All enzymes for which data are available have been found to comprise at least three components: 1 multisubunit binuclear iron active centre-containing terminal oxygenase, where oxygen ac
Trang 1common reaction pathways and component interactions among bacterial hydrocarbon oxygenases
William L J Fosdike1, Thomas J Smith1,2and Howard Dalton1
1 Department of Biological Sciences, University of Warwick, Coventry, UK
2 Biomedical Research Centre, Sheffield Hallam University, UK
Alkene monooxygenase (AMO) (EC 1.14.13.69), of
Rhodococcus rhodochrous (formerly Nocardia corallina)
B-276 belongs to a family of soluble multicomponent
oxygenases that possess a binuclear iron active centre
[1–5] This group of enzymes includes AMOs from two
other bacterial sources [6–8], the soluble methane
monooxygenases (sMMOs) (EC 1.14.13.25) produced
by certain methanotrophic bacteria [9] and a range of
oxygenases that perform aromatic ring hydroxylations with a variety of specificities [3,10] All enzymes for which data are available have been found to comprise
at least three components: (1) multisubunit binuclear iron active centre-containing terminal oxygenase, where oxygen activation and substrate oxygenation occur; (2) an NAD(P)H-dependent reductase that sup-plies electrons to the active centre of the terminal
Keywords
alkene monooxygenase; alkyne; component
interactions; peroxide shunt reaction;
turnover-dependent inhibition
Correspondence
H Dalton, Department of Biological
Sciences, University of Warwick, Coventry
CV4 7AL, UK
Fax: +44 24 76523568
Tel: +44 24 76523552
E-mail: h.dalton@warwick.ac.uk
Website: http://www.bio.warwick.ac.uk/res/
frame.asp?id=4;
http://www.shu.ac.uk/schools/sci/biomed/
bmrc/tomsmith.htm
(Received 24 January 2005, revised
14 March 2005, accepted 21 March 2005)
doi:10.1111/j.1742-4658.2005.04675.x
Alkene monooxygenase (AMO) from Rhodococcus rhodochrous (formerly Nocardia corallina) B-276 belongs to a family of multicomponent nonheme binuclear iron-centre oxygenases that includes the soluble methane mono-oxygenases (sMMOs) found in some methane-oxidizing bacteria The enzymes catalyse the insertion of oxygen into organic substrates (mostly hydrocarbons) at the expense of O2 and NAD(P)H AMO is remarkable in its ability to oxidize low molecular-mass alkenes to their corresponding epoxides with high enantiomeric excess sMMO and other well-character-ized homologues of AMO exhibit two adventitious activities: (1) turnover-dependent inhibition by alkynes and (2) activation by hydrogen peroxide in lieu of oxygen and NAD(P)H (the peroxide shunt reaction) Previous stud-ies of the AMO had failed to detect these activitstud-ies and opened the possi-bility that the mechanism of AMO might be fundamentally different from that of its homologues Thanks to improvements in the protocols for culti-vation of R rhodochrous B-276 and purification and assay of AMO, it has been possible to detect and characterize turnover-dependent inhibition of AMO by propyne and ethyne and activation of the enzyme by hydrogen peroxide These results indicate a similar mechanism to that found in sMMO and also, unexpectedly, that the enantiomeric excess of the chiral epoxypropane product is significantly reduced during the peroxide shunt reaction Inhibition of the oxygen⁄ NADH-activated reaction, but not the peroxide shunt, by covalent modification of positively charged groups revealed an additional similarity to sMMO and may indicate very similar patterns of intersubunit interactions and⁄ or electron transfer in both enzyme complexes
Abbreviations
AMO, alkene monooxygenase; sMMO, soluble methane monooxygenase; sulfo-NHS-acetate, sulfosuccinimidyl acetate.
Trang 2oxygenase; and (3) a small component, known as the
coupling or gating protein, which is also required for
full activity [3–5] The terminal oxygenase components
of soluble methane monooxygenases [9,11], aromatic
monooxygenases [10] and the AMO of Xanthobacter
sp strain Py2 [12] all have an (abc)2 quaternary
struc-ture, whereas the AMOs from R rhodochrous B-276
[13] and Mycobacterium sp [8] lack the c subunit In
addition, the aromatic monooxygenases and AMOs
from Xanthobacter Py2 and Mycobacterium sp also
possess a Rieske iron-sulfur protein that appears to
pass electrons between the reductase and terminal
oxygenase [12,14–16]
The binuclear iron-centre monooxygenases are
char-acterized by close biochemical and, apparently,
struc-tural similarities There is complete conservation of
the protein ligands to the binuclear iron site (four
glu-tamyl and two histidyl residues) and all catalyse
similar hydrocarbon monooxygenation reactions at the
expense of NADH and dioxygen [3–5] The enzymes
show a gamut of substrate ranges and regio- and
stereo-selectivities For instance, the substrate range of
AMO from R rhodochrous B-276 is restricted almost
exclusively to alkenes [17], whereas sMMOs have a
remarkably wide range of substrates that includes
alka-nes, alkenes and aromatic compounds [18] Consistent
with their different enzymatic activities, the terminal
oxygenase components of AMOs are termed
epoxygen-ases as their products are epoxides; the equivalent
components of the other enzymes add hydroxyl groups
to their natural substrates and are therefore known as
hydroxylases There are also important differences in
the enantiopurity of products obtained from
oxygen-ation of alkene substrates: AMO from R rhodochrous
B-276 epoxygenates propene to R-epoxypropane
with high enantiomeric excess (83%) [1,13], whereas
sMMO produces a nearly racemic mixture of products
with the same substrate (S.E Slade, T.J Smith and
H Dalton, unpublished observations)
In addition to these oxygenation reactions, several
well characterized binuclear iron-centre
monooxygen-ases have been found to exhibit two adventitious
reac-tions: turnover-dependent inhibition by alkynes and
the so-called peroxide shunt reaction Terminal alkynes
such as ethyne have been shown to act as suicide
sub-strates of sMMO [19], soluble butane monooxygenase
[20], several aromatic monooxygenases [21,22] and the
AMO from Xanthobacter sp Py2 [12,23], presumably
by oxygenation to ketenes that then covalently modify
and inactivate the enzymes [19] Irreversible inhibition
of the heme active-site monooxygenases of the
cyto-chrome P450 family, which are not related to the
binu-clear iron centre-containing enzymes, may also proceed
via similar ketene intermediates [24] The peroxide shunt reaction allows the terminal oxygenase compo-nent to perform oxygenation reactions in the absence
of other protein components and NAD(P)H, if the oxidant is hydrogen peroxide rather than dioxygen The peroxide shunt has been observed in sMMO [25,26] and toluene 2-monooxygenase [27], as well as unrelated monooxygenases of the cytochrome P450 family [28] In sMMO it has been shown that the whole-complex (i.e NADH-dependent) and peroxide-activated activities can be functionally separated by covalently modifying positively charged groups on the hydroxylase, which inhibits the whole-complex reaction but not the peroxide shunt [29]
Neither of these adventitious activities has to our knowledge previously been reported in the rhodococcal AMO The rhodococcal AMO was previously found to
be inhibited by propyne, but the inhibition appeared
to be competitive because Km for propene oxygen-ation was increased by the inhibitor but Vmax was unchanged [1] This, together with the lack of any pub-lished account of the peroxide shunt reaction in the rhodococcal AMO, opened the possibility of funda-mental differences in reaction mechanism and⁄ or sub-strate selectivity between AMO and its homologues In addition to their possible mechanistic significance, these inferences had potential implications for com-mercial application of the enzyme An AMO that was not destroyed by alkynes would be tolerant of alkyne contamination of the alkene feedstock, whereas one that did not exhibit the peroxide shunt reaction could not be economically employed in a cell-free system without a means for regeneration of NADH In the context of a whole-cell biocatalyst that would bypass problems with coenzyme regeneration, we began the present study of AMO by investigating the effect of alkynes on AMO-dependent growth of R rhodochrous B-276 Subsequently, by refining the purification, inhi-bition and activity assay protocols we have been able
to produce large amounts of pure high-activity AMO and to perform a more thorough investigation of its interaction with alkynes and hydrogen peroxide than has previously been possible
Results
Alkynes strongly inhibit growth of
R rhodochrous B-276 on propene The effect of alkynes on whole-cell systems expressing AMO was investigated by monitoring the growth of flask cultures of R rhodochrous B-276 in the presence
of a range of alkynes (Fig 1) A fivefold molar excess
Trang 3of propene over the alkyne was used as the carbon and
energy source to ensure that cell growth was dependent
on AMO The observed complete inhibition of growth
in the presence of propyne and but-1-yne was
qualita-tively a much more severe effect than was expected from
the relatively low level (£ 70%) of competitive inhibition
that the previous data implied [1] and suggested either
that these alkenes were inhibiting AMO to a greater
extent than had been observed in the purified enzyme
experiments or that some other essential metabolic
pro-cess was inhibited by alkynes Even more remarkably,
ethyne (which barely inhibited purified AMO in the
previous study [1]), caused significantly delayed growth
and reduced growth rate relative to the control
Preincubation of AMO with alkyne and NADH
allowed turnover-dependent inhibition to be
observed
In order to explain the unexpectedly large effect of
alkynes on the growth of AMO-expressing R
rhodo-chrous B-276 cells on propene, the effect of alkynes
on the activity of purified AMO was re-examined
Previously, the inhibition properties of AMO were
investigated in reactions in which propene and the
alkyne were present simultaneously and were added
before NADH, which is essential for turnover of
the AMO complex [1,13] Hence, although the effect
of propyne in causing an increase in Km with respect
to propene without significant change in Vmax was
consistent with reversible competitive inhibition,
turn-over-dependent and -independent events could not
be distinguished Consequently, the nature of the
inhibition could not be unambiguously assigned To
resolve this uncertainty, purified AMO components
and NADH were preincubated aerobically in the pres-ence of ethyne or propyne (5% v⁄ v in the headspace) and subsequent assay of the propene oxygenation activity after removal of the alkyne clearly demonstra-ted irreversible inhibition at 80% (Table 1) It was clear that this inhibition was predominantly turnover-dependent because omission of NADH during the pre-incubation phase completely abolished it Conversely, when a much higher concentration of ethyne (50%
v⁄ v, corresponding to an increase in calculated liquid-phase concentration from 1.8 mm to 18 mm) and pro-pene were added at the same time, before NADH, no inhibition was observed during a 10-min assay A sim-ilar assay using propyne (35% v⁄ v, corresponding to
an increase in calculated liquid-phase concentration from 3.1 mm to 22 mm) in place of ethyne resulted
in only a 50% reduction in epoxide formation over a 10-min assay, relative to the control in which nitrogen was substituted for the alkyne The fact that the pres-ence of propene protects against inhibition by the alky-nes is consistent with the alkyalky-nes’ acting as suicide substrates which compete for the same active site on the enzyme as the natural substrate propene
Residual AMO activity was measured as a function
of the time between the start of the reaction (addition
of NADH) and removal of the alkyne by flushing with nitrogen First-order decay of enzyme activity was observed, from which first order decay constants for the inactivation of the enzyme by propyne and ethyne under these conditions could be calculated (Fig 2) It
is likely that the linear part of the graph in Fig 2 does not cross the ordinate at the position corresponding to the uninhibited enzyme activity because the measured reaction times do not include the time taken to remove the alkyne during the flushing process and are there-fore uniformly underestimated The positive deviation
of the latest data points from the extrapolated linear
Fig 1 Effect of alkynes on the growth of R rhodochrous B-276
using propene as the growth substrate Cultures were incubated
aerobically in the presence of propene plus nitrogen (solid symbols,
solid line), ethyne (solid symbols, dotted line), propyne (open
sym-bols, solid line) and but-1-yne (open symsym-bols, dotted line).
Table 1 Turnover-dependent inhibition of AMO by alkynes Preincubation before assay a Percent activity b
a
Reactions contained 8 l M of epoxygenase and 12 l M each of reductase and GST-coupling protein fusion in a total volume of
100 lL; the headspace concentration of alkyne was 11% (v ⁄ v) and the reaction was preincubated for 10 min before removal of the alkyne and assaying with propene as the substrate, in the presence
of O2 and NADH, as described in the Experimental procedures.
b Specific activities are given as percentages of the uninhibited activity of 192 nmolÆmin)1Æ(mg of epoxygenase))1.
Trang 4portion possibly reflects the exhaustion of alkyne later
in the reaction, if a number of turnovers are required
per inactivation event
Improved epoxygenase preparations allowed
detection of the AMO-catalysed peroxide shunt
reaction
Investigation of the effect of hydrogen peroxide on
AMO was facilitated by the improved protocols for
growth of R rhodochrous B-276 and purification of the
binuclear iron site-containing epoxygenase component
described in the Experimental procedures Use of pro-pene as the sole carbon and energy source ensured a high level of AMO expression by making cell growth dependent on AMO The subsequent purification pro-tocol yielded > 95% pure epoxygenase with a specific activity of at least 145 nmolÆmin)1Æ[mg of protein])1, which was more than twice as active as previous pre-parations produced from cells grown in rich medium [1,13] In addition, the use of smaller assay reaction volumes (0.1 mL rather than 0.5 mL used previously) enabled extensive investigations to be carried out at high epoxygenase concentrations Thanks to these improvements, the peroxide shunt reaction was found
to be detectable and easily quantifiable at 40 lm epoxygenase after only 3 min reaction time, using
500 mm hydrogen peroxide and propene at 37% (v⁄ v)
in the headspace gas The specific activity of the hydrogen peroxide-activated reaction under these con-ditions was 32 nmolÆmin)1Æ[mg of epoxygenase])1, i.e 22% of the NADH-dependent reaction catalysed by the whole AMO complex The amount of epoxypro-pane produced was unchanged when the reaction was purged of oxygen by flushing with oxygen-free nitro-gen for 5 min before addition of the hydronitro-gen per-oxide, showing that the reaction did not require molecular oxygen Formation of product was depend-ent on the presence of hydrogen peroxide and, more importantly, active epoxygenase Omission or prior heat-denaturation (100C, 5 min) of the epoxygenase completely abolished formation of epoxypropane under otherwise identical conditions
The rate of epoxypropane formation via the peroxide shunt reaction was linear with epoxygenase concentra-tion between 20 and 60 lm (data not shown) When a reaction time of 3 min was used, the reaction rate was linear with hydrogen peroxide concentration up to 1.0 m, the highest concentration tested, suggesting that the Kmfor hydrogen peroxide was considerably greater than 1 m This contrasts with the lower value of Kmfor hydrogen peroxide of 66 mm estimated from experi-ments with the corresponding hydroxylase component
of sMMO [25] When longer reaction times were used, the average rate did not increase beyond about 500 mm hydrogen peroxide (Fig 3), probably because of pro-gressive inactivation of the enzyme by higher concentra-tions of hydrogen peroxide in a manner that was also observed with sMMO [25] The AMO epoxygenase-catalysed peroxide shunt reaction showed moderate inhibition by the coupling protein component of AMO (29 ± 8% inhibition relative to an activity of
30 ± 2 nmolÆmin)1Æ[mg of epoxygenase])1at a coupling protein⁄ epoxygenase molar ratio of 3 : 1), again similar
to the result obtained with sMMO [25]
Fig 2 Kinetics of inactivation of AMO by (A) propyne and (B)
ethyne Alkynes were added to the headspace at 6.25% (v ⁄ v), which
was calculated to give equilibrium aqueous-phase concentrations
of propyne and ethyne of 3.9 and 2.2 m M , respectively Residual
AMO activity was measured after removal of the alkyne using
pro-pene as the substrate and are derived from single-timepoint
meas-urements of the product after 10 min reaction time; 100% activity
corresponded to 153 nmolÆmin)1Æ(mg of epoxygenase))1 Error bars
show standard deviation from three experiments First order decay
constants during the exponential decay periods were 0.083 min)1
and 0.13 min)1for propyne and ethyne, respectively The zero-time
measurement is derived from enzyme that was not exposed to the
alkyne.
Trang 5The peroxide shunt reaction in AMO showed low
stereoselectivity even in the presence of the
coupling protein
When epoxypropane produced via the whole-complex
reaction was subjected to chiral analysis, the
R-enantio-mer predominated with an enantioR-enantio-meric excess >80%,
consistent with the 83% previously observed [1] In
contrast, epoxypropane produced by the AMO
epoxy-genase via the peroxide shunt reaction, whilst still
showing a predominance of the R-enantiomer, had an
enantiomeric excess of only 25%
In sMMO from Methylosinus trichosporium OB3b
[30], it has been observed that the protein B
compo-nent, equivalent to the coupling protein of AMO,
influences the regioselectivity of the enzyme If the
coupling protein influenced product distribution in
AMO also, it was possible that the low
stereoselecti-vity observed via the peroxide shunt was due to the
absence of the coupling protein It was found that the
presence of the coupling protein (up to a threefold
molar excess relative to the epoxygenase) had no
signi-ficant effect on the chiral composition of the
epoxypro-pane product in the peroxide shunt reaction, although
the possibility that the coupling protein was damaged
by the high concentration of hydrogen peroxide used
in this experiment cannot be excluded
Modification of positively charged residues on
the surface of the epoxygenase allowed
functional separation of the whole-complex
and peroxide shunt activities
As treatment of the hydroxylase component of
sMMO (equivalent to the epoxygenase of AMO)
with reagents specific for positively charged moieties
completely inhibited the whole-complex reaction but not the peroxide shunt [29], covalent modification of AMO afforded an additional test of its biochemical similarity to sMMO When the epoxygenase of AMO was reacted with the primary amine-specific reagent sulfo-NHS-acetate, the whole complex reac-tion was abolished whilst the activity via the per-oxide shunt was unaffected, as in the sMMO system The control experiment described in the Experimental procedures confirmed that the specific inactivation of the whole-complex reaction was due solely to the effect of the sulfo-NHS-acetate on the epoxygenase component and not due to the effect of any carried over reagent during the assay reaction A similar functional separation of the peroxide shunt and whole-complex reactions was observed by using the arginine-specific reagent p-hydroxyphenylglyoxal, which did not significantly inhibit the peroxide shunt reac-tion but resulted in progressive inactivareac-tion of the whole-complex reaction (data not shown) These results suggested that accessible positively charged residues were necessary for interactions between the enzyme components but not per se for substrate oxy-genation at the active site Consistent with the hypo-thesis that protein–protein interactions between the AMO components require positive charges on the epoxygenase, chemical modification of the coupling protein with sulfo-NHS-acetate or p-hydroxyphenyl-glyoxal had no effect on its activity in the whole-complex propene oxygenation reaction (data not shown)
Discussion
The observations that the rhodococcal AMO, like other binuclear iron-centre monooxygenases, exhibits turnover-dependent inhibition by alkynes and can be activated by hydrogen peroxide support a unified mechanism for oxygenation reactions catalysed by this family of enzymes The results of Gallagher et al [1], where it was observed using partially purified AMO that propyne increased the apparent Km with respect
to propene but left Vmax unchanged, can now be reinterpreted as showing that competition between the substrate propene and the inhibitor propyne not only alleviates inhibition at high substrate concentration by preventing propyne from blocking the active site but in
so doing also protects the enzyme from the irreversible inhibition that would result from turnover of the alkyne The relatively small amount of inhibition of growth and AMO activity seen when ethyne was pre-sent at the same time as propene are consistent with the conclusion that the two-carbon ethyne competes
Fig 3 Kinetics of peroxygenation of propene catalysed by the
AMO epoxygenase Activity was measured by quantifying
epoxy-propane formation as described in the Experimental procedures,
using 40 l M epoxygenase and reaction times of 3 min (solid line),
10 min (dotted line) and 15 min (dashed line) Error bars show
standard deviations from three experiments.
Trang 6less well with propene for the active site than the
three-carbon propyne The approximately twofold
dif-ference in solubility of the two alkynes would not be
expected to account for the gross difference in
inacti-vation that was observed in competition with the
pro-pene substrate
As it is now clear that the adventitious activities of
AMO are broadly similar to those of other binuclear
iron-centre monooxygenases, the range of activities
exhibited by this family of enzymes can be explained
by differences in substrate binding and the ability of
the highly oxygenating species at the active site to
acti-vate high energy C–H bonds such as those in benzene,
methane and other alkanes The fact that AMO can be
activated via hydrogen peroxide is consistent with the
presence of a binuclear iron–peroxo intermediate
sim-ilar to that found during the catalytic cycle of sMMO
[31,32] Whether, as previously proposed [33], AMO is
unable to oxidize methane because it lacks the
charac-teristic and probably diferryl (FeIV
2) intermediate Q observed in sMMO must await future studies
Whatever the structural differences that underlie the
difference in active-site reactivity of the binuclear
iron-centre monooxygenases, it appears to be relatively
subtle as far as the immediate environment of the
binuclear iron site is concerned Not only are the
ligat-ing residues perfectly conserved throughout the group
[3] but also the recent structure of the hydroxylase
component of toluene⁄ o-xylene monooxygenase has
indicated that the only appreciable difference between
the active-centre ligation environments of this enzyme
and sMMO, which share only 24% identity in their
a-subunits, are relatively minor differences in active
site hydrogen bonding patterns and the coordinating
nitrogen atom of one histidine ligand [34]
The results obtained with the reagents specific for
positively charged groups reveal an additional level
of similarity between AMO and sMMO that allows
functional separation of the whole-complex and
per-oxide shunt reaction in both enzyme systems In
both systems, dioxygen activation and⁄ or functional
interactions between the enzyme components require
accessible positively charged moieties on the terminal
oxygenase, whereas the actual process of oxygen
insertion into the substrate does not There may be
at least two positively charged moieties, perhaps
unidentified conserved residues, that are
independ-ently necessary for electron transfer and⁄ or
intersub-unit interactions in each enzyme because primary
amine- and arginine-specific reagents have similar
effects in both systems
The diminished enantiomeric excess of
epoxypro-pane production observed when AMO was activated
via the peroxide shunt reaction is reminiscent of the altered substrate specificity and product distribution seen in the peroxide shunt reaction in sMMO [25,30] In sMMO from Methylosinus trichosporium OB3b, the coupling protein component has been shown to influence the regioselectivity of oxygenation reactions [30] and mutagenesis and modelling studies have suggested that the coupling protein interacts directly with the hydroxylase active site and controls substrate entry [35] The binding site for the coup-ling protein of AMO may be similarly positioned on the epoxygenase However, unless the coupling pro-tein is damaged by the high concentration of hydro-gen peroxide used, presence of the coupling protein
is clearly not the sole determinant of enantioselective catalysis in AMO because addition of the coupling protein to the AMO epoxygenase-catalysed peroxide shunt reaction reduced the overall reaction rate but did not increase the enantiomeric excess of the prod-uct Whilst the operation of the peroxide shunt reaction in AMO presents an opportunity for devel-opment of an AMO-based biocatalyst that is inde-pendent of reduced coenzyme, the low product enantiomeric excess obtained via the peroxide shunt presents an important new question about the origin
of enantioselectivity in this enzyme and a challenge
to novel applications of AMO for chiral synthesis
Experimental procedures
Bacterial strains and growth conditions
R rhodochrousB-276 was cultivated at 30C in nitrate min-imal salts liquid medium or on nitrate minmin-imal salts agar [36], using propene as the sole carbon and energy source Batch cultures for analysis of the effect of alkynes on growth were performed in 1-L cultures in 2-L conical flasks
(Quick-fit, Fisher, Loughborough, UK), sealed with Subaseal rub-ber seals (W Freeman, Barnsley, UK) After inoculation,
600 mL of headspace gas was removed and replaced by
500 mL of propene, plus 100 mL of the appropriate alkyne
or nitrogen, as stated for each experiment Growth was monitored over a 5-day incubation at 30C with shaking (180 r.p.m.) by measuring the OD600 of samples removed through the seal with a hypodermic syringe Large-scale con-tinuous growth of R rhodochrous B-276 for purification of the AMO epoxygenase and reductase component was achieved by using a 2000 Series fermentor (LH Engineering, Stoke Poges, UK) with a working volume of 4 L and a dilu-tion rate of 0.035 h)1 The culture was gassed with a 1 : 10 (v⁄ v) propene ⁄ air mixture at a flow rate of 1 LÆh 1, agitated
at an imepellor speed of 450 r.p.m and maintained at
pH 7.0 The Escherichia coli strain used to produce the
Trang 7recombinant glutathione S-transferase (GST)-coupling
pro-tein fusion was cultivated as described previously [37]
Purification of the AMO components
All protein purification procedures were conducted at
0–4C For purification of the AMO epoxygenase (i.e
ter-minal oxygenase) and reductase components, cells from
20 L of propene-grown R rhodochrous B-276 culture at an
OD600of 12–15 were harvested at 3000 g using a Westfalia
continuous centrifuge (Northvale, NJ, USA), washed by
centrifugation (10 000 g, 10 min) and resuspension in
10 mm MgSO4, 5% (v⁄ v) glycerol, 25 mm Mops pH 7.5
and resuspended in a minimal volume of the same buffer
containing benzamidine (1 mm), dithiothreitol (1 mm) and
a total glycerol concentration of 15% (v⁄ v) (buffer B)
Deoxyribonuclease I (Sigma, Gillingham, UK) was added
to 20 lgÆmL)1 and the cells were broken by passing twice
through a high-pressure cell disrupter (172 MPa; Constant
Systems, Warwick, UK), after which the cell-free extract
was centrifuged (48 000 g, 1 h) and the supernatant (the
soluble extract) was removed
The soluble extract was applied to a DE52 anion
exchange column (Whatman, Maidstone, UK; 5 cm·
15 cm), previously equilibrated with buffer B After
wash-ing with buffer B, the column was eluted with a step
gradi-ent of 150 and 250 mm NaCl in buffer B The reductase
was purified from the 250-mm NaCl fraction as described
previously [13] The 150-mm NaCl fraction was
concentra-ted to a volume of 200 mL using a 900 cm2 surface-area
spiral-wound ultrafiltration cartridge (Amicon, Stonehouse,
UK) with a molecular size cut-off of 10 kDa MgSO4was
added to a final concentration of 0.5 m and then the
epoxygenase-containing solution was applied to a Phenyl
Sepharose high-performance column
(Amersham-Pharma-cia, Little Chalfont, UK; 2.6· 12 cm) previously
benzamidine and dithiothreitol, each at 1 mm) containing
0.5 m MgSO4 Proteins were eluted with a linear gradient
of 0.5–0 m MgSO4 in buffer D Fractions containing
epoxygenase activity, which eluted at 0 mm MgSO4, were
adjusted to an MgSO4concentration of 10 mm and
concen-trated using a 30 kDa molecular size cut-off membrane
(Amicon) The phenyl Sepharose column was washed with
0.7% (w⁄ v) sodium cholate after use to remove residual
protein and prevent loss of epoxygenase yield during
subse-quent preparations The concentrated epoxygenase was
applied to a Mono Q anion exchange column
(Amersham-Pharmacia; 1 · 10 cm) that had been equilibrated with
25 mm Mops pH 7.5 containing 15% (v⁄ v) glycerol and
10 mm MgSO4 and then proteins were eluted with a
lin-ear gradient of 0–400 mm NaCl in the same buffer The
pure epoxygenase eluted at 300 mm NaCl
The coupling protein used throughout this study, which
was a recombinant GST-coupling protein fusion that is
fully active with the GST tag attached, was produced in Escherichia coli and purified by affinity chromatography [37]
AMO assays and inhibition studies
AMO assays and alkyne inhibition reactions were per-formed in 100-lL reaction volumes in 1.7-mL crimp-seal glass vials For measurement of activity via the whole-complex AMO reaction, the three AMO components were mixed with 25 mm Mops pH 7.5 containing 10 mm MgSO4
to give 8 lm epoxygenase, 12 lm reductase and 12 lm coupling protein The vial was then sealed and 0.7 mL of the headspace gas was removed and replaced with 0.7 mL
of propene, after which the vial was preincubated at 30C for 30 s The reaction was initiated by adding NADH (to
100 lm) and the vial was shaken (180 r.p.m.) at 30C for a further 3 min, unless otherwise stated, before the epoxypro-pane formed was quantified by gas chromatography of 0.5-mL gas phase samples, as described previously [1] Activity assays of the individual AMO components during purification were performed in the presence of an excess of the other two components Peroxide shunt assays were per-formed in a similar manner to the whole-complex reactions except that reductase and (except where stated otherwise) the coupling protein were omitted and the reaction was started by adding hydrogen peroxide instead of NADH The reaction time and concentrations of protein compo-nents and hydrogen peroxide are stated for each experi-ment
Inhibition of the AMO complex by alkynes was achieved as follows Epoxygenase (8 lm), reductase (12 lm) and coupling protein (12 lm) were mixed and the reaction vial was sealed Headspace gas was removed and replaced with an equal volume of the alkyne, to give the alkyne concentration stated for each experiment Concen-trations of alkynes in the aqueous phase were calculated using Henry’s law and Henry’s constants of 0.068 mÆatm)1 [38] and 0.037 mÆatm)1 (http://www.mpch-mainz.mpg.de/
sander/res/henry.html), respectively, for propyne and eth-yne The vial was preincubated with shaking at 30C for
30 s and then, except where stated otherwise, NADH was added to 100 lm Turnover-dependent inhibition was then allowed to proceed under the same incubation conditions, for 10 min unless stated otherwise Unreacted alkyne was removed by flushing with nitrogen for 5 min; the vial was then flushed with air and the remaining AMO activity was assayed by replacing 0.7 mL of the headspace gas with 0.7 mL of propene and incubating at 30C for a fur-ther 10 min, before removing 0.5 mL of the headspace gas for quantitation of the epoxypropane product by gas chro-matography as above Residual propene oxidation activity was measured after 10 min reaction with the alkyne, at
a range of concentrations, in the presence of NADH (100 lm) The decay of enzyme activity as a function of
Trang 8time was analysed by plotting the natural logarithm of the
residual activity against time, to yield the apparent
first-order decay constant, k_app, from the slope of the linear
portion of the graph [39]
Chiral analysis
Reactions for analysis of chiral composition were scaled up
to 1-mL volume and incubated at 30C until the
epoxypro-pane concentration was about 2 mm Epoxyproepoxypro-pane was
extracted using 200–300 lL of diethyl ether, which was
dried using molecular sieve and analysed by means of
capil-lary GC using a Phillips 4500 GC apparatus fitted with a
Chiraldex (20 m· 0.25 mm) a-cyclodextrin trifluoroacetate
column (Advanced Separation Technologies, Whippany,
NJ, USA) The injection volume was 1 lL, the split ratio
was 100 : 1 and the carrier gas (nitrogen) flow rate was
1.3 mLÆmin)1 The injector and flame ionization detector
were maintained at 150C and the column temperature
was 30C
Modification of positively charged groups
Covalent modification of the AMO epoxygenase and
coup-ling protein was effected using protocols based on those
used with sMMO [29], as follows Accessible primary
amine groups were modified by reacting the epoxygenase or
coupling protein (10 mgÆmL)1) with a 13-fold molar excess
of sulfosuccinimidyl acetate (sulfo-NHS-acetate; Pierce,
Rockford, IL, USA) at room temperature for 30 min
Un-reacted sulfo-NHS-acetate was removed by buffer exchange
via three cycles of ultrafiltration [using a Microcon
centrifu-gal concentrator (Amicon)] and dilution In the case of the
epoxygenase, a 30 kDa cut-off membrane was used and
each time the sample was diluted with a volume equal to
the original sample volume of 25 mm Mops pH 7.5
con-taining 15% (v⁄ v) glycerol In the case of the coupling
pro-tein, the membrane had a 10 kDa cut-off and glycerol was
omitted from the dilution buffer The reaction between the
epoxygenase and sulfo-NHS-acetate abolished the activity
of the epoxygenase in the AMO whole-complex reaction
and so a control was performed to confirm that the
observed inhibition was due to reaction of the
sulfo-NHS-acetate with the epoxygenase and not due to reaction of
carried over reagent with the other AMO components
Here, the epoxygenase was not added to the reaction until
after the sulfo-NHS-acetate had been removed by buffer
exchange No inhibition of the reaction was observed,
con-firming that the buffer exchange procedure was effective in
removing the sulfo-NHS-acetate and showing that
inhibi-tion required contact between the epoxygenase and
sulfo-NHS-acetate Modification of accessible arginyl side-chain
guanidinium groups was effected by reacting the
epoxyge-nase or coupling protein (10 mgÆmL)1) with
p-hydroxyphe-nylglyoxal (12 mm) for 20 min at room temperature, after
which the reaction was quenched by adding arginine to
60 mm
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