Mutagenesis of these active site residues had no effect on recombinant pro-tein yield, and all mutants were purified in FAD-bound form.. furiosus PRODH to oxidize multiple amine com-pound
Trang 1hyperthermophilic L-proline dehydrogenase from
Pyrococcus furiosus related to dimethylglycine
Phillip J Monaghan, David Leys and Nigel S Scrutton
Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, UK
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
tran-scription 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-carboxy-late (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 interme-diate P5C is hydrolyzed to glutamate 5-semialdehyde, which is then oxidized to glutamate by the P5C dehydrogenase domain, with NAD+acting as electron
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 4 January 2007, revised 9
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 reduc-tion, 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 (pKavalues 7.0 ± 0.2 and 7.6 ± 0.2) for kcat⁄ Kmas a function
of pH was observed The pH dependence of kcatis 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 dehy-drogenases 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 mVÆC)1), and extra-polation to physiologic growth temperature for P furiosus (100C) yields
a value of ) 407 ± 5 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
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.
Trang 2acceptor (Fig 1) The structure of a truncated form of
PutA comprising the FAD-containing proline
dehy-drogenase 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,
dis-tinct 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
p53-mediated 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
hetero-tetramer, contains 2 moles of FAD per mole of
enzyme, and is bifunctional, having proline
dehydro-genase and dye-linked NADH dehydrodehydro-genase activities
[10,11] The enzyme is a complex iron–sulfur
flavopro-tein, with the a subunit containing a 2Fe)2S center
Additionally, the c subunit contains a broad
absorp-tion peak around 420 nm typical of an 8Fe)8S
ferre-doxin 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
dye-linked 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 resi-dues (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 crys-tal structure of a related enzyme from Pyrococcus hori-koshii 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 hyperthermo-philic 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 hexafluorophos-phate, but not molecular oxygen, NAD+, or pyrococ-cal 4Fe)4S ferredoxin This article represents the first detailed report of the kinetic and spectroscopic proper-ties of this novel type of PRODH
Results Analysis of a and b subunit sequences Sequence analysis of both subunits suggests the pres-ence of an ADP-binding motif in the N-terminal region of the a subunit, with the 11 participating
Fig 1 Reactions catalyzed by the
bifunc-tional PutA protein of enteric bacteria.
Hydrolysis of P5C is nonenzymatic In
euk-aryotes, distinct enzymes encoded by
separ-ate genes catalyze these reactions.
Trang 3residues satisfying the physicochemical parameters of
the consensus [17] The a subunit also contains a
con-served 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
NADPH-binding 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 dimethyl-glycine dehydrogenase (Fig 2), residues that are pre-sent in a number of sarcosine dehydrogenase-like proteins Given these sequence similarities, we conjec-tured that the protein encoded by the two identified
A
B
Fig 2 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 Corynebac-terium 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.
Trang 4ORFs in Pyrococcus furiosus encode a new type of
amine dehydrogenase⁄ oxidase, a hypothesis that we
addressed through detailed characterization of the
pro-tein as a new type of (PRODH), described below
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
crystall-ogenesis and X-ray diffraction studies, and was
puri-fied 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
subpopula-tion (approximately 13%) of the a subunit was
trun-cated The sequence MKVQRQ was obtained for the
truncated a subunit N-terminus, indicating that
trunca-tion 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
1 : 1 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
pre-dicted 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
pro-tein yield, and all mutants were purified in
FAD-bound form Despite the close proximity of both
His225 and Tyr251 to the isoalloxazine ring moiety of
FAD, no major perturbations in the absorption prop-erties 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
A
B
Fig 3 (A) SDS ⁄ PAGE analysis of the purification of recombinant wild-type PRODH from E coli strain Rosetta(DE3)pLysS trans-formed 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 clarifica-tion by centrifugaclarifica-tion Lane 4: pooled fracclarifica-tions 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 Condi-tions: 100 m M potassium phosphate buffer, pH 7.5; 25 C; enzyme concentration 16 l M
Trang 5the 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
posi-tively 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
well-characterized 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
sarco-sine 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
dehydro-genase from Mus musculus [21] Given these sequence
similarities, amine compounds were analyzed as
potential substrates by mixing with enzyme (19.4 lm)
at 80C under anaerobic conditions to preclude potential oxidase chemistry Enzyme–substrate reactiv-ity 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⁄ 2¼ 105 s), followed by l-pipecolic acid (t1⁄ 2¼ 110.5 s; a structural analog of l-proline), and sarcosine (t1⁄ 2¼ 654 s) Glycine betaine, glycine, dimethylgly-cine 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 com-pounds is in stark contrast to the catalytic properties reported for dye-linked proline dehydrogenase 1 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 fea-ture 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 cat-alytic 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 andL-pipecolic acid
In developing a suitable and continuous turnover assay for wild-type enzyme at an elevated temperature
FAD FAD
FMN ATP
B A
Fig 4 Preliminary P furiosus PRODH crys-tal structure (A) Omit maps (in blue) super-imposed 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.
Trang 6(80C), 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
tempera-ture Assays were performed in 100 mm potassium
phosphate buffer (pH 7.5), which has a low
tempera-ture 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 80C 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
hyper-bolic plots of initial velocity as a function of substrate
concentration yielded apparent Kmvalues for the wild-type enzyme of 30.8 ± 1.1 mm and 212.3 ± 17.0 mm for l-proline and l-pipecolic acid, respectively The cor-responding apparent kcatvalues were 18.1 ± 0.2 s)1and 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)1Æmm)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 sin-gle-turnover assays, reinforcing functional differences between the P furiosus and E coli enzymes Exogenous FMN did not act as electron acceptor in steady-state
A
B
C
D
Fig 5 MALDI-TOF MS of flavin cofactor released from PRODH (FAD-bound form) (A) Authentic FAD showing positively charged quasi-molecular 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 repre-sents 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 1 mgÆmL)1stock solutions in double deionized H2O, and filtered using a 0.22 lm Acro-disc; PRODH was exchanged into double deionized H 2 O.
Trang 7anaerobic 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
tem-perature range 40–90C Plots of initial velocity as
a function of l-proline concentration were hyperbolic
at all temperatures, and kinetic parameters were
calcu-lated by fitting to the Michaelis–Menten equation
Both kcatand Kmincrease 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)
Thermo-dynamic parameters were obtained by fitting to the
Eyring equation (Eqn 1)
Inðk=TÞ ¼ In kB=hþ DSz=R DHz=RT ð1Þ
where kBand 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
DH¼ 83.4 ± 2.9 kJÆmol)1, DS¼ 27.2 ± 1.0 JÆmol)1Æ
K)1, and DG¼ 73.3 kJÆmol)1(at 373 K) Incubation of
PRODH at elevated temperatures prior to activity assay showed that the enzyme is extremely stable, with no loss
of activity being evident up to 100C Above this tem-perature, thermal denaturation of PRODH is apparent, with complete loss of activity after 10 min of incubation
in glycerol buffer at temperatures ‡ 115 C (data not shown) Thus, PRODH from P furiosus is the most ther-mostable PRODH described to date
E
Fig 6 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 l M ) with sarcosine (20 m M ) (B) As for (A), but with L -pipecolic acid (20 m M ) (C) As for (A), but with L -proline (20 m M ) (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 m M
potassium phosphate buffer, pH 7.5; 80 C (E) Structural formulae for the three secon-dary amine molecules, sarcosine, L -pipecolic acid, and L -proline, which show substrate reactivity with PRODH Boxed areas illus-trate the common moiety suggested to be important for binding in the PRODH active site.
Table 1 Steady-state kinetic parameters for the reaction of PRODH with L -proline determined at different temperatures Condi-tions: 100 m M potassium phosphate buffer, pH 7.5, at each assay temperature.
Temperature (C) K m (m M ) k cat (s)1) k cat ⁄ K m (s)1Æm M )1)
Trang 8For mechanistic analyses, steady-state turnover assays were performed aerobically with l-proline at
60C 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)
kcat⁄ Km was found to be bell-shaped, and fitting of the data using Eqn (4) (see Experimental proce-dures) 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 depend-ence of kcat exhibited a simple sigmoid behavior that, when analyzed by fitting to Eqn (3) (see Experimen-tal 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 stabiliza-tion 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]
B
A
Fig 7 Temperature dependence and Eyring analysis of initial
velo-city data for wild-type PRODH reacting with L -proline (A)
Three-dimensional plot showing initial velocity (y-axis) versus time (x-axis)
versus temperature (z-axis) Reactions were performed in the
pres-ence of saturating L -proline (200 m M ) over the temperature range
40–90 C The dimension of time demonstrates any potential loss
of activity due to enzyme thermal denaturation at elevated
temper-atures (not observed in the case of PRODH-catalyzed oxidation of
L -proline) The three-dimensional plot was generated using
SIGMA-PLOT 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
elici-ted 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
col-lection Conditions: 100 m M 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
Thermody-namic parameters derived from fitting of data to the Eyring
equa-tion are DH ¼83.4 ± 2.9 kJÆmol)1, DS ¼ 27.2 ± 1.0 JÆmol)1ÆK)1
and DG z
373 ¼73.3 kJÆmol)1at 100 C.
Table 2 Steady-state kinetic parameters determined for the reac-tion of PRODH with L -proline at different pH values and at constant ionic strength Assays were performed at 60 C.
pH kcat(s)1) Km(m M ) kcat⁄ K m (s)1Æm M )1)
8.5 16.36 ± 0.46 19.54 ± 1.73 0.84 ± 0.10 9.0 16.36 ± 0.25 42.35 ± 1.56 0.39 ± 0.02 9.5 16.54 ± 0.41 87.21 ± 4.19 0.19 ± 0.01 10.0 18.00 ± 1.14 137.97 ± 15.11 0.13 ± 0.02
Trang 9Properties of mutant enzymes altered in the
active site
From analysis of the preliminary crystal structure of
P furiosus PRODH (Fig 4A; a more complete
struc-tural 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
sub-strate-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
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)
v¼ Vmax
1þ Km
½S þK½S
i
ð2Þ
where v is the initial velocity, Vmax is the maximum value of the initial velocity, [S] is the substrate concen-tration, 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 sub-strate 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 analy-sis 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 sig-moidal for both the H225A and H225Q mutant forms, and when fitted to Eqn (3) (see Experimental proce-dures) produced a macroscopic pKa value of 7.1 ± 0.1 for each mutant The Y251F mutant enzyme was sta-ble over the entire pH range of study, with kcatvalues again showing a simple sigmoidal dependence on solu-tion pH; fitting data to Eqn (3) (see Experimental pro-cedures) 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 deter-mined from fitting of the kcatdata plots for wild-type and mutant enzymes, the macroscopic pKa of 7.7 for
A
B
Fig 8 Dependence of steady-state kinetic parameters on solution
pH for the PRODH-catalyzed oxidation of L -proline (A) pH
depen-dence of kcat⁄ K m following ionizations in the free enzyme and
sub-strate Fitting of data to Eqn (4) gave two pK a values of 7.0 ± 0.2
and 7.6 ± 0.2 (B) pH dependence of kcatfollowing the pKaof the
enzyme–substrate complex Fitting of data to Eqn (3) showed a
simple sigmoid relationship, giving a pK a 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.
Table 3 Steady-state kinetic parameters determined for the H225A, H225Q and Y251F mutant PRODH forms Conditions: buffer composed of Mes, TAPSO and diethanolamine at final concentra-tions of 0.052, 0.052 and 0.1 M , respectively, pH 7.5, at an assay temperature of 60 C.
Mutant k cat (s)1) K m (m M ) k cat ⁄ K m (s)1Æm M )1) H225A 1.40 ± 0.01 19.67 ± 0.75 0.07 ± 0.003 H225Q 8.97 ± 0.12 14.46 ± 0.80 0.62 ± 0.042 Y251F 37.17 ± 1.16 1.95 ± 0.23 19.11 ± 2.887
Trang 10wild-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
activa-tion mechanisms observed in TMADH [25] and
mono-meric sarcosine oxidase [29] Given this finding and the
absence of an active site residue that acts as base
dur-ing 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
develop-ment 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)1Æm)1, and analysis of the stoichiometry of
con-version indicated that the ratio of l-proline oxidized to
o-aminobenzaldehyde–P5C formed was 0.59, the
spon-taneous hydrolysis of P5C to
glutamate–5-semialde-hyde at the elevated assay temperature used (60C)
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
corres-ponding to a mass⁄ charge ratio of 114.1 was observed,
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-amino-benzaldehyde 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 ± 3 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 elec-trons 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 7.5–31C (the limits imposed by the performance of the electrode), and a
‘normal’ linear temperature dependence was found (Fig 10C) The temperature dependence of the mid-point potential was calculated to be ) 3.1 ± 0.05 mVÆC)1 from the plot gradient Extrapolation to physiologic temperature (i.e 100C for P furiosus) indicated an operational midpoint potential for PRODH of ) 407 ± 5 mV Thermodynamic para-meters for the reduction of PRODH by sodium dithi-onite were calculated from the temperature dependence
of the midpoint potential, and shown to be DH¢ ¼ ) 127.6 kJÆmol)1, DS¢ ¼) 290.4 JÆmol)1ÆK)1, and DG¢298¼) 41.1 kJÆmol)1 Potentiometric redox titra-tions of the H225A, H225Q and Y251F mutants at
25C 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 ± 3 mV (H225A), ) 155 ± 3 mV (H225Q), and ) 157 ± 3 mV (Y251F) (supplementary Fig S4),