evident in the a subunit This motif has been found in both FAD-binding and NADPH-binding proteins, where it forms the fourth b-strand of the Rossman fold and the connecting loop In flavoproteins, the ATG motif has a defined function, in that it is always present at the junction with the substrate-binding domain, and not within a domain, as in NADPHbinding proteins [18] The b subunit also contains an ADP-binding motif, and shares 27% sequence identity with the b subunit of tetrameric sarcosine oxidase from Corynebacterium sp P-1, and 26% sequence identity with the N-terminal half of human dimethylglycine dehydrogenase Of particular note is the finding that the b subunit of PRODH shows sequence conservation with active site residues His225, Tyr259 and Gly270 of DMGO from A globiformis and mouse lung dimethylglycine dehydrogenase (Fig 2), residues that are present in a number of sarcosine dehydrogenase-like proteins Given these sequence similarities, we conjectured that the protein encoded by the two identified A B Fig Multiple sequence alignments of amino acid sequences of the a and b subunits of PRODH (A) Multiple sequence alignment for the a subunit, showing 18% sequence identity with the N-terminal region of the a subunit of tetrameric sarcosine oxidase from both Corynebacterium sp P-1 [20] and Arthrobacter sp 1-IN [13] The 11 residues that comprise the ADP-binding motif are highlighted in bold, and shaded where residues are conserved All 11 residues in PRODHa obey the physicochemical requirements established by Wierenga et al [17] The conserved GG doublet and ATG motif are also shaded (B) Multiple sequence alignment deduced for the b subunit of PRODH, showing 24% sequence identity with DMGO from A globiformis [13] and 26% sequence identity with the cDNA translation product of dimethylglycine dehydrogenase from M musculus lung tissue [21] The N-terminal ADP-binding motif is highlighted in bold, and shaded where residues are conserved Again, all 11 residues satisfy the consensus sequence, with the exception of the glutamate residue at position 1, although this hydrophilic residue has the correct physicochemical requirements for this position DMGO active site residues His225, Tyr259 and Gly270, identified from the crystal structure, align with conserved residues in both dimethylglycine dehydrogenase and PRODHb, and are highlighted with bold type and shading Additional conserved residues are marked with an asterisk 2072 FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS L-proline P J Monaghan et al ORFs in Pyrococcus furiosus encode a new type of amine dehydrogenase ⁄ oxidase, a hypothesis that we addressed through detailed characterization of the protein as a new type of (PRODH), described below dehydrogenase from P furiosus A Purification of recombinant enzyme and general properties Recombinant wild-type enzyme was expressed at high levels in Escherichia coli strain Rosetta(DE3)pLysS transformed with either plasmid pPRODH1 or plasmid pPRODH2 The enzyme was purified to homogeneity in three steps and in the oxidized form (Fig 3A) The holoenzyme form of PRODH was selected for crystallogenesis and X-ray diffraction studies, and was purified as previously described [19] PRODH for mechanistic studies was further exchanged into 100 mm potassium phosphate buffer (pH 7.5) This treatment releases the ATP and FMN cofactors from the protein, but leaves the FAD-bound form This is a convenient form of the enzyme for simplified analysis of FAD reduction (see below) The protein yield was typically $ 10 mg of purified enzyme per liter of recombinant culture The purified enzyme was found to be yellow in color, and had a typical flavoprotein absorbance spectrum characterized by two flavin peaks with absorption maxima at 367 and 450 nm (Fig 3B) N-terminal sequence analysis of the a and b subunits purified from pPRODH1 indicated that a subpopulation (approximately $ 13%) of the a subunit was truncated The sequence MKVQRQ was obtained for the truncated a subunit N-terminus, indicating that truncation in the a subunit is located 83 amino acids from the initiating methionine in the full-length a subunit Enzyme expressed from plasmid pPRODH2 lacked a truncated a subunit, consistent with removal of the internal ribosome-binding site by mutagenesis The a and b subunits were coexpressed in a molar ratio of $ : from pPRODH2, as judged by SDS ⁄ PAGE peak area image scanning Analysis of purified enzyme by MALDI-TOF MS gave a molecular mass of 42 437.5 Da for the b subunit, comparable to the predicted molecular mass of 42 481.2 Da from the gene sequence Electrospray mass data for the b subunit gave a molecular mass of 42 474.0 Da Mass data for the larger a subunit could not be obtained using the MALDI-TOF or electrospray methods Purification of H225A, H225Q and Y251F mutant enzymes was as described for wild-type PRODH Mutagenesis of these active site residues had no effect on recombinant protein yield, and all mutants were purified in FADbound form Despite the close proximity of both His225 and Tyr251 to the isoalloxazine ring moiety of B Fig (A) SDS ⁄ PAGE analysis of the purification of recombinant wild-type PRODH from E coli strain Rosetta(DE3)pLysS transformed with pPRODH2 Lane 1: molecular mass marker (97, 66, 45, 30 and 20.1 kDa from top to bottom of the gel) Lane 2: cell lysate Lane 3: sample after heat denaturation at 80 °C and clarification by centrifugation Lane 4: pooled fractions following anion exchange chromatography (Q-Sepharose) Lane 5: pooled fractions following size-exclusion chromatography (Superdex 75) showing the pure a and b subunits of PRODH (B) UV-visible absorption spectrum and reductive titration of recombinant wild-type PRODH with sodium dithionite The absorption spectrum recorded between 300 and 600 nm is typical of a flavoprotein spectrum Flavin peaks are at 367 and 450 nm, and the arrow indicates the direction of absorption change A single isosbestic point was observed at 340 nm Inset: a plot of absorbance at 450 nm versus electron equivalents, which demonstrates that reduction of FAD-bound PRODH is complete following addition of two electrons Conditions: 100 mM potassium phosphate buffer, pH 7.5; 25 °C; enzyme concentration 16 lM FAD, no major perturbations in the absorption properties of the enzyme-bound flavin were evident as a consequence of mutagenesis Holoenzyme cofactor content Our preliminary crystallographic analysis of the enzyme indicates a heterooctomer (ab)4 structure for PRODH, as initially suggested from the computed self-rotation of diffraction data [19] It is evident from FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS 2073 L-proline dehydrogenase from P furiosus P J Monaghan et al A B H225β Y251β FAD FAD FMN ATP the preliminary X-ray crystal structure of PRODH that one molecule of ATP cofactor is bound in the ADP-binding motif of the a subunit (Fig 4A) This cofactor has no obvious function from a mechanistic perspective, but may play a stabilizing role under the harsh physiologic conditions that P furiosus is subject to The ADP-binding motif in the b subunit binds one molecule of noncovalent FAD (Fig 4B) FMN is located at the interface of the a and b subunits (Fig 4A) Reductive titration of PRODH highlighted a single isosbestic point at 340 nm, with no evidence for a semiquinone species obtained during titration with sodium dithionite (Fig 3B) FAD-bound PRODH for mechanistic studies was confirmed by MALDI-TOF MS after heat treatment to remove cofactor A mass ⁄ charge peak at 787 corresponded to the positively charged quasimolecular ion ([M + H]+) of FAD Partial hydrolysis of FAD during heat treatment was revealed by mass ⁄ charge peaks at 348 and 458, assigned to AMP and FMN ([M + H]+) hydrolysis products, respectively ATP cofactor was not detected (Fig 5), although the preliminary crystal structure of PRODH indicates its presence in the enzyme Reduction with amine substrates Alignment of the a and b subunit sequences with wellcharacterized enzymes suggests that the purified enzyme is an amine-specific oxidoreductase (Fig 2) In particular, the a subunit shows 18% identity with the N-terminal region of the a subunit of tetrameric sarcosine oxidase from Corynebacterium sp P-1 [20] and Arthrobacter sp 1-IN [13] The b subunit shows 24% identity with DMGO from A globiformis [13] and 26% sequence identity with dimethylglycine dehydrogenase from Mus musculus [21] Given these sequence similarities, amine compounds were analyzed as 2074 Fig Preliminary P furiosus PRODH crystal structure (A) Omit maps (in blue) superimposed on the bound FMN (green sticks), FAD (yellow sticks) and ATP (magenta sticks) cofactors of the heterotetrameric PRODH (represented by gray ribbons) The electron density map is contoured at 3r (B) Position of the active site residues His225b and Tyr251b with respect to the FAD isoalloxazine group potential substrates by mixing with enzyme (19.4 lm) at 80 °C under anaerobic conditions to preclude potential oxidase chemistry Enzyme–substrate reactivity was established by following bleaching of the flavin spectrum on addition of the amine compound (20 mm) The enzyme was found to oxidize only secondary amine compounds, namely sarcosine, l-proline, and l-pipecolic acid (Fig 6); l-proline was most effective as reducing substrate (t1 ⁄ ¼ $ 105 s), followed by l-pipecolic acid (t1 ⁄ ¼ $ 110.5 s; a structural analog of l-proline), and sarcosine (t1 ⁄ ¼ $ 654 s) Glycine betaine, glycine, dimethylglycine and d-proline did not lead to significant flavin reduction The common structural link between these identified substrates for PRODH is that they are all secondary a-amino acids (Fig 6) The ability of P furiosus PRODH to oxidize multiple amine compounds is in stark contrast to the catalytic properties reported for dye-linked proline dehydrogenase of Pyrococcus horikoshii OT-3, which has been shown to act exclusively on l-proline, with l-pipecolic acid and sarcosine being inert as substrates [16] A spectral feature is apparent at $ 550 nm upon kinetic reduction of PRODH with each of the three identified amine substrates This signal may represent a minor transient population of a charge-transfer species during the catalytic reaction Addition of sodium sulfite (50 mm) to purified enzyme did not perturb the flavin absorption spectrum, indicating that a flavin–N5–sulfite adduct does not form This suggests that the enzyme is not a flavoprotein oxidase, as reactivity with sulfite is a characteristic of this class of flavoenzyme [22] Steady-state turnover analysis with L-proline and L-pipecolic acid In developing a suitable and continuous turnover assay for wild-type enzyme at an elevated temperature FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS L-proline P J Monaghan et al dehydrogenase from P furiosus A B C D Fig MALDI-TOF MS of flavin cofactor released from PRODH (FAD-bound form) (A) Authentic FAD showing positively charged quasimolecular ion of FAD ([M + H]+) corresponding to the m ⁄ z peak of 787 and the FAD–Na+ adduct with an m ⁄ z peak at 809 (B) Authentic FMN showing both [M + H]+ ion and FMN–Na+ adduct m ⁄ z peaks at 458 and 480, respectively (C) Authentic AMP showing both the [M + H]+ ion and AMP–Na+ adduct m ⁄ z peaks at 348 and 370, respectively (D) Released cofactor of heat-denatured PRODH showing the [M + H]+ ion of FAD cofactor with an m ⁄ z peak of 787 identical to that of the authentic FAD standard The m ⁄ z peak at 825 represents the K+ adduct of released FAD cofactor The m ⁄ z peaks of 348 and 458 represent the [M + H]+ ion of both AMP and FMN, respectively, which result from partial hydrolysis of FAD cofactor during protein heat treatment The m ⁄ z peak of 496 represents the K+ adduct of the FAD cofactor heat hydrolysis product, FMN K+ ions are present from the purification buffers Conditions: samples of authentic FAD, FMN and AMP were prepared as mgỈmL)1 stock solutions in double deionized H2O, and filtered using a 0.22 lm Acrodisc; PRODH was exchanged into double deionized H2O (80 °C), a number of issues were taken into account A continuous assay was chosen, because coupled assays are usually inappropriate, owing to a lack of suitable accessory enzymes that are stable at elevated temperature Assays were performed in 100 mm potassium phosphate buffer (pH 7.5), which has a low temperature coefficient [d(pKa) ⁄ dT ¼ ) 0.0028] [23,24], and ferricenium hexafluorophosphate (200 lm) was used as electron acceptor Steady-state assays were performed at 80 °C with both l-proline and l-pipecolic acid as substrate A comparison of steady-state turnover under aerobic and anaerobic conditions indicated that oxygen did not affect turnover reaction rates, consistent with the enzyme not being of the oxidase class Consequently, steady-state kinetic parameters were determined under aerobic conditions Analysis of hyperbolic plots of initial velocity as a function of substrate concentration yielded apparent Km values for the wildtype enzyme of 30.8 ± 1.1 mm and 212.3 ± 17.0 mm for l-proline and l-pipecolic acid, respectively The corresponding apparent kcat values were 18.1 ± 0.2 s)1 and 0.4 ± 0.02 s)1 for l-proline and l-pipecolic acid, respectively, and the calculated specificity constants (kcat ⁄ Km) were 0.59 ± 0.03 s)1Ỉmm)1 (l-proline) and 0.002 ± 0.0002 s)1 mm)1 (l-pipecolic acid) We infer that l-proline is the preferred substrate, and that the enzyme therefore represents a new member of the class of PRODHs that is distinct from E coli PRODH Unlike what was observed for the E coli enzyme, we were unable to show any NAD+ reduction activity for P furiosus PRODH in either multiple-turnover or single-turnover assays, reinforcing functional differences between the P furiosus and E coli enzymes Exogenous FMN did not act as electron acceptor in steady-state FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS 2075 L-proline dehydrogenase from P furiosus P J Monaghan et al B A D C E Fig Absorption changes as a function of reaction time accompanying reduction of wild-type PRODH with sarcosine, L-pipecolic acid, and L-proline (A) Absorption changes following reduction of PRODH (19.4 lM) with sarcosine (20 mM) (B) As for (A), but with L-pipecolic acid (20 mM) (C) As for (A), but with L-proline (20 mM) (D) Plot of absorbance change at 450 nm as a function of time for each of the spectral changes shown in (A)–(C) Symbols: d, sarcosine; m, L-pipecolic; , L-proline Conditions: 100 mM potassium phosphate buffer, pH 7.5; 80 °C (E) Structural formulae for the three secondary amine molecules, sarcosine, L-pipecolic acid, and L-proline, which show substrate reactivity with PRODH Boxed areas illustrate the common moiety suggested to be important for binding in the PRODH active site anaerobic assays Additionally, we were unable to show electron transfer from l-proline-reduced PRODH to P furiosus ferredoxin under anaerobic turnover conditions, either in the presence or in the absence of exogenous FMN Steady-state assays were performed over the temperature range 40–90 °C Plots of initial velocity as a function of l-proline concentration were hyperbolic at all temperatures, and kinetic parameters were calculated by fitting to the Michaelis–Menten equation Both kcat and Km increase with temperature (Table 1) The temperature dependence of PRODH-catalyzed l-proline oxidation was investigated at a saturating l-proline concentration (200 mm) (Fig 7A) Thermodynamic parameters were obtained by fitting to the Eyring equation (Eqn 1) Table Steady-state kinetic parameters for the reaction of PRODH with L-proline determined at different temperatures Conditions: 100 mM potassium phosphate buffer, pH 7.5, at each assay temperature Ink=Tị ẳ In kB =h ỵ DSz =R DH z =RT PRODH at elevated temperatures prior to activity assay showed that the enzyme is extremely stable, with no loss of activity being evident up to 100 °C Above this temperature, thermal denaturation of PRODH is apparent, with complete loss of activity after 10 of incubation in glycerol buffer at temperatures ‡ 115 °C (data not shown) Thus, PRODH from P furiosus is the most thermostable PRODH described to date ð1Þ where kB and h are the Boltzmann and Planck constants, respectively Initial velocity was strongly dependent on temperature (Fig 7B), and analysis of the data using the Eyring plot gave thermodynamic parameters DH ẳ 83.4 2.9 kJặmol)1, DS ẳ 27.2 1.0 Jặmol)1ặ K)1, and DG ẳ 73.3 kJặmol)1 (at 373 K) Incubation of 2076 Temperature (°C) Km (mM) kcat (s)1) 40 45 50 55 60 65 70 75 80 5.6 5.6 5.3 8.0 9.9 11.6 19.9 25.9 30.8 0.4 0.8 1.3 2.5 4.0 5.9 10.4 14.8 18.01 ± ± ± ± ± ± ± ± ± 0.4 0.2 0.4 0.4 0.4 1.0 2.3 1.3 1.1 ± ± ± ± ± ± ± ± ± kcat ⁄ Km (s)1ỈmM)1) 0.005 0.005 0.02 0.02 0.04 0.1 0.4 0.2 0.2 0.08 0.1 0.2 0.3 0.4 0.5 0.5 0.6 0.6 ± ± ± ± ± ± ± ± ± 0.006 0.005 0.02 0.02 0.02 0.05 0.08 0.04 0.03 FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS L-proline P J Monaghan et al dehydrogenase from P furiosus Table Steady-state kinetic parameters determined for the reaction of PRODH with L-proline at different pH values and at constant ionic strength Assays were performed at 60 °C A pH 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 B Fig Temperature dependence and Eyring analysis of initial velocity data for wild-type PRODH reacting with L-proline (A) Threedimensional plot showing initial velocity (y-axis) versus time (x-axis) versus temperature (z-axis) Reactions were performed in the presence of saturating L-proline (200 mM) over the temperature range 40–90 °C The dimension of time demonstrates any potential loss of activity due to enzyme thermal denaturation at elevated temperatures (not observed in the case of PRODH-catalyzed oxidation of L-proline) The three-dimensional plot was generated using SIGMAPLOT v9.0 for Windows Curve-fitting used the Loess transformation to smooth data based on local regression, which applies a tricube weight function to elicit trends from noisy data [45] The trend elicited from the smoothing process was then used to extrapolate data back to time-point zero to compensate for the time lag between addition of enzyme to initiate the reaction and the start of data collection Conditions: 100 mM potassium phosphate buffer, pH 7.5 (pH corrected at each assay temperature) (B) Eyring plot of initial velocity data for PRODH with L-proline as substrate Thermodynamic parameters derived from fitting of data to the Eyring equation are DHà ¼83.4 ± 2.9 kJặmol)1, DS ẳ 27.2 1.0 Jặmol)1ặK)1 z and DG 373 ẳ73.3 kJặmol)1 at 100 C kcat (s)1) 1.10 1.67 2.40 4.08 6.83 11.68 16.36 16.36 16.54 18.00 ± ± ± ± ± ± ± ± ± ± kcat ⁄ Km (s)1ỈmM)1) Km (mM) 0.06 0.03 0.02 0.04 0.14 0.25 0.46 0.25 0.41 1.14 9.96 5.76 1.08 1.27 1.81 5.82 19.54 42.35 87.21 137.97 ± ± ± ± ± ± ± ± ± ± 2.20 0.51 0.08 0.11 0.32 0.65 1.73 1.56 4.19 15.11 0.11 0.29 2.22 3.21 3.76 2.01 0.84 0.39 0.19 0.13 ± ± ± ± ± ± ± ± ± ± 0.03 0.03 0.18 0.31 0.74 0.27 0.10 0.02 0.01 0.02 For mechanistic analyses, steady-state turnover assays were performed aerobically with l-proline at 60 °C over the pH range 5.5–10.0, to identify kinetically influential ionizations Ionic strength across the pH range was kept constant using a three-component buffer system (see Experimental procedures) Kinetic parameters were calculated by fitting to the Michaelis–Menten equation (Table 2) For wild-type PRODH, the pH dependence of kcat ⁄ Km was found to be bell-shaped, and fitting of the data using Eqn (4) (see Experimental procedures) yielded macroscopic pKa values of 7.0 ± 0.2 (acid limb) and 7.6 ± 0.2 (alkali limb) (Fig 8A) Assuming no change in rate-limiting step across the pH range, these macroscopic pKa values most likely represent ionization of residues in the free enzyme By analogy with other amine oxidases ⁄ dehydrogenases that share similarity at the sequence level with PRODH (Fig 2), we speculated that the pKa of 7.0 ± 0.2 may be attributed to ionization of the conserved His225, a potential active site base residue However, this proposal was later refuted in light of kinetic analyses performed with both H225A and H225Q mutant forms (see below) The pH dependence of kcat exhibited a simple sigmoid behavior that, when analyzed by fitting to Eqn (3) (see Experimental procedures) (Fig 8B), produced a macroscopic pKa value of 7.7 ± 0.1 The pKa value for the protonation of free proline is 10.6, but this might be lowered on binding to enzyme in the Michaelis complex by ) 2.9 pH units A precedent for stabilization of the free base form of amine substrates at physiologic pH values is available from studies with trimethylamine dehydrogenase (TMADH) [25], and is consistent with mechanistic proposals that require the unprotonated amine substrate species to react with the enzyme-bound flavin [26] FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS 2077 L-proline dehydrogenase from P furiosus P J Monaghan et al Table Steady-state kinetic parameters determined for the H225A, H225Q and Y251F mutant PRODH forms Conditions: buffer composed of Mes, TAPSO and diethanolamine at final concentrations of 0.052, 0.052 and 0.1 M, respectively, pH 7.5, at an assay temperature of 60 °C A Mutant kcat (s)1) Km (mM) kcat ⁄ Km (s)1ỈmM)1) H225A H225Q Y251F 1.40 ± 0.01 8.97 ± 0.12 37.17 ± 1.16 19.67 ± 0.75 14.46 ± 0.80 1.95 ± 0.23 0.07 ± 0.003 0.62 ± 0.042 19.11 ± 2.887 showed major perturbations in the apparent kinetic parameters calculated for each mutant in comparison to wild-type PRODH (Table 3) Unlike those for the wild-type enzyme, initial velocities recorded for the Y251F mutant enzyme were subject to inhibition at high l-proline concentrations in the acid-to-neutral solution pH region (supplementary Fig S1) The apparent kinetic parameters of the Y251F mutant enzyme for l-proline were derived by fitting data to a steady-state rate expression that incorporates substrate inhibition (Eqn 2) B v¼ Fig Dependence of steady-state kinetic parameters on solution pH for the PRODH-catalyzed oxidation of L-proline (A) pH dependence of kcat ⁄ Km following ionizations in the free enzyme and substrate Fitting of data to Eqn (4) gave two pKa values of 7.0 ± 0.2 and 7.6 ± 0.2 (B) pH dependence of kcat following the pKa of the enzyme–substrate complex Fitting of data to Eqn (3) showed a simple sigmoid relationship, giving a pKa of 7.7 ± 0.1 This value is tentatively assigned to deprotonation of the substrate L-proline Conditions: three-component buffer system comprising 0.052, 0.052 and 0.1 M Mes, TAPSO, and diethanolamine, respectively; 60 °C Properties of mutant enzymes altered in the active site From analysis of the preliminary crystal structure of P furiosus PRODH (Fig 4A; a more complete structural analysis is to be published elsewhere), residues His225 and Tyr251 are situated on the re face of the isoalloxazine ring of FAD, forming part of the substrate-binding site (Fig 4B) To assess the potential role of these two residues as active site bases, pH dependence studies were performed with H225A, H225Q and Y251F mutant enzymes, as described for wild-type PRODH Initial steady-state experiments using the three-component buffer system at pH 7.5 2078 Vmax ½SŠ ỵ Km ỵ K ẵS i 2ị where v is the initial velocity, Vmax is the maximum value of the initial velocity, [S] is the substrate concentration, Km is the substrate concentration at half the maximal velocity, and Ki is the equilibrium constant for inhibitor binding Marked inhibition has also been reported in studies of mutant forms of the flavoprotein morphinone reductase under conditions of high substrate concentration [27,28] pH dependence studies revealed that the H225A mutant enzyme was unstable and precipitated from solution below pH 7.0 This consequently compromised the accuracy of data analysis in the acid solution pH region The H225Q mutant was somewhat more stable, displaying activity down to solution pH 6.0 The pH dependence of kcat was sigmoidal for both the H225A and H225Q mutant forms, and when fitted to Eqn (3) (see Experimental procedures) produced a macroscopic pKa value of 7.1 ± 0.1 for each mutant The Y251F mutant enzyme was stable over the entire pH range of study, with kcat values again showing a simple sigmoidal dependence on solution pH; fitting data to Eqn (3) (see Experimental procedures) gave a macroscopic pKa value of 7.3 ± 0.1, which compares favorably with the values determined for the wild-type and His225 mutant In light of the high degree of similarity between pKa values determined from fitting of the kcat data plots for wild-type and mutant enzymes, the macroscopic pKa of 7.7 for FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS P J Monaghan et al wild-type PRODH has been tentatively attributed to ionization of l-proline in the Michaelis complex Like wild-type PRODH, all mutant enzyme forms displayed a bell-shaped dependence of kcat ⁄ Km as a function of solution pH, and analysis of the data by fitting to Eqn (4) (see Experimental procedures) gave macroscopic pKa values of 6.8 ± 0.1 and 9.9 ± 0.1 (H225A), 6.8 ± 0.1 and 9.4 ± 0.2 (H225Q), and 6.0 ± 0.1 and 7.4 ± 0.1 (Y251F) (supplementary Fig S2) The initial idea that the pKa of 7.0 ± 0.1 determined for the wild-type enzyme might represent ionization of the conserved His225 active site residue in the free enzyme has been rejected, as mutant pH dependence data reveal that this ionization is not lost, but is apparent from the acid limb of the bell-shaped fits in the kcat ⁄ Km plots for both H225A and H225Q mutant forms This analysis has revealed that His225 and Tyr251 are not active site base residues, and are not essential for catalysis The results obtained suggest that PRODH stabilizes the deprotonated form of l-proline substrate in the Michaelis complex, analogously to the substrate activation mechanisms observed in TMADH [25] and monomeric sarcosine oxidase [29] Given this finding and the absence of an active site residue that acts as base during oxidation of l-proline, the data suggest that PRODH-catalyzed amine oxidation may occur by addition of a deprotonated l-proline at the C4 position of the FAD cofactor and abstraction of a substrate proton by the N5 atom of the flavin, a contemporary mechanism of flavoprotein-catalyzed amine oxidation proposed for TMADH [30] Identification of product The product of the enzyme-catalyzed oxidation of l-proline was determined by monitoring the development of the o-aminobenzaldehyde–P5C complex at 443 nm [31] and by MS The rate constant for the reaction of P5C with o-aminobenzaldehyde was a direct function of o-aminobenzaldehyde concentration The value of the second-order rate constant was 42.4 s)1Ỉm)1, and analysis of the stoichiometry of conversion indicated that the ratio of l-proline oxidized to o-aminobenzaldehyde–P5C formed was 0.59, the spontaneous hydrolysis of P5C to glutamate–5-semialdehyde at the elevated assay temperature used (60 °C) and rapid polymerization of free o-aminobenzaldehyde accounting for the remaining 0.41 fraction not detected as o-aminobenzaldehyde–P5C chromophore MALDI-TOF MS was employed for direct analysis of product Following enzyme turnover, a peak corresponding to a mass ⁄ charge ratio of 114.1 was observed, L-proline dehydrogenase from P furiosus corresponding to the positively charged ion [M + H]+ of P5C (supplementary Fig S3) Additionally, we also analyzed the product of o-aminobenzaldehyde reaction with P5C using electrospray MS In this case, a single peak with a mass ⁄ charge ratio of 217 was observed, corresponding to the positive ion of the P5C–o-aminobenzaldehyde condensation product (Fig 9) Reduction potential of the enzyme-bound FAD at physiologic temperature The midpoint potential (Em) of FAD–PRODH was determined by potentiometric redox titration with sodium dithionite at ambient temperature and pH 7.0 During the course of reductive titration, the oxidized flavin was reduced directly to the dihydroflavin form, without a visible population of a flavin semiquinone species, indicating that the potential of the oxidized ⁄ semiquinone flavin couple is much lower than that of the semiquinone ⁄ hydroquinone couple (Fig 10A) Data were fitted to the two-electron Nernst function (Eqn 5) (see Experimental procedures) by least-squares regression analysis, and gave a midpoint two-electron potential value of ) 192 ± mV and a corresponding unrestricted RT/nF value of 28.9 ± 0.4 mV (where R is gas constant, T is temperature, n is number of electrons and F is Faraday constant), consistent with the expected value (29.5 mV) for two-electron reduction of the enzyme-bound FAD (Fig 10B) The temperature dependence of the two-electron midpoint potential was measured within the range 7.5–31 °C (the limits imposed by the performance of the electrode), and a ‘normal’ linear temperature dependence was found (Fig 10C) The temperature dependence of the midpoint potential was calculated to be ) 3.1 ± 0.05 mVỈC°)1 from the plot gradient Extrapolation to physiologic temperature (i.e 100 °C for P furiosus) indicated an operational midpoint potential for PRODH of ) 407 ± mV Thermodynamic parameters for the reduction of PRODH by sodium dithionite were calculated from the temperature dependence of the midpoint potential, and shown to be DH°¢ ẳ ) 127.6 kJặmol)1, DS ẳ ) 290.4 Jặmol)1ặK)1, and DGÂ298 ẳ ) 41.1 kJặmol)1 Potentiometric redox titrations of the H225A, H225Q and Y251F mutants at 25 °C all showed reduction of oxidized flavin directly to the dihydroflavin form without a visible population of a flavin semiquinone species Mutant datasets were fitted to the two-electron Nernst function (Eqn 5) (see Experimental procedures) by least-squares regression analysis, and gave midpoint potential values of ) 169 ± mV (H225A), ) 155 ± mV (H225Q), and ) 157 ± mV (Y251F) (supplementary Fig S4), FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS 2079 L-proline dehydrogenase from P furiosus P J Monaghan et al Fig Electrospray MS analysis of the o-aminobenzaldehyde adduct of the catalytic reaction product P5C generated during the oxidation of + L-proline by PRODH The ionization mode used produced the positively charged quasimolecular ion ([M + H] ) The electrospray spectrum showing an m ⁄ z peak of 217 represents the stable adduct formed from the reaction of P5C with o-aminobenzaldehyde Inset: the proposed mechanism for the reaction of P5C (1a) and o-aminobenzaldehyde (2) Following addition of activated P5C (1b) C4 carbon to the aldehyde function of o-aminobenzaldehyde, redolent of a Knoevenagel condensation whereby the imine moiety of activated P5C is comparable to the carbonyl in a conventional ketone–aldehyde reaction, the adduct precursor (3), following condensation and elimination of water, forms a stable adduct (4) The reaction is analogous to that of P5C with pyridoxal phosphate [46] which compares with ) 174 ± mV for the wild-type enzyme at the same temperature (25 °C) Discussion The mechanism of substrate oxidation by flavoprotein amine dehydrogenase ⁄ oxidases remains contentious In recent years, however, substrate oxidation by members of this enzyme class has been shown to occur by quantum mechanical tunneling [30,32] We and others have demonstrated that analysis of the temperature dependence of kinetic isotope effects suggests that H-transfer by quantum tunneling occurs 2080 during substrate C–H bond cleavage, and that the temperature effects are consistent with contemporary environmentally coupled tunneling models [33] A major restriction in the use of variable temperature to study tunneling reactions is the limited temperature range available in experimental studies For this reason, in this study we have targeted a hyperthermophilic amine dehydrogenase, with a view to extending in future work the temperature window available for detailed physicochemical analysis of the enzyme chemistry Thus, we have cloned and expressed two ORFs from P furiosus that show some sequence similarity with bacterial amine-specific flavoprotein FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS L-proline P J Monaghan et al Fig 10 Redox potentiometric titration of wild-type monoflavinylated PRODH with sodium dithionite, and temperature dependence of the midpoint potential (A) Spectral changes accompanying reductive titration of PRODH with sodium dithionite at 25 °C (B) Plot of absorbance at 450 nm versus the observed potential (corrected against the normal hydrogen electrode) The data are fitted to the Nernst equation (Eqn 5) for a two-electron reduction process, giving a midpoint reduction potential, Em, of ) 192 ± mV, and an RTF value of 28.9 ± 0.4 mV (C) Plot of Em versus temperature, illustrating the linear dependence in the range 7.5–31 °C; gradient, ) 3.1 ± 0.05 mVỈC°)1) The plot extrapolates to an operational midpoint potential at physiologic temperature (100 °C for P furiosus) of ) 407 ± mV Conditions: 100 mM potassium phosphate buffer, pH 7.0 Mediator dyes used: methyl viologen (0.3 lM), benzyl viologen (1 lM), 2-hydroxy-1,4-napthaquinone (7 lM), and phenazine methosulfate (2 lM) dehydrogenase from P furiosus A Absorbance 0.8 0.6 0.4 0.2 300 400 500 600 Wavelength (nm) B 0.9 Abs (450 nm) 0.8 0.7 0.6 0.5 0.4 0.3 0.2 -400 -300 -200 -100 100 (Potential versus NHE) (mV) C -100 Midpoint Potential (mV) oxidases ⁄ dehydrogenases, including tetrameric sarcosine oxidase and DMGO Conservation of residues His225 and Tyr251 between PRODH and DMGO provoked a mutagenesis strategy for PRODH to target these residues, which are suspected to prime substrate during DMGO catalysis through deprotonation of the substrate amine function prior to FAD reduction [34] PRODH is a heterooctomer (ab)4, as confirmed by determination of a preliminary crystal structure Purification of PRODH for mechanistic and redox studies enabled the isolation of a form that contains a single FAD cofactor noncovalently bound to the b subunit of the protein, and that shows reactivity with l-proline, generating P5C as product l-pipecolic acid and sarcosine are slow substrates, and are not physiologic substrates of PRODH, as judged from the observed kinetic parameters The enzyme is not an oxidase, and does not use nicotinamide cofactors as electron acceptors It also does not show any sequence homology with the PRODH (PutA protein) of E coli [7] The enzyme therefore represents a new member of a recently identified class of PRODHs of hyperthermophilic origin We have shown that the PRODH active site is situated in the b subunit and that these residues, as suggested from structural and kinetic studies of A globiformis DMGO [14], play a role in the mechanism of amine substrate oxidation but not participate as catalytic base residues The presence of iron coordination in the Cys-clustered domain of the a subunit could not be confirmed from the structure (Fig 4A) The anaerobic environment that P furiosus inhabits led to the possibility that any Fe–S cluster in the PRODH a subunit may be aerobically labile, so wild-type enzyme was expressed and purified under -120 -140 -160 -180 -200 10 15 20 25 30 35 Temperature (°C) strict anaerobic conditions (see Experimental procedures), and anaerobic assays were repeated with P furiosus ferredoxin and NADP+ to measure electron transfer to putative physiologic acceptors via the Fe–S cluster No evidence of an Fe–S cluster was apparent from the UV-visible absorption spectrum of anaerobic PRODH, and no activity towards ferredoxin and ⁄ or NADP+ was observed Our kinetic studies indicate a strong pH dependence on the kinetic parameters kcat and kcat ⁄ Km A single FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS 2081 L-proline dehydrogenase from P furiosus P J Monaghan et al macroscopic pKa of 7.7 can be seen in the plot of kcat versus pH, and maximum activity is realized on the alkaline side of this ionization This is consistent with kinetic studies of other amine-oxidizing flavoproteins, such as TMADH [25], where the ionization has been attributed to the deprotonation of the cationic form of the substrate This implies that the unprotonated form of the substrate is reactive, consistent with proposed mechanisms of amine substrate oxidation by flavoprotein enzymes [26] On binding of substrate, the pKa for the substrate ionization is sufficiently perturbed to favor formation of the free base form at physiologic pH In the case of TMADH, stopped-flow studies indicate a shift in pKa of about ) 3.5 pH units (from pH 9.8 to $ pH 6.5) for substrate ionization on formation of the enzyme–substrate complex [25] Our data for P furiosus PRODH suggest that a similar shift in pKa of ) 2.9 pH units (from 10.6 to 7.7) occurs on binding of l-proline to the enzyme, although unequivocal demonstration of this must await detailed stopped-flow studies with protiated and deuterated substrates A significant change in the pKa value for the ionization of the alkaline limb of the kcat ⁄ Km plot is seen following mutagenesis (Fig and supplementary Fig S2) At this stage, we are unable to tentatively assign this ionization to a functional group, but its location is most likely in the active site Mutagenesis could affect the pKa of this neighboring group substantially At this stage, we cannot unequivocally rule out changes in the rate-limiting step, or contributions from other ionizable groups, as the origin of the observed dependence of kcat on solution pH Mutagenesis studies of TMADH have indicated a role for residues His172 and Tyr60 in perturbing the pKa of the substrate, and structural studies of DMGO suggest that residues His225 and Tyr259, which are conserved in PRODH, might also facilitate deprotonation of substrate close to physiologic pH values In this regard, it is important to note that the value of the Michaelis constant for l-proline is based on the total concentration of substrate (i.e cationic, zwitterionic and deprotonated anionic forms) At pH 7.5, the Km for substrate expressed in terms of the unprotonated form only is 24.5 lm Concluding remarks We have identified a new member of a recently identified class of PRODHs of hyperthermophilic origin In terms of cofactor content and redox chemistry, this flavoprotein is simpler than the heterotetrameric dyelinked PRODH from the hyperthermophilic archaeon, T profundus, an analog of which is also encoded in the genome sequence of P furiosus Our work demon2082 strates structural diversity among l-proline dehydrogenase ⁄ oxidase enzymes Our studies are now focused on: (a) delineating the role of the two types of PRODH in P furiosus; and (b) addressing the mechanisms of substrate oxidation and electron transfer Experimental procedures Cloning and expression of genes encoding a novel flavoenzyme amine dehydrogenase Two ORFs [gi_18977617 and gi_18977618; protein extraction description and analysis tool (pedant) database] were identified in the genome of P furiousus DSM 3638 that encode a putative flavoenzyme amine dehydrogenase ⁄ oxidase These genes were amplified by PCR using the oligonucleotides 5¢-GTG AGA AAC TTG AGG CCA CTA GAC TTA ACG G-3¢ and 5¢-TCA ACC CAT TTG AAG AGC AAC AGT TCT TAA TTC TCC C-3¢ The PCR product was purified by agarose gel electrophoresis, and used as template DNA in a second PCR reaction to incorporate flanking restriction sites (5¢ XbaI and 3¢ BamHI) for directional cloning, and a 5¢-ribosome-binding site to allow expression of the cloned DNA Primers used during this PCR reaction were: 5¢-GGG GGG TCT AGA AAG GAG ATA AAG AGA TGA GAA ACT TGA G-3¢, and 5¢-GGG GGG GGA TCC TCA ACC CAT TTG AAG AGC A-3¢ The PCR product was digested with XbaI and BamHI, ligated with vector pET11d, previously made end-compatible with the same restriction endonucleases, and transformed into Novablue cells (Novagen, Merck Chemicals Ltd., Nottingham, UK) Restriction analysis confirmed positive recombinants, and DNA sequencing confirmed the correct sequence of the recombinant clone, designated pPRODH1 A silent mutation was incorporated into pPRODH1 to eradicate an internal ribosome-binding site responsible for translation initiation within the gene, forming a truncated population of the a subunit in E coli The following primers were used in the mutagenesis protocol marketed by Stratagene (Amsterdam, the Netherlands) (QuikChange): 5¢-GGT GTC GAT GCT AGG AAA ACA AAA GTT AAA GAT GGA ATG AAA GTA C-3¢, and 5¢-GTA CTT TCA TTC CAT CTT TAA CTT TTG TTT TCC TAG CAT CGA CAC C-3¢ The underlined letters indicate the mismatch with the template DNA The new expression construct was designated pPRODH2 The H225Ab, H225Qb and Y251Fb mutant enzymes (mutations on b subunit) were also isolated using the QuikChange mutagenesis protocol, using the pPRODH2 expression construct as template for primers: 5¢-CCA ATT GAG CCC TAC AAG GCT CAA GCA GTG ATA ACC-3¢ and 5¢-GGT TAT CAC TGC TTG AGC CTT GTA GGG CTC AAT TGG-3¢ (H225A); 5¢-CCA ATT GAG CCC FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS P J Monaghan et al TAC AAG CAG CAA GCA GTG ATA ACC-3¢ and 5¢-GGT TAT CAC TGC TTG CTG CTT GTA GGG CTC AAT TGG-3¢ (H225Q); and 5¢-CAA GTA TGG TCA CGC TTT TTT AAC ACA AAC TGC GC-3¢ and 5¢-GCG CAG TTT GTG TTA AAA AAG CGT GAC CAT ACT TG-3¢ (Y251F) The underlined letters indicate the mismatch with the template DNA All mutant constructs were identified by DNA sequencing, which also confirmed sequence integrity Wild-type PRODH was expressed and purified as described previously [19] Mutant forms were purified using the purification protocol described for the wild-type enzyme The protein concentration was determined by the method of Bradford [35] Anaerobic expression and partial purification of wild-type PRODH Anaerobic 2xYT media containing 20 mm sodium fumarate was supplemented with ampicillin (50 lgỈmL)1) and chloramphenicol (34 lgỈmL)1), and inoculated with E coli Rosetta(DE3)pLysS cells transformed with plasmid pPRODH2 Cells were grown at 37 °C until the attenuance at 600 nm reached $ 0.8 Cells were induced with isopropyl thio-b-d-galactoside at a final concentration of mm, and grown for a further 48 h at 37 °C Cells were harvested under anaerobic conditions, resuspended in 50 mm anaerobic Mops buffer (pH 7.9), and sealed in an airtight centrifuge tube Cells were lysed anaerobically by three cycles of freeze–thaw treatment, and cell debris was removed by centrifugation (12 100 g for 50 using a Beckman Avanti J-25 centrifuge, rotor type JA 25.50) following DNA hydrolysis The supernatant was transferred to an airtight tube inside a glovebox, and the solution was subjected to a heat denaturation step at 80 °C for h, with denatured protein being removed by centrifugation (27 200 g for 30 using a Beckman Avanti J-25 centrifuge, rotor type JA 25.50) The supernatant was loaded onto an anaerobic DE52 anion exchange column equilibrated with anaerobic 50 mm Mops buffer (pH 7.9), and anaerobic wild-type PRODH was eluted with a 0–0.5 m NaCl gradient N-terminal protein sequence analysis and MS N-terminal sequence analysis was performed using a ABI 476 Protein Sequencer (Applied Biosystems, Foster City, CA, USA) Protein samples were electrophoresed by SDS-PAGE, and electroblotted onto a poly(vinylidene difluoride) membrane Protein was stained with Coomassie Brilliant Blue R250 to identify protein bands prior to excision from the membrane for automated N-terminal sequence analysis MALDI-TOF MS of the purified enzyme was performed with a Kratos Kompact MALDI-TOF III mass spectrometer (Kratos, Manchester, UK), using sinapinic acid as matrix Electrospray MS was performed using a Waters Micromass LCT TOF mass spectrometer (Waters Ltd., Elstree, UK) L-proline dehydrogenase from P furiosus Preparation of anaerobic samples Buffers were made anaerobic by bubbling humidified oxygen-free argon gas at lbỈin)2 through solutions for $ h with stirring Solutions were then transferred to an anaerobic glovebox, which was left open overnight to remove residual oxygen Samples of PRODH were made anaerobic by passing them through a 10-DG column (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) equilibrated with anaerobic buffer in an anaerobic glovebox Solutions of substrates, dithionite, potassium ferricyanide and mediator dyes were made by dissolving the appropriate solid in anaerobic buffer All anaerobic experiments were performed in a Belle Technology (Portesham, UK) glovebox under a positive pressure atmosphere of nitrogen, with residual oxygen levels being maintained at < 0.05 p.p.m with a BASF (Cheadle, UK) R3-11 oxygen-scavenging catalyst Identification of flavin cofactor in PRODH Following release from the enzyme, the chemical identity of the flavin cofactor in PRODH was determined by MALDITOF MS Cofactor was released from the enzyme by heat denaturation, and precipitated protein was removed by centrifugation (17 000 g for 10 at °C in the dark using a Microcentrifuge Fisherbrand accuSpin Micro 17R) Cofactor was mass analyzed in double deionized H2O alongside authentic FAD, FMN, ATP, ADP and AMP treated in the same way Analysis was performed using a Bruker Biflex mass spectrometer Bruker Daltonics Ltd., Coventry, UK, calibrated with a combination of peptides of known mass, and the matrix was dihydroxybenzoic acid Optical titrations with reducing substrates and steady-state kinetic analysis PRODH (800 lL; 19.4 lm in 100 mm potassium phosphate buffer, pH 7.5) was incubated in a quartz cuvette at 80 °C for 10 to allow temperature equilibration Anaerobic stocks (2 m) of dimethylglycine, sarcosine, glycine, glycine betaine, l-proline, d-proline, l-pipecolic acid and sodium sulfite were mixed with enzyme to a final concentration of 20 mm Enzyme reduction was monitored by time-dependent spectral acquisition in the region 300–600 nm Steady-state kinetic measurements with identified substrates were performed spectrophotometrically in a cm light path in 100 mm potassium phosphate buffer (pH 7.5), at 80 °C in a final reaction cell volume of 800 lL Ferricenium hexafluorophosphate was used as electron acceptor Buffer and substrate were equilibrated at the assay temperature for 10 prior to addition of ferricenium (200 lm) followed by addition of 75 nm enzyme to initiate the reaction Initial velocities were measured by following reduction of ferricenium (De300 ¼ 4300 m)1Ỉcm)1 [36]), and expressed as the concentration of ferricenium reduced FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS 2083 L-proline dehydrogenase from P furiosus P J Monaghan et al per unit time (lmỈmin)1) Initial velocities as a function of substrate concentration were analyzed by fitting to the Michaelis–Menten equation Similar reaction conditions were employed when studying the activity of PRODH with P furiosus 4Fe)4S ferredoxin (the ferredoxin was supplied by W Hagen, Delft University) Reduction of the 4Fe)4S ferredoxin was monitored at 390 nm in an anaerobic glovebox, as described previously for aldehyde ferredoxin oxidoreductase activity [37], but using l-proline as substrate For the acquisition of temperature-dependent steadystate data, enzyme activity was measured in a continuous assay using the ferricenium ion as an artificial electron acceptor Substrate concentrations were maintained at 10 times Km Reaction temperatures were recorded by direct measurement inside the cuvette The temperature was recorded during temperature equilibration prior to the reaction at both the top and the bottom of the cuvette to detect any temperature gradient in the assay mixture Temperatures were also recorded immediately after assay completion, and reactions were repeated if temperature fluctuations exceeded 0.1 C° Assays were initiated by addition of microliter volumes of enzyme to ensure that there was no significant effect on overall reaction temperature Initial velocity data were plotted as a complete dataset of rate versus time versus temperature, analogously to the studies performed on thermophilic enzymes by Peterson et al [38], who described an equilibrium model to determine the thermal parameter Teq that represents a submillisecond timescale-reversible temperature-dependent equilibrium between active enzyme and inactive (or less active) forms The effect of a decrease in enzyme activity above the optimal temperature occurring due to a shift in Teq is up to two orders of magnitude greater than the contribution of thermal denaturation It is important to note, however, that application of this proposed equilibrium model may be restricted to monomeric enzymes, as the model does not account for the complicating effects of thermally induced subunit dissociation of oligomeric enzymes The function of time in the three-dimensional dataset is necessary to detect thermal inactivation ⁄ denaturation of enzyme at temperatures up to and exceeding the source (evolved) temperature For studies on pH dependence, steady-state assays were performed over the pH range 5.5–10.0 in increments of 0.5 pH units, at an assay temperature of 60 °C To keep the ionic strength of the assay solutions constant over the experimental pH range, a three-component buffer system was employed, comprising Mes (pKa 6.02), 3-{[tris(hydroxymethyl)-methyl]amino}-2-hydroxypropane sulfonic acid (TAPSO; pKa 7.49), and diethanolamine (pKa 8.88), at final concentrations of 0.052, 0.052 and 0.1 m, respectively [39] pH profiles for the kinetic parameters kcat and Km were constructed, and the data were fitted to Eqn (3) and Eqn (4), respectively, to obtain the relevant pKa values 2084 kcat ¼ EH 10pHị ỵ E 10pKa ị 10pHị ỵ 10pKa ị kcat Tmax ẳ Km ỵ 10pKa1 pHị þ 10ðpHÀpKa2 Þ ð3Þ ð4Þ where EH and E are the catalytic activities of the protonated and unprotonated forms of the ionization group, respectively, and Tmax is the theoretical maximal value of kcat ⁄ Km Product identification and quantification The product of the enzyme-catalyzed oxidation of l-proline was identified using a modified method described for PutA protein of enteric bacteria [31] and by MS Recombinant enzyme (75 nm) was used to oxidize l-proline (200 mm) in a final reaction volume of 800 lL, using limiting ferricenium ion (200 lm) as artificial electron acceptor in 100 mm potassium phosphate buffer (pH 7.5) Assays were performed at 60 °C, and allowed to progress until complete reduction of ferricenium The assay mixture was then incubated at °C, and 100 lL of this mixture was added to o-aminobenzaldehyde (0.5–4 mm) in the same assay buffer Formation of the o-aminobenzaldehyde–P5C chromophore was followed at 443 nm at 25 °C o-Aminobenzaldehyde was made as a 50 mm stock solution in 20% ethanol, and stored at ) 20 °C The extinction coefficient used for quantifying the complex was e443 ẳ 2.71 mm)1ặcm)1 Baseline controls were performed with each assay component in the absence of enzyme The o-aminobenzaldehyde–P5C chromophore was analyzed using electrospray MS Ten-microliter samples were injected via a Rheodyne valve into a mobile phase of methanol flowing at 0.2 mLỈmin)1 into the electrospray source The source temperature was maintained at 80 °C, and the needle voltage was $ 3.0 kV The sample cone was operated at 20 V, and nitrogen was used as the desolvation and sheath gas at 600 and 100 LỈh)1, respectively The spectrometer was calibrated with a solution of sodium iodide Direct analysis of enzyme reaction product was also performed using MALDI-TOF MS Following enzyme turnover, a lL sample of reaction mixture was plated for analysis on a Bruker Biflex mass spectrometer in positive ion mode, using dihydroxybenzoic acid as matrix A mgỈmL)1 standard solution of authentic l-proline was also prepared in double deionized H2O and filtered through a 0.22 lm Millex-GP Acrodisc filter (Millipore Ltd., Watford, UK) for MALDI-TOF MS analysis The spectrometer was externally calibrated using a combination of peptides of known mass Redox potentiometry Anaerobic redox titrations were performed between and 31 °C in 100 mm potassium phosphate buffer (pH 7.0) FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS P J Monaghan et al L-proline Enzyme (5 mL; $ 16.3 mgỈmL)1) was electrochemically titrated following the method of Dutton [40], using sodium dithionite as reductant and potassium ferricyanide as oxidant The mediator dyes methyl viologen (0.3 lm), benzyl viologen (1 lm), 2-hydroxy-1,4-napthaquinone (7 lm) and phenazine methosulfate (2 lm) were added immediately prior to titration to facilitate equilibration across the range + 100 mV to ) 480 mV [41,42] Equilibration times after microliter addition of reductant were typically between 10 and 15 min, after which the potential was noted and the corresponding absorbance spectra were measured between 280 and 800 nm Complete titration curves typically consisted of $ 40 different potential measurements Plots of absorbance against redox potential were fitted to Eqn (5) [42] Aobs ẳ a ỵ b10E12 Eị=29:5 ị ỵ 10E12 Eị=29:5 5ị In Eqn (5), Aobs is the absorbance value at the peak for oxidized flavin at the electrode potential E, and a and b are the absorbance values of the fully oxidized and reduced enzyme at this wavelength, respectively E12 is the midpoint potential for the concerted two-electron reduction of the flavin Data manipulation and analysis were performed using origin software package version 6.0 (Microcal, OriginLab Corporation, Northampton, MA, USA) All redox potentials are given relative to the standard hydrogen electrode Structure determination Crystals were obtained as described previously [19] Molecular replacement using the PRODH from Pyrococcus horikoshii OT-3 as a model (75% and 90% identical to P furiosus PRODH a and b subunits, respectively) was performed using AMoRe [43] Initial electron density maps were calculated using refmac5.0 [44], following several rounds of positional refinement with strict noncrystallographic symmetry restraints imposed, leading to a R ⁄ Rfree ˚ ratio of 24.7 ⁄ 28.9 for all data (30–3.25 A) Materials Redox mediators, amine substrates, FAD, FMN, ATP, ADP, AMP, sodium fumarate and buffer components were obtained from Sigma-Aldrich (Gillingham, UK) Sodium dithionite was obtained from FSA Laboratory Supplies (Loughborough, UK), and complete protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Burgess Hill, UK) Isopropyl thio-b-d-galactoside was obtained from Melford Laboratories (Ipswich, UK) Oxygen-free nitrogen and Pureshield brand argon were purchased from the British Oxygen Company (BOC gases, Guildford, UK) Broth components were purchased from Oxoid (Basingstoke, UK) The plasmid pET11d and E coli strains Novablue and Rosetta (DE3)pLysS were 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Dependence of steady-state kinetic parameters on solution pH for the oxidation of l-proline substrate catalyzed by the H225A, H225Q and Y251F PRODH enzyme forms Fig S3 MALDI-TOF MS identifying the mass of P5C produced from the oxidation of substrate l-proline catalyzed by PRODH of P furiosus DSM 3638 Fig S4 Redox potentiometric titration of H225A, H225Q and Y251F mutant PRODH forms with sodium dithionite This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 2070–2087 ª 2007 The Authors Journal compilation ª 2007 FEBS 2087 ... MALDI-TOF MS identifying the mass of P5C produced from the oxidation of substrate l-proline catalyzed by PRODH of P furiosus DSM 3638 Fig S4 Redox potentiometric titration of H225A, H225Q and Y251F... Authors Journal compilation ª 2007 FEBS L-proline P J Monaghan et al dehydrogenase from P furiosus A B C D Fig MALDI-TOF MS of flavin cofactor released from PRODH (FAD-bound form) (A) Authentic... ion and AMP–Na+ adduct m ⁄ z peaks at 348 and 370, respectively (D) Released cofactor of heat-denatured PRODH showing the [M + H]+ ion of FAD cofactor with an m ⁄ z peak of 787 identical to that