We also report the crystal structure of 2-electron reduced PETN reductase in complex with the steroid inhibitor and discuss its implications for the binding and reduction of steroid subs
Trang 1pentaerythritol tetranitrate reductase
Structure of the reduced enzyme-progesterone complex and the roles of residues Tyr186, His181 and His184
Huma Khan1, Terez Barna1, Neil C Bruce2, Andrew W Munro1,*, David Leys1,† and
Nigel S Scrutton1,†
1 Department of Biochemistry, University of Leicester, UK
2 CNAP, Department of Biology, University of York, UK
Pentaerythritol tetranitrate (PETN) reductase was
ori-ginally purified from a strain of Enterobacter cloacae
(strain PB2) on the basis of its ability to utilize nitrate
ester explosives such as PETN and glycerol trinitrate
(GTN) as sole nitrogen source Sequence analysis [1]
and structural studies [2] have indicated that PETN reductase is a flavoprotein member of the Old Yellow Enzyme (OYE) family [3] Other well-defined members include bacterial morphinone reductase (MR) from Pseudomonas putida M10 [4], estrogen binding protein
Keywords
crystallography; flavoprotein mechanism;
kinetics; Old Yellow Enzyme; PETN
reductase
Correspondence
N S Scrutton, Faculty of Life Sciences,
University of Manchester, Stopford Building,
Oxford Road, Manchester, M13 9PT, UK
Fax: +44 161 2755586
Tel: +44 161 2755632
E-mail: nigel.scrutton@manchester.ac.uk
Present addresses
*Manchester Interdisciplinary Biocentre,
School of Chemical Engineering and
Ana-lytical Science, University of Manchester,
The Mill, PO Box 88, Manchester,
M60 1QD, UK
†Manchester Interdisciplinary Biocentre and
Faculty of Life Sciences, Faculty of Life
Sciences, University of Manchester,
Stopford Building, Oxford Road,
Manchester, M13 9PT, UK
(Received 13 June 2005, revised 15 July
2005, accepted 21 July 2005)
doi:10.1111/j.1742-4658.2005.04875.x
The roles of His181, His184 and Tyr186 in PETN reductase have been examined by mutagenesis, spectroscopic and stopped-flow kinetics, and by determination of crystallographic structures for the Y186F PETN reductase and reduced wild-type enzyme—progesterone complex Residues His181 and His184 are important in the binding of coenzyme, steroids, nitro-aromatic ligands and the substrate 2-cyclohexen-1-one The H181A and H184A enzymes retain activity in reductive and oxidative half-reactions, and thus do not play an essential role in catalysis Ligand binding and catalysis is not substantially impaired in Y186F PETN reductase, which contrasts with data for the equivalent mutation (Y196F) in Old Yellow Enzyme The structure of Y186F PETN reductase is identical to wild-type enzyme, with the obvious exception of the mutation We show in PETN reductase that Tyr186 is not a key proton donor in the reduction of a⁄ b unsaturated carbonyl compounds The structure of two electron-reduced PETN reductase bound to the inhibitor progesterone mimics the catalytic enzyme-steroid substrate complex and is similar to the structure of the oxidized enzyme-inhibitor complex The reactive C1-C2 unsaturated bond
of the steroid is inappropriately orientated with the flavin N5 atom for hydride transfer With steroid substrates, the productive conformation is achieved by orientating the steroid through flipping by 180, consistent with known geometries for hydride transfer in flavoenzymes Our data highlight mechanistic differences between Old Yellow Enzyme and PETN reductase and indicate that catalysis requires a metastable enzyme-steroid complex and not the most stable complex observed in crystallographic studies
Abbreviations
EBP, estrogen binding protein; GTN, glycerol trinitrate; MR, morphinone reductase; OYE1, Old Yellow Enzyme 1; PETN, pentaerythritol tetranitrate; TNT, trinitrotoluene.
Trang 2(EBP) from Candida albicans [5], glycerol trinitrate
reductase from Agrobacterium radiobacter [6], the
xenobiotic reductases of Pseudomonas species [7] and
12-oxophytodienoic acid reductase from tomato [8]
and Arabidopsis thaliana [9] These enzymes reduce a
variety of cyclic enones, including 2-cyclohexen-1-one
and steroids Some steroids act as substrates, whereas
others are potent inhibitors of these enzymes PETN
reductase is also related to the more complex bile-acid
inducible flavoenzymes Bai H and Bai C from
Eubacte-rium species [10], the bacterial Fe⁄ S flavoenzymes
tri- and dimethylamine dehydrogenases [11,12], the
his-tamine dehydrogenase from Nocardiodes simplex [13]
and the NADH oxidase of Thermoanaerobium brockii
[14] These latter enzymes utilize diverse substrates, but
the catalytic framework has clearly evolved from a
common progenitor [15]
PETN reductase is unusual in its ability to degrade
major classes of explosive, including nitroaromatic
compounds, e.g trinitrotoluene (TNT), and nitrate
esters (GTN and PETN) [16–18] Degradation of TNT
involves reductive hydride addition to the aromatic
nucleus [16,19], and key residues involved in this
pro-cess have been discerned [20] The catalytic cycle of
PETN reductase comprises two half-reactions In the
reductive half-reaction, enzyme is reduced by NADPH
to yield the dihydroquinone form of the enzyme-bound
FMN, a reaction known to proceed by quantum
mechanical tunneling [21] In the oxidative
half-reac-tion, the flavin is oxidized by nitro-containing
explo-sive substrates or, in common with related enzymes,
cyclic enone substrates such as 2-cyclohexen-1-one A
detailed kinetic mechanism based on stopped-flow data
has been proposed [19]
Studies with OYE have established a role for Tyr196
in proton donation during the reduction of a⁄ b
unsat-urated carbonyl compounds [22] This residue is
con-served in PETN reductase as Tyr186 [1], and X-ray
structural and NMR analyses of PETN reductase in
complex with a number of steroid ligands have
sugges-ted this residue may likewise function as the key
pro-ton donor during the reduction of a⁄ b unsaturated
carbonyl compounds by PETN reductase [2] This
act-ive site tyrosine is not conserved in MR, where it is
replaced by cysteine [23,24] Recent mutagenesis
stud-ies have failed to identify the key proton donor in
MR, and suggest solvent water is the source of the
proton required for reduction of a⁄ b unsaturated
car-bonyl compounds [21,24,25] Herein, we report
solu-tion studies of three mutant forms of PETN reductase
We show that Tyr186 is not the key proton donor in
the oxidative half-reaction of PETN reductase, which
contrasts with reported studies with the highly
homo-logous OYE We demonstrate that residues His181 and His184 are determinants for substrate⁄ ligand bind-ing as suggested by the crystal structure of PETN reductase, consistent with a similar role for conserved residues in OYE [26] and MR [25] We also report the crystal structure of 2-electron reduced PETN reductase
in complex with the steroid inhibitor and discuss its implications for the binding and reduction of steroid substrates Our studies demonstrate a probable role for water in proton donation in PETN reductase and mul-tiple binding modes for steroid ligands in the active site The work emphasizes the need for (i) detailed evaluation of mechanism, and (ii) caution in inferring mechanistic similarities in structurally highly related enzymes
Results
Properties of the H181A and H184A enzymes The structure of PETN reductase solved in complex with prednisone, progesterone, 1,4-androstadiene (Fig 1A) and 2-cyclohexen-1-one indicate that these ligands bind above the si face of the FMN isoalloxa-zine ring and are held in position by hydrogen bond interactions with His181 and His184 [2] These residues also form interactions with the hydroxyl group of 2,4-dinitrophenol (an inhibitor) and picric acid (a sub-strate) [19] In OYE1 the counterpart residues are His191 and Asn194, and are known to have an important role in the binding of phenolic compounds
in the active site [3,26] Likewise, in MR the counter-part residues His186 and Asn189 form key interactions with reducing nicotinamide coenzyme and the oxi-dizing substrate 2-cyclohexen-1-one [25] Furthermore, NMR and kinetic studies have ruled out a role for His186 in proton donation in the oxidative half-reac-tion of MR [25] PETN reductase is unusual in having two histidine residues (rather than the His-Asn pair seen in OYE, MR and some of the other member proteins; Fig 1B) in the active site for ligand and substrate binding For those members that contain a His-His pair, there has been no report of a systematic analysis of the contribution of each histidine residue to binding and catalysis Thus, to ascertain the role of each histidine residue, and to identify any differential contribution to binding and catalysis, we isolated the two mutant enzymes H181A and H184A
Both the H181A and H184A enzymes were purified
to homogeneity as described and, as for wild-type enzyme [19], UV-visible spectra indicated stoichiometric assembly with the FMN cofactor Ligand binding titra-tions revealed a substantially increased dissociation
Trang 3constant for enzyme-ligand complexes, consistent with
a key role for both residues in the binding of substrates
and inhibitors (Table 1; Fig 2) Anaerobic
stopped-flow studies of FMN reduction by NADPH in H181A
and H184 enzymes indicated reduction of the FMN,
but unlike for wild-type enzyme [19] there was no evi-dence at 560 nm for an oxidized enzyme-NADPH charge-transfer intermediate prior to FMN reduction (Fig 3A) Plots of observed rate constant vs NADPH concentration were hyperbolic for the mutant enzymes, and limiting rate constants for FMN reduction were elevated modestly compared with wild-type PETN reductase (Fig 3A, inset; Table 2) The exchange of His181 and His184 residues by alanine, however, com-promised binding of reducing coenzyme in the active site (Table 2) Stopped-flow studies in which 2-electron reduced enzyme (obtained by titration against dithio-nite) was mixed with 2-cyclohexen-1-one indicated that both the H181A and H184A enzymes are able to trans-fer electrons to 2-cyclohexen-1-one (Fig 3B) In both cases, single wavelength stopped-flow studies at 450 nm established that observed rate constants for FMN oxi-dation were hyperbolically dependent on 2-cyclohexen-1-one concentration (Fig 3B) Kinetic parameters derived from fitting to a standard hyperbolic expression are given in Table 2 The limiting rate constants for oxidation of FMNH2 by 2-cyclohexen-1-one in the H181A and H184A enzymes are substantially less than that measured for the wild-type enzyme We suggest that mutation perturbs the binding geometry such that
A
B
Fig 1 (A) Superposition of steroid com-plexes bound to wild-type oxidized penta-erythritol tetranitrate (PETN) reductase The bound steroids are prednisone, progester-one and 1,4-androstadiene-3,17-diprogester-one (B) Sequence of PETN reductase and some PETN reductase related enzymes in the region of His181 and His184 The proteins are PETN (PETN reductase from Entero-bacter cloacae), MR (MR from Pseudomo-nas putida), OYE1 (Old Yellow Enzyme 1 from brewer’s bottom yeast), OYE2, OYE3 (two isoforms of Old Yellow), EBP1 (estro-gen binding protein from Candida albicans), NER A (glycerol trinitrate reductase from Agrobacterium radiobacter), NEM A (N-ethyl-maleimide reductase from Escherichia coli), OPDA (12-oxophytodienoate reductase from Arabidopsis thaliana), BAIH (bile acid-indu-cible protein from Eubacterium species), NADH (NADH oxidase from Thermobacillus brockii) The arrows indicate the positions of histidine residues in PETN reductase inferred to be involved in ligand binding and counterpart residues in related enzymes and also the location of the tyrosine residue implicated as proton donor in OYE.
Table 1 Dissociation constants for enzyme-ligand complexes
calcu-lated from equilibrium titrations Enzymes (each 10 l M ) were
titra-ted with picric acid, progesterone and 2,4-dinitrophenol in 50 m M
potassium phosphate buffer, pH 7.0, in a 1 mL quartz cuvette.
Spectra were recorded after the addition of ligand to the enzyme.
From the resultant spectra (examples shown in Fig 2) the
absorp-tion changes at 518 nm were plotted as a funcabsorp-tion of ligand
con-centration and fitted to a hyperbolic or quadratic function from
which the dissociation constants were determined Dissociation
constants for wild-type enzyme-ligand complexes are taken from
[2,19].
Dissociation constant (K d , l M )
Picric acid 2,4-Dinitrophenol Progesterone
Wild-type PETN reductase 5.4 ± 1.1 0.95 ± 0.10 0.07 ± 0.03
H181A PETN reductase 92 ± 12 56 ± 7 16 ± 5
H184A PETN reductase 73 ± 16 34 ± 6 15 ± 3
Y186F PETN reductase 11 ± 1 1.9 ± 0.5 0.05 ± 0.09
Trang 4the reducible carbon double bond of the substrate is
less optimally positioned with respect to the flavin N5
atom compared with the geometry in the wild-type
enzyme
Stopped-flow studies were extended to include the
nitroaromatic compound TNT as oxidizing substrate
TNT is a known substrate for the wild-type enzyme,
and the kinetics of FMN oxidation with wild-type
PETN reductase have been reported [19] With
wild-type enzyme, reduction of TNT follows two parallel
pathways; the first pathway involves direct reduction of
the nitro group (the nitroreductase pathway), whereas
the second pathway involves hydride transfer from
FMNH2 to the aromatic nucleus of the substrate to
form a hydride-Meisenheimer product (further details
in [19,20]) The hydride-Meisenheimer product is
readily detected in stopped-flow studies using a photo-diode array detector as it has a strong absorption band around 560 nm [19] Photodiode array studies of the oxidative half-reaction of the H181A and H184A enzymes indicated that the hydride-Meisenheimer com-plex is not formed However, TNT was able to oxidize the flavin resulting in recovery of the oxidized FMN absorption spectrum (Fig 3C) Single wavelength stud-ies at 453 nm indicated a hyperbolic dependence of the flavin oxidation rate on TNT concentration (Fig 3D); limiting rate constants and reduced enzyme-TNT disso-ciation constants are presented in Table 2 The reduc-tion potentials of the FMN centres in the H184A and H181A enzymes were determined to be )266 ± 5 mV and )229 ± 5 mV, respectively, which compares with
a value of)267 mV for wild-type enzyme [20]
Fig 2 Analysis of ligand binding by equilibrium titration studies (A) UV-visible analysis of the titration of H181A PETN reductase with 2,4 dinitrophenol Inset, detail of the absorbance change around 518 nm used to calculate the dissociation constant for the complex (B) UV-visi-ble analysis of the titration of H181A PETN reductase with progesterone (C) Plot of absorbance change as a function of 2,4 dinitrophenol concentration for the data shown in (A) (D) Plot of absorbance change as a function of progesterone concentration for the data shown in (B) Conditions for panels A and B: 50 m M potassium phosphate buffer pH 7.0, 25 C Similar plots were generated for other enzyme-ligand combinations Dissociation constants for the enzyme–ligand complexes are given in Table 1 Arrows indicate direction of absorption change with time.
Trang 5Properties of the Y186F PETN reductase
Equilibrium binding measurements with picric acid,
2,4-dinitrophenol and progesterone were performed as
described for wild-type and the H181A and H184A
enzymes Spectral changes were similar to those
repor-ted above and absorption changes at 518 nm were
used to determine enzyme-ligand dissociation constants
(Table 1) Enzyme-ligand dissociation constants for
nitroaromatic ligands are elevated approximately
two-fold compared with wild-type enzyme, whereas the
binding of progesterone is essentially unaffected by
mutation (Table 1)
Stopped-flow studies of flavin reduction by NADPH
produced mono exponential reaction traces at 464 nm
and observed rate constants were independent of coen-zyme concentration (concentration range 200–1300 lm NADPH) The rate constant for flavin reduction (9 s)1) is similar to that measured for wild-type enzyme (12 s)1) at 5C Absorption changes at 560 nm showed a rapid increase in absorption followed by a slower decay, consistent with the formation and sub-sequent collapse of an oxidized enzyme–NADPH charge-transfer complex (Fig 4A) The rapid forma-tion of the charge-transfer intermediate prevented accurate analysis of the rate constant for its formation
by the stopped-flow method; the rate constant for col-lapse of the charge-transfer species was similar to that measured for hydride transfer at 464 nm, indicating that both processes are kinetically equivalent
Waveleng h t ( n m ) 0
0
5
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1
.
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5
.
0
2
.
0
[ βNADPH ] ( M )
-1 )
0 0 0 0 0 0
[ - Cycl h 2 o exen n o e ] ( mM)
-1 )
0
1 0
2 0
3 0
4 0
5 0
[ N T T ] ( µ ) M
-1 )
0 5 0 5 0
Waveleng h t m) ( n 0
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Fig 3 Stopped-flow kinetic analysis of FMN reduction and oxidation in the H181A and H184A PETN reductases (A) spectral changes accompanying the reduction of H184A PETN reductase (20 l M ) by NADPH (200 l M ) Four hundred spectra were recorded over a period of one second One in every 25 of the spectra is displayed Conditions: 50 m M potassium phosphate buffer, pH 7.0, at 5 C Inset: plot of observed rate constant for FMN reduction as a function of NADPH concentration for H181A (filled squares) and H184A (filled circles) (B) dependence of the observed rate for the oxidation of reduced FMN on 2-cyclohexen-1-one concentration for the H181A (filled squares) and H184A (filled circles) PETN reductases (C) Spectral changes accompanying the oxidation of 2-electron reduced H184A PETN reductase (20 l M ) by TNT (200 l M ) (D) dependence of the observed rate for the oxidation of reduced FMN on TNT concentration for the H181A ( )
and H184A (d) PETN reductases Kinetic parameters are given in Table 2 Arrows indicate direction of absorption change with time.
Trang 6Stopped-flow studies of the oxidative half-reaction
of Y186F PETN reductase (reduced at the 2 electron
level with dithionite) with 2-cyclohexen-1-one revealed
a hyperbolic dependence of the rate of flavin oxidation
on 2-cyclohexen-1-one concentration (Fig 4D) The
limiting rate constant for flavin oxidation is twofold
less than that for wild-type enzyme (Table 2)
Stopped-flow studies of the oxidative half-reaction of Y186F
PETN reductase with TNT indicated formation and
subsequent decay of the hydride Meisenheimer
com-plex (Fig 4B,C), indicating that residue Y186 does not
influence strongly the partitioning along the hydride
transfer and nitroreductase pathways The rate of
Meisenheimer complex formation measured at 560 nm
was shown to be equivalent to that for FMN oxidation
at 464 nm, indicating that both processes are
kinetic-ally equivalent Potentiometric titrations indicated that
the reduction potential for the concerted 2-electron
)265 ± 5 mV, which is comparable to that measured
previously for wild-type enzyme ()267 mV; [20])
Structures of the Y186F PETN reductase, and
reduced wild-type enzyme in complex with
progesterone
The 1.0 A˚ Y186F structure is essentially identical to
the wild-type structure (PDB code 1VYR) with the
obvious exception of the mutation A bound
thio-cyanide ion and iso-propanol molecule can be seen
occupying the substrate-binding site adjacent to the
FMN (Fig 5) PETN reductase can reduce a⁄ b
unsaturated steroids with a double bond located between C1 and C2; the double bond between C4 and C5 is not susceptible to reduction, and thus progesterone and related steroids (e.g 4-androstene-3,17-dione), which lack a double bond between C1 and C2, are inhibitors of PETN reductase (Fig 6) The overall structure of the 1.05 A˚ reduced PETN-progesterone complex is virtually identical to the oxidized complex (PDB code 1H60) Two molecules
of isopropanol could be resolved and are bound between the progesterone and the protein (Fig 7A)
In comparison to the oxidized structures, the FMN isoalloxazine ring is less planar, with both the N5 atom and the C8-C7 methyl groups moving to signi-ficantly out-of-plane positions The progesterone sub-strate is bound in a similar manner, albeit shifted by approximately 0.4 A˚ towards the Thr26 side chain This places the progesterone C4—C5 double bond in close proximity to the FMN N5 atom (distances 3.47 and 3.61 A˚ for C4-N5 and C5-N5, respectively) While this distance is ideal for reduction of the dou-ble bond by FMN, the N10-N5-C5 angle value of 92 (Fig 7B) is distinct form the range of angles (125 to 170) observed in flavoenzyme-substrate complexes [31] Any putative motion of the pro-gesterone molecule required to increase the N10-N5-C5 angle to within or close to the 125 to 170 range causes severe steric clashes of the C6 carbon atom with the Thr26 side chain, explaining why progesterone is an inhibitor rather than a substrate for this enzyme The presence of Cb atom at posi-tion 26 therefore causes the substrate specificity of
Table 2 Kinetic parameters for the reductive and oxidative half-reactions of wild-type, H181A, H184A and Y186F PETN reductases Kinetic data are shown in Figs 3 and 4 Parameters were determined by fitting to a standard hyperbolic expression to obtain values for the limiting rate of flavin reduction or oxidation (k lim ) and the enzyme-substrate dissociation constants (K d ) All reactions were performed in 50 m M potas-sium phosphate buffer, pH 7.0 ND, not determined Owing to the very rapid formation of the charge-transfer intermediate and the small absorption changes it was not possible to evaluate the dissociation constant for the oxidized enzyme-NADPH charge-transfer species from analysis of its rate of formation as a function of NADPH concentration.
Oxidative half-reaction
a Data taken from [19].
Trang 7the enzyme to be limited to enones with a primary
or secondary b-carbon such as steroids with a double
bond located between C1 and C2 However, crystal
structures of the oxidized enzyme in complex with
C1-C2 unsaturated steroids reveal a mode of binding
similar to that observed for progesterone, placing the
C4-C5 bond rather than the reactive C1-C2 bond in
close proximity of the N5 atom A putative flipping
motion of the progesterone molecule along an axis
parallel to the FMN plane aligns the C1-C2 with the
plane of the isoalloxazine ring and in close proximity
to the N5, with possibility of adopting a conforma-tion with an appropriate N10-N5-C1 angle for cata-lysis Previous NMR data and modelling suggested that the preferred orientation of steroid substrates (with a C1-C2 double bond) might be different dependent on the oxidation state of the protein [2] Similar behaviour was also proposed recently for Escherichia coli nitroreductase in complex with the antibiotic nitrofurazone [32] Our present data
Time (s) 1
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[2-Cyclohexenone] (mM)
-1 )
0 2 4 6 8 0 2 4 6
Fig 4 (A) Typical stopped-flow transient obtained for the reductive half-reaction of Y186F PETN reductase at 464 nm The absorption trace was measured by mixing NADPH (200 l M ) with Y186F PETN reductase (20 l M ) in 50 m M potassium phosphate buffer, pH 7.0, at 5 C Inset: the small absorption change observed at 560 nm indicating formation of an oxidized enzyme-NADPH charge-transfer species over 0.02 s Measurements over longer time periods indicate the absorption decreases with a rate constant similar to that observed at 464 nm, which indicates reduction of the FMN Conditions: 50 m M potassium phosphate buffer, pH 7.0, at 5 C (B) Time-dependent spectral chan-ges following the reaction of dithionite-reduced Y186F PETN reductase and TNT, using stopped-flow spectroscopy Dithionite-reduced Y186F PETN reductase (40 l M ) was mixed with (400 l M ) TNT at 25 C, in 1% acetone, potassium phosphate buffer, pH 7.0, under anaer-obic conditions Spectral changes are shown over 2 s, and indicate re-oxidation of the flavin and the formation of the hydride-Meisenheimer complex of TNT (C) As for panel B except the spectral changes are recorded over an extended time period and show degradation of the hydride-Meisenheimer complex which generates the oxidized form of the enzyme (D) The concentration-dependence of the rate of hydride transfer from dithionite-reduced Y186F PETN reductase to 2-cyclohexen-1-one Anaerobic single wavelength spectroscopy at 464 nm was performed with 20 l M enzyme and a range of 2-cyclohexen-1-one concentrations, in 50 m M potassium phosphate buffer, pH 7.0 at 25 C The solid line shows the fit of the data to a hyperbolic function Kinetic parameters are given in Table 2 Arrows indicate direction of absorp-tion change with time.
Trang 8strongly indicate that the preferred steroid binding
mode is independent of enzyme redox state and is in
a nonproductive conformation for C1-C2 unsaturated
steroids The slow observed turnover values with
these steroids are therefore a likely consequence of
the low concentration of enzyme complexes in the
productive conformation with the C1-C2 bond
optimally aligned for hydride transfer There are two models for productive binding of steroid substrates:
in the first, a (small) sub population of enzyme binds directly the steroid in the reactive conformation, with the remainder of the enzyme binding the unreactive conformation Rescue of the unreactive conformation involves release of steroid and re-binding in the reactive conformation In the second model, the ster-oid flips its conformation from the unreactive to reactive binding mode whilst resident in the enzyme active site At this stage, we cannot categorically rule out either model However, flipping within the active site would require a change in steroid conformation
A
B
Fig 7 Structure of the reduced PETN reductase-progesterone complex (A) Active site of the reduced protein–progesterone com-plex Progesterone binds to reduced enzyme in the same con-formation as observed for the oxidized enzyme, i.e with the nonreducible C4-C5 double of the steroid positioned close to the flavin N5 The reduced protein-progesterone atoms are displayed in coloured sticks, with the oxidized protein-progesterone overlayed for comparison and displayed in white sticks The sigmaA weighted
F 0 -F c density for the progesterone molecule is displayed in blue (B) schematic displaying the steric clashes between enones with tertiary Cb atoms and the Cb atom at position 26 for N10-N5-Cb angles in the range of 125 to 170.
R
A
C
B
O
O
O
O
O
1
2
3
4
5
6
7
8
9
0
1
2
3
4 5 6 7
g r
P e s t e r o e
A P H + H + A P +
i
d
t
s
o
r
d
a
-4
,
1 e e 3 , 7 - d i o e 4 - a d r o s t e e - 3 , 7 - d i o e
Fig 6 Steroid nomenclature and chemical structures (A)
Nomen-clature for atom labelling in 3-oxo steroids; (B) chemical structure
of the inhibitor progesterone; (C) reaction catalysed with the
sub-strate 1,4-androstadiene-3,17-dione.
Fig 5 Superposition of the thiocyanide complex structures for
Y186F PETN reductase and wild-type PETN reductase The
struc-ture of Y186F PETN reductase (shown in atom coloured sticks) is
similar to the wild-type enzyme (shown in white), confirming that
the mutation of Tyr186 to Phe186 does not grossly perturb the
overall framework of the enzyme or the active site The active sites
of the enzymes are shown in stick format with the sigmaA
weigh-ted 2F 0 -F c density for the Y186F mutant displayed in blue.
Trang 9and is hindered by a number of unfavourable steric
interactions with the protein We suggest therefore
that flipping does not occur within the active site
and that the productive conformation is formed
directly by the binding of free steroid from solution
Discussion
A number of crystal structures are now available for
members of the Old Yellow Enzyme family [2,3,24,33–
35] Each of these indicates that the counterpart
resi-dues of His181 and His184 of PETN reductase are
implicated in ligand binding Solution studies with MR
[25] and OYE1 [26], which contain a His-Asn pair at
this position support this inferred role, and the data
reported in this paper indicate that the His181 and
His184 pair are likewise determinants for the binding
of coenzyme, nitroaromatic ligands, steroids and
2-cyclohexen-1-one In this regard, the structural
simi-larity seen in the Old Yellow Enzyme family members
is consistent also with similar functional roles An
unexpected finding, however, is that mutation of
Tyr186 to phenylalanine in PETN reductase does not
prevent reduction of 2-cyclohexen-1-one In OYE1, the
counterpart residue (Tyr196) has been shown to be the
key proton donor required for reduction of
2-cyclo-hexen-1-one and related substrates, and its mutation
leads to inactivation of the oxidative half-reaction [22]
Clearly, in PETN reductase proton transfer is not from
Tyr186 despite (i) the very similar structural
architec-ture of OYE1 and PETN reductase, and (ii) geometry
for the binding of steroid compounds and
2-cyclohe-xen-1-one [2,19] The structure of the Y186F enzyme is
essentially identical to that of wild-type PETN
reduc-tase, which therefore rules out major structural change
as a result of mutation that might have been
respon-sible for the recruitment of a surrogate proton donor
in the oxidative half-reaction The overall structural
similarities of OYE1 and PETN reductase previously
led us to propose that Tyr186 (PETN reductase) might
function in a role analogous to that of Tyr196 (OYE1)
[2] We now conclude, however, that as with MR
[25,36], proton transfer is most likely from water This
therefore highlights subtle mechanistic differences
between family members despite their overall structural
similarity No significant differences in position of
Y186⁄ 196 and nearby residues can be observed for
PETN reductase and available OYE structures (PDB
codes 1OYA, 1OYB) Detailed comparison of water
molecules in or near the active site is precluded due to
either lack of corresponding complexes with identical
molecules bound in the active site or sufficiently high
resolution data for the OYE complexes
The structure of the 2-electron reduced form of PETN reductase bound to progesterone is very similar
to that of the oxidized enzyme-progesterone complex Moreover, in these inhibitor complexes the steroid is bound in the active site in a similar manner to the bind-ing of steroid substrates in oxidized PETN reductase
We noted previously from structures of oxidized PETN reductase in complex with steroid substrates that the C1-C2 reactive bond of the steroid substrate is inappro-priately aligned with the flavin N5 atom to enable hydride transfer from the flavin in the reduced form of the enzyme [2] This led us to propose that steroid sub-strates would need to flip by 180, such that the react-ive bond was optimally aligned with the flavin N5 atom
in the catalytically relevant 2-electron reduced form of the enzyme-steroid complex We conjectured that the reduced form of the enzyme might direct binding of the steroid in the flipped conformation, thus facilitating catalysis Our structure of the 2-electron reduced form
of PETN reductase in complex with progesterone, how-ever, indicates this is not the case, and that the reduc-tion state of the flavin does not ‘signal’ a change in the binding geometry Shifts in substrate position following reduction of a cofactor have been postulated in other enzyme systems, including cytochrome P450 BM3 [37] and nitroreductase [32], in an attempt to reconcile the apparent conflict between the postulated structure of the catalytically active reduced enzyme-substrate com-plex and the observed crystallographic structures of the oxidized enzyme-substrate complex However, in some cases, the substrates studied are not closely related to the physiological substrates and one cannot assume such compounds are good mimics of the physiological substrate It is probable that in both redox states the majority of substrate-enzyme complexes adopt the crystallographically determined structure and that the catalytically active conformation is populated to only a small extent and therefore represents a less sta-ble form of the reduced enzyme-substrate complex The preferred binding mode that we observed in our crystal-lographic studies is catalytically incompetent, and ster-oid must either be released from the active site to allow binding in the active configuration, or flipping of the steroid must occur whilst resident in the active site An
in situ flipping mechanism seems unlikely given the extensive steric clashes and change in steroid conforma-tion that must occur to allow rotaconforma-tion through 180, and we therefore favour a ‘release-and-rebinding’ mechanism The low occupancy of the catalytically competent form of the enzyme-substrate complex no doubt contributes to the very low turnover numbers for the PETN reductase-catalysed reduction of steroid substrates [2]
Trang 10Experimental procedures
Chemicals
All chemicals were of analytical grade where possible
Com-plex bacteriological media were from Unipath Ltd
(Basing-stoke, UK), and all media were prepared as described in
Sambrook et al [27] Mimetic Orange 2 affinity
chromato-graphy resin was from Affinity Chromatochromato-graphy Ltd
(Cam-bridge, UK) Q-Sepharose resin was from Amersham
Biosciences (Piscataway, NJ, USA) NADPH, glucose
6-phosphate dehydrogenase, glucose 6-phosphate, benzyl
viologen, methyl viologen, 2-hydroxy-1,4-naphthaquinone,
phenazine methosulfate and 2,4 dinitrophenol were from
Sigma (St Louis, MO, USA) 2-cyclohexen-1-one was from
Acros Organics (Geel, Belgium) S Nicklin (UK Defence and
Evaluation Research Agency) supplied TNT The following
extinction coefficients were used to calculate the
concentra-tion of substrates and enzyme: NADPH (e340¼ 6.22 · 103
m)1 cm)1); PETN reductase (e464¼ 11.3 · 103 m)1 cm)1)
Stock solutions of TNT (600 mm) were made up in acetone
Dilutions were then made into 50 mm potassium
phos-phate buffer, pH 7.0, and the acetone concentration was
maintained at 1% (v⁄ v) The presence of acetone in buffers
at 1% (v⁄ v) was shown not to affect enzyme activity
Mutagenesis and purification of enzymes
Site-directed mutagenesis was achieved using the
Quik-change mutagenesis method (Stratagene, La Jolla, CA,
USA) and the following oligonucleotides: 5¢-TTCACTCTG
CGCACGGTTTTCTGCTGCATCAGTTC-3¢ (Y186F
for-ward primer), 5¢-GAACTGATGCAGCAGAAAACCGTG
CGCAGAGTGAA-3¢ (Y186F, reverse primer), 5¢-CTTCG
ACCTGGTTGAGCTTGCGTCTGCGCACGGTTACCTG-3¢ (H181A forward primer), 5¢-CAGGTAACCGTGCGCG
ACGCAAGCTCAACCAGGTCGAAG-3¢ (H181A, reverse
primer), 5¢-GTTGAGCTTCACTCTGCGGCGGGTTACC
TGCTGCATCAG-3¢ (H184, forward primer) and 5¢-CTG
ATGCAGCAGGTAACCCGCCGCAGAGTGAAGCTCA
AC-3¢ (H184, reverse primer) Plasmid pONR1 [1] was used
as template for mutagenesis reactions All mutant genes
were completely sequenced to ensure that spurious changes
had not arisen during the mutagenesis reaction The
expres-sion and purification of the wild-type and mutant PETN
reductase enzymes was as described previously for wild-type
enzyme [1] Owing to poor retention by the Mimetic
Orange 2 affinity chromatography resin used for
purifica-tion of wild-type PETN reductase, the H181A and H184A
enzymes were purified by Q-Sepharose ion exchange
chro-matography (pre-equilibrated with 50 mm Tris⁄ HCl buffer,
pH 7.5; buffer A) The enzymes were subsequently eluted
using a salt gradient (0–200 mm NaCl contained in 500 mL
buffer A) Following an initial purification using
Q-Seph-arose resin a second chromatography step using the same resin was required (gradient of 15–150 mm NaCl contained
in 1 L buffer A) to obtain pure protein
Redox potentiometry, ligand binding and stopped-flow kinetic analyses
Redox titrations and the determination of redox potential
of the enzyme-bound FMN were performed as described previously for wild- type PETN reductase [19] Ligand binding studies were also as performed previously with wild-type PETN reductase [19], relying on the perturbation
of the flavin electronic absorption spectrum on binding lig-and in the active site of PETN reductase Data were collec-ted in the UV-visible region (250–600 nm), and the absorption at 518 nm plotted as a function of ligand con-centration Data for the wild-type enzyme were analysed by fitting to the quadratic function (Eqn 1)
DA¼DAmax 2ET
ðLTþ ETþ KdÞ ðL Tþ ETþ KdÞ2 ð4LTETÞ0:5
ð1Þ where DAmax is the maximum absorption change at
518 nm, LT is the total ligand concentration and ET the total enzyme concentration Data for mutant enzymes were calculated by fitting to the standard hyperbolic expression (Eqn 2)
DA¼DAmaxLT
Kdþ LT
ð2Þ
Rapid reaction kinetic experiments using single wavelength absorption and photodiode array detection were performed Table 3 Crystallographic data and refinement statistics.
Red enzyme– progesterone complex Crystal properties
Spacegroup P212121 P212121 Cell dimensions (A ˚ ) a ¼ 56.6
b ¼ 68.6
c ¼ 88.6
a ¼ 58.2
b ¼ 68.6
c ¼ 88.4 Data collection
Completeness (%) 98.2 (84) 99.1 (98.4)
Resolution 15–1.0 (1.03–1.0) 15–1.05 (1.08–1.05) Refinement
RMSD bond lenghts (A ˚ ) 0.017 0.016