The enzyme does not show significant NADH peroxidase activity and is not active in the reduction of glutathi-one, cystine, or 3¢ dephospho-CoA disulfide unlike the staphylococcal enzyme, w
Trang 1reductase from Pyrococcus horikoshii
Implications for the disulfide metabolism of anaerobic
hyperthermophiles
Dennis R Harris*, Donald E Ward1, Jeremy M Feasel2, Kyle M Lancaster2, Ryan D Murphy2,
T Conn Mallet3and Edward J Crane III2
1 Genencor International, Palo Alto, CA, USA
2 Department of Chemistry, Pomona College, Claremont, CA, USA
3 Center for Structural Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA
While surveying the genomes of hyperthermophilic
and thermophilic Archaea for homologues of the
flavoprotein disulfide reductases, many homologues
with a high degree of identity to the branch of this
family represented by glutathione reductase were
found [1] Most of the homologues appear to belong
to the subfamily that depend on a redox-active single cysteine, analogous to the NADH oxidase and per-oxidase of Enterococcus and the coenzyme A disulfide reductase (CoADR; EC 1.8.1.14) of Staphylococcus
Correspondence
E J Crane III, Department of Chemistry,
Pomona College, 645 North College
Avenue, Claremont, CA 91711, USA
Fax: +1 909 607 7726
Tel: +1 909 607 9634
E-mail: ej.crane@pomona.edu
Website: http://www.userwebs.
pomona.edu/ejc14747/EJ_web.htm
*Present address
Department of Biochemistry, University of
Wisconsin-Madison, Madison, WI, USA
(Received 20 July 2004, revised 7 December
2004, accepted 4 January 2005)
doi:10.1111/j.1742-4658.2005.04555.x
We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P furiosus, and purified the recombinant form of the P horikoshii enzyme to homogeneity from Escherichia coli Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H+fi 2CoA-SH + NAD(P)+) The P horikoshii enzyme shows a kcat app of 7.2 s)1 with NADPH at 75C While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal kcats, while the Kmfor NADPH is roughly eightfold lower than that for NADH The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephos-pho-CoA Anaerobic reductive titration of the enzyme with NAD(P)H pro-ceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2form
of the enzyme Addition of a second of NADPH results in the formation
of an apparent FAD-NAD(P)H complex The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source P furio-suscells were assayed for small molecular mass thiols and found to contain 0.64 lmol CoAÆg dry weight)1 (corresponding to 210 lm CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents
Abbreviations
CoADR, coenzyme A disulfide reductase (EC# 1.8.1.14); pfCoADR, P furiosus coenzyme A disulfide reductase; phCoADR, P horikoshii coenzyme A disulfide reductase; DTNB, 5,5¢ dithiobis(2-nitrobenzoic acid); EH 2 , two-electron reduced enzyme; EH 4 , four-electron reduced enzyme; HEPPS, N-(2-hydroxyethyl)piperazine-N¢-3-propanesulfonic acid; NOX, NADH oxidase; NPX, NADH peroxidase; TCA, trichloroacetic acid.
Trang 2aureus [2,3] These enzymes are proposed to be
involved in the robust oxygen-defense systems of
aerobic and facultatively anaerobic organisms [4,5]
and would not be expected to be present in the
mostly strictly anaerobic hyperthermophiles Evidence
is mounting, however, for the presence of a vigorous
oxidative stress response in Pyrococcus, including the
discovery of both a novel peroxide-producing
super-oxide reductase [6] and an NADH oxidase [1]
Addi-tionally, an oxidative stress response has been
characterized in the strictly anaerobic bacterium
Clos-tridium perfringens [7]
Microorganisms in the genus Pyrococcus are strictly
anaerobic hyperthermophiles (Topt¼ 100 C) isolated
from marine hydrothermal vents [8–10] The genomes
of P horikoshii, P furiosus and P abyssii each contain
at least two NADH oxidase homologues
Characteriza-tion of one of these homologues (NOX1) from
P furiosus has been described previously [1] NOX1
shows a novel H2O and H2O2producing NADH
oxid-ase activity in both the absence and presence of
exo-genous FAD The second NOX homologue examined
(from P horikoshii, previously referred to as NOX2)
showed a slow NAD(P)H oxidase activity in the
pres-ence of high concentrations of substrate-level FAD,
i.e., in addition to the enzyme bound FAD The results
described here demonstrate that this enzyme is not
likely to act as an NADH oxidase in vivo, instead
act-ing as a CoADR This is only the second
demonstra-ted CoA reductase activity, and the first appearance of
this activity in both the Archaea and in a strict
anaer-obe While the best known small molecular mass thiol
is probably glutathione, a number of novel thiols such
as mycothiol, c-glutamyl cysteine, and trypanithione
have been found in microorganisms [11] The function
of these thiols appears to be the maintenance of a
reducing intracellular environment Due to the
pres-ence of a CoADR homologue in all three pyrococcal
genomes, P furiosus cells were assayed for the
pres-ence of small molecular mass thiols in order to better
understand the role of this enzyme and thiol⁄ disulfide systems in pyrococcal metabolism The results presen-ted below provide an insight into the use of a small molecular mass thiol system for the maintenance of the internal redox environment in an anaerobic hyper-thermophile
Results
Characterization of the recombinant CoADR The recombinant CoADR from P horikoshii (phCo-ADR) was purified 15-fold with a yield of 58% and
a specific activity of 3.26 UÆmg)1 (oxidase activity) (Table 1) and 8.3 UÆmg)1 (CoADR activity) The phCoADR had a subunit m¼ 50 k as determined by SDS⁄ PAGE, and was shown by both HPLC and con-ventional size-exclusion chromatography to be a tetra-meric enzyme of m ¼ 198 k The enzyme obtained from the overexpression host is approximately 20% holoenzyme After reconstitution with FAD the enzyme contains 0.92 flavin per subunit based on the ratio of protein concentration to flavin concentration (as determined at 460 nm) blast and tfasta analysis
of the phCoADR revealed a significant level of identity
to putative NADH oxidases from hyperthermophiles and bacterial NADH oxidases from mesophilic sources (Fig 1) Of particular interest was the identity to the well characterized NOXs from mesophilic organisms, with the highest levels of identity found with the NADH oxidases from E faecalis (28%), Streptococcus mutans (26%), and Brachyspira (Serpulina) hyodysente-riae(24%) The CoADR from S aureus is 26% identi-cal to the P horikoshii CoADR As shown in Fig 1, the P furiosus NOX1 and the pf and ph CoADRs con-tain a cysteine which corresponds to the single redox active cysteine of the E faecalis NOX and NPX, as well as considerable identity in areas that have been shown to be important for NADH and FAD binding
in these enzymes
Table 1 Purification of the recombinant coenzyme A disulfide reductase from P horikoshii For the purposes of this table, purification was monitored by the FAD-dependent NADH oxidase activity of the enzyme, with a unit equal to the amount of enzyme required to oxidize
1 lmol NADH in 1 min in the presence of 100 lm NADH, 100 lm FAD, in 50 mm potassium phosphate, pH 7.50 at 75 C The specific activity of the purified enzyme in terms of coenzyme A disulfide reductase activity is 8.3 UÆmg)1.
Fraction Total units Total protein (mg)
Specific activity (unitsÆmg)1) Purification (fold) Yield (%)
Heat-treated extract 134 77.2 1.73 7.83 68.0
Size-exclusion 114 35.0 3.26 14.8 57.8
Trang 3In order to determine the extinction coefficient of
the enzyme-bound FAD, the FAD was released from
the holoenzyme by trichloroacetic acid (TCA)
precipi-tation of the protein The e460 of the enzyme-bound
FAD was determined to be 10 200 m)1Æcm)1 It is
interesting to note that attempts to remove the FAD
from the enzyme by treatment in 6.0 m guanidine⁄ HCl
were unsuccessful, even with overnight incubation at
90C This result is consistent with the extreme stabil-ity of this enzyme, and consistent with the observation that thermostable proteins, including the NOX1 from
P furiosus, are frequently stable in organic solvents and in the presence of denaturants [1] The visible spectrum of the enzyme as purified (Fig 2) has the same distinct shoulder in the area of 470 nm as the mesophilic staphylcoccal enzyme [12]
Fig 1 Multiple sequence alignment of
P horikoshii and P furiosus CoADRs to
known NADH oxidases ⁄ NADH peroxidase ⁄
CoADR The alignment was performed with
CLUSTAL W The GenBank accession numbers
for the other enzymes are as follows:
Staphylococcus aureus CoADR (AF041467),
E faecalis NOX (P37061), E faecalis NPX
(P37062), P furiosus NOX (PF_1430634).
Trang 4CoADR activity of the recombinant P furiosus
CoADR homologue
The CoADR from P horikoshii has a homolouge in
P furiosus that is 92% identical (Fig 1), indicating
that this gene product was also likely to be a CoADR
The recombinant P furiosus CoADR (pfCoADR) was
expressed in E coli Heat-treated FAD-reconstituted
extracts of the putative pfCoADR showed a strong
CoADR activity corresponding to a specific activity of
11 UÆmg)1 of heat-treated extract This result
com-pares favorably with the specific activity obtained
dur-ing the production of the recombinant CoADR from
P horikoshii Like the phCoADR, the pfCoADR is
active with both NADH and NADPH CoADR
activ-ity was also detected in crude extracts of P furiosus,
however, the level of activity was low and difficult to
distinguish from background reactions
Steady-state kinetics of the CoADR and oxidase
reactions
The kinetic constants for the CoADR and oxidase
activities of phCoADR are listed in Table 2 The
enzyme does not show significant NADH peroxidase
activity and is not active in the reduction of
glutathi-one, cystine, or 3¢ dephospho-CoA disulfide (unlike the
staphylococcal enzyme, which has a 40 lm Kmfor the
dephospho substrate) The enzyme also does not show
significant 5,5¢ dithiobis(2-nitrobenzoic acid) (DTNB)
reductase in a standard aerobic steady-state kinetic
assay; however, in the anaerobic assays of free thiols
discussed below the enzyme does show DTNB turn-over with NADPH at a very high concentration of enzyme (30–60 lm) While NADPH is the preferred substrate for the CoADR activity based on Km, the two nucleotide substrates have almost identical kcats This result is quite distinct from the substrate specifi-city observed with the staphylococcal enzyme, which shows a marked preference for NADPH [12]
While phCoADR shows a low level of NAD(P)H oxidase activity (in the absence of CoA disulfide) in the absence of substrate level FAD (i.e., FAD added
in addition to that present in the enzyme-bound form),
a significant amount of oxidase activity can be observed in the presence of additional substrate-level FAD (Table 2) The kcat app obtained in the presence
of 100 lm NADH and FAD and 115 lm O2 is 8.2 s)1, which correlates well with the catalytic constant observed for the CoADR reaction
Thermostability and thermoactivity of the CoADR phCoADR is stable for months at both )80 C and )20 C, and has half-lives of > 100 and 39 h at 85 and 95C, respectively Figure 3 shows the dependence
of the oxidase and phCoADR activities on tempera-ture While both activities show the preference for high temperature expected of an enzyme from a hyper-thermophile, at temperatures above 75C both activit-ies appear to plateau slightly, rather than increasing all the way to the optimal growth temperature for Pyro-coccus
Anaerobic reduction with NAD(P)H and redox state of the proposed cysteine nonflavin redox center
As shown in Fig 2, when phCoADR is titrated anaero-bically at 60C with NADPH the titration shows two main phases, each corresponding to the addition of
300
0.6
0.4
0.2
0
Wavelength, nm
0
NADPH, eq
A600.7200.040 0.080
Fig 2 Anaerobic reductive titration with NADPH at 60 C 56 l M
phCoADR in 50 m M potassium phosphate at pH 7.50 was titrated
with NADPH Spectra are shown at 0 (—), 1.1 (- - -) and 2.0 (ÆÆÆÆ)
NADPH Inset, Change in absorbance at 600 (s) and 720 (d) nm
during titration.
Table 2 Michaelis constants for the P horikoshii CoADR, deter-mined at 75 C in 50 m M potassium phosphate, pH 7.50.
Activity-substrate Km app(l M ) kcat app(s)1) CoADR-NADHa 73 8.1 CoADR-NADPH a < 9.0 7.2 CoADR-CoANaCl ⁄ CitoA b 30 7.1 Oxidase-NADHc 73 8.2 Oxidase-NADPH c 13 2.0 Oxidase-FAD d 22 5.9
a Determined at 200 lm CoA-S-S-CoA, b determined at 100 lm NADPH, cdetermined at 100 lm FAD, ddetermined at 100 l M
NADH.
Trang 5roughly one equivalent of NAD(P)H At 720 nm the
majority of the change occurs during the addition of
the first equivalent of the reducing agent as seen in
Fig 2 (inset) Little change is observed at 361 nm
dur-ing addition of the first equivalent of NADPH, while
an increase is observed during the addition of the
sec-ond and subsequent equivalents of NADPH With the
exception of the expected increase at 340 nm from the
addition of free NADPH, little additional change is
seen in the spectra when NADPH is added to a total of
six equivalents No reduction of the FAD is observed
during addition of excess NAD(P)H Anaerobic
titra-tion of phCoADR with NADH shows very similar
spectral results to those obtained with NADPH (results
not shown) During anaerobic reduction with dithionite
(data not shown) the FAD becomes reduced upon the
addition of a second equivalent of dithionite, consistent
with initial reduction of a nonflavin redox center,
fol-lowed by reduction of the FAD by the strongly
redu-cing dithionite
While the spectral changes upon addition of
NADPH appear to correlate well with enzyme states
corresponding to the addition of roughly 1 and 2
equivalents of NADPH, it is difficult to determine
directly from the spectrum the fate of the reduced
pyr-idine nucleotide During the addition of the first
equiv-alent of NADPH, there is a blue shift in the 380 nm
peak and subsequent small increase in absorbance at
340 nm, although the increase is much less than that
expected for the amount of NADPH added (the
increase corresponds to the addition of 15 lm
NADPH, when 50 lm NADPH had been added)
When the titration is performed at room temperature,
no additional absorbance is observed at 340 nm during the addition of the first equivalent of NADPH con-firming that the pyridine nucleotide is consumed during the addition of the first equivalent (data not shown)
Determination of free thiol content
In order to characterize the redox state of the pro-posed active site cysteine on the enzyme as purified and after addition of 0.8 equivalent of NADPH, as well as any small molecular mass thiols released by the enzyme, we used the thiol specific reagent DTNB Because the enzyme contains only one cysteine residue, there is only one possible reactive thiol on the enzyme,
in addition to any small molecular mass thiol trapped
in the form of a mixed-disulfide with the cysteine (Cys-S-S-R) The results of these experiments are shown in Table 3 The enzyme as purified contains 0.00 equiva-lents of DTNB reactive thiol Following anaerobic reduction with NADPH, less than 0.01 equivalent of small molecular mass thiol was detected, indicating that little if any of the enzyme is purified in the mixed disulfide form If the NADPH reduced enzyme is kept anaerobic and assayed for thiol content, 0.85 equival-ent of thiol is detected, indicating that reduction by NADPH produces a reactive thiol If the enzyme is exposed to air following reduction, this thiol becomes unreactive, suggesting that it is rapidly oxidized to the sulfenic acid
Determination of CoA levels and relative stability
in P furiosus
To determine whether CoA might play a role as a pool
of reducing equivalents in Pyrococcus, as suggested by the presence of a CoADR homologue in all three pyrococcal genomes, cells of P furiosus were assayed for small molecular thiols CoA was present at a con-centration of 0.64 lmol CoAÆg dry weight)1, which corresponds roughly to 210 lm CoA in the cell (assu-ming a wet⁄ dry weight ratio of 3 : 1 [13]) Glutathione was not detected CoA was present almost entirely
100
75
50
25
0
Temperature
Fig 3 NADH oxidase (d) and CoA disulfide reductase (j) activity
of phCoADR at varying temperatures Activities at 81.3 (oxidase)
and 85.0 C (disulfide reductase) were set at 100%.
Table 3 Equivalents of free thiol, as detected by DTNB.
Equivalents
of free thiol phCoADR, as purified 0.00 NADPH reduced phCoADR, anaerobic 0.85 Small molecular mass thiol
released upon NADPH reduction
0.01 NADPH reduced, exposed to air 0.00
Trang 6in the thiol form, as there was < 5% detected in the
disulfide form, as determined by the concentration of
(thiol + disulfide)–(thiol)
CoA has previously been shown to be four times
more stable than glutathione and 180 times more
sta-ble than cysteine to the Cu2+-catalyzed oxidation to
the disulfide form in the presence of oxygen [14]
Because CoA is significantly more complex than either
cysteine or glutathione, we were interested in
determin-ing whether the increased stability to overoxidation
extends to higher temperatures For this reason, the
oxidation of thiols at a high temperature was assayed
It was found that CoA oxidizes approximately 4.4
times slower than glutathione at 88C in 50 mm
imi-dazole buffer, 1.0 lm Cu2+ pH 7.50, as shown in
Fig 4 (under these conditions cysteine oxidizes too
quickly to be measured using conventional techniques)
Under conditions in which the copper and any trace
contaminating metals were chelated with EDTA and
phosphate (50 mm potassium phosphate, 0.50 mm
EDTA, pH 7.50), no significant oxidation of CoA was
observed over 100 min at 88C
Discussion
The phCoADR is only the second CoADR
character-ized to date, and is the first from a hyperthermophile
and Archaeon Moreover, the enzyme displays distinct
differences from the S aureus CoADR The enzyme is
quite distinct from the staphylococcal enzyme in its
ability to use both NADPH and NADH as substrates
With the S aureus enzyme, substitution of NADH as
the reducing nucleotide gives approximately 17% of the rate observed with NADPH [12], while the pyro-coccal enzyme gives almost identical kcat values with either reduced nucleotide substrate The Kmappof < 9 and 73 lm for NADPH and NADH, respectively, do differ significantly, with NADPH appearing to be the more efficient substrate
The reduced nucleotide concentrations have been determined for P furiosus [15], and while the total concentration of NAD(P)(H) was found to be about half the value seen in the mesophilic Salmonella typhimurium, the pyrococcal NADP(H)⁄ NAD(H) ratio was found to be more than twice that found in
S typhimurium This result is not surprising, given the presence of several unique NADP+-dependent cata-bolic enzymes present in Pyrococcus [15] The kcat of 7.2 s)1determined for the phCoADR with NADPH as the reducing substrate is similar to the kcat of 27 s)1 observed for the staphylococcal enzyme, although it is lower [12]
Stabilization of enzyme intermediates Different enzymes within the disulfide reductase family stabilize different intermediates upon anaerobic reduc-tion with their nucleotide substrates The H2 O-produ-cing NADH oxidases are reduced by their nucleotide substrates by four electrons to an EH4 or EH4ÆNAD+ form, as shown in Scheme 1 [16,17] In this case the first reducing equivalent reduces the active site cysteine residue, which serves as a nonflavin redox center, from the oxidized sulfenic acid to a reduced thiol species This 2e– reduced form of the enzyme is referred to as the EH2 species [17] Addition of a second equivalent
of reduced nucleotide substrate results in reduction
of the enzyme-bound FAD, forming the EH4 or
EH4ÆNAD(P)+species In the case of a true oxidase, it makes sense for the enzyme to stabilize the EH4 form, since the reduced flavin is reactive with O2
Other enzymes in the family, such as the NADH peroxidase or glutathione reductase, tend to stabilize the enzyme-reduced nucleotide complex referred to
as EH2ÆNAD(P)H (Scheme 1) [18,19] On the EH2Æ NADH complex of the NADH peroxidase from
E faecalis[20] (Npx: NADH + H++ H2O2fi NAD+ + 2H2O), the reducing equivalents of NADH are held
in the nicotinamide ring on the re-side of the flavin isoalloxazine ring with the C4 position of the nicotina-mide ring located 3.49 A˚ from N5 of the isoalloxazine ring The active site cysteine thiolate is 3.48 A˚ from N5 on the si-side of the flavin, which allows for oxida-tion of this residue by peroxide, followed by reducoxida-tion
by NADH via the flavin This configuration ensures
0
0.4
0.6
0.8
1
Time (min)
Fig 4 Autoxidation of glutathione (d) and CoA (s) at high
tem-perature (88 C) in the presence of Cu 2+ The observed rates of
oxi-dation correspond to pseudo first order rate constants of 5.7 · 10)3
and 1.3 · 10)3min)1for glutathione and CoA, respectively.
Trang 7that NADH is available to be used ‘on demand’ when
a substrate appears while at the same time avoiding
the stabilization of a reduced flavin intermediate that
could react undesirably with O2
In Fig 2, the redox state of the enzyme-bound FAD
is indicated by the peaks at 380 and 460 nm, with
for-mal reduction and⁄ or charge transfer to the
isoalloxa-zine ring resulting in a loss of absorbance at these
wavelengths During the addition of the first equivalent
of NADPH there is a decrease in absorbance at
460 nm with a concomitant increase in long
wave-length (> 510 nm) absorbance The observed spectral
changes are consistent with the reduction of a
nonfla-vin redox center and the formation of an EH2 species,
with charge transfer possibly developing between the
FAD and the conserved active site cysteine thiol or
thiolate
During the anaerobic reduction of the S aureus
enzyme there is loss of roughly 50% of the absorbance
at 452 nm In that case, the loss of absorbance is
attributed to an asymmetric reduction of the dimeric
enzyme, with the net result of reduction by the first
equivalent being an oxidized flavin⁄ oxidized cys
resi-due in the form of a mixed disulfide on one subunit
and a reduced flavin⁄ reduced cys in the form of a thiol
on the other subunit [12] The decrease of 460 nm
absorbance during titration of the phCoADR
corres-ponds to at most a 24% loss, which is inconsistent
with the scheme proposed for the S aureus CoADR
It is interesting to note, however, that the overall
shape of the titration curve at 452 nm for the
sta-phylococcal enzyme and 460 nm for the phCoADR is
nearly identical, with most of the decrease in
absorb-ance occurring during addition of the first reducing
equivalent
Addition of a second equivalent of NADPH to the
EH2 form of phCoADR results in an increase in
absorbance in the regions between 507 and 700 nm
and 400 and 500 nm There is also an increase and
blue shift in the peak which corresponds to the 380 and 360 nm peaks of the E and EH2 forms, respect-ively This result is inconsistent with reduction of the FAD and consistent with the formation of an EH2Æ NADPH complex, although further characterization is currently underway to determine definitively the nature
of this enzyme species It is apparent, however, that lit-tle if any of the enzyme is stabilized in the FADH2 form It was this finding that led us to originally con-clude that this enzyme was not likely to act as an NAD(P)H oxidase in vivo
The difference in the reductive half reaction, inclu-ding the apparent lack of subunit asymmetry, and the difference in quaternary structure (the S aureus enzyme is a dimer) suggest that the mesophilic and thermophilic enzymes, which operate at very different temperatures, may use different mechanisms These differences are currently being investigated
Redox state of the single cysteine Based on homology to the single cysteine members of the disulfide reductase family, it seems likely that the CoADR uses its single cysteine to catalyze its redox chemistry As purified, the CoADR cysteine is not reactive with DTNB The observation that the cysteine becomes reactive following reduction with NADPH, along with the absence of small molecular mass thiols released upon reduction, leads to the conclusion that the enzyme is likely to be purified in the sulfenic acid form (Scheme 1) This result is not surprising, given that most of the enzyme is obtained in the apo form from the overexpression host It is also not particularly surprising that this enzyme, whose physiological func-tion appears to be to serve as a CoADR, is easily oxidized to the sulfenic acid form, when the anaerobic nature of Pyrococcus is taken into account While Pyrococcus may encounter some oxidative stress, it seems unlikely that it would be regularly subjected to
Scheme 1 Possible redox states for
single-cysteine containing disulfide reductases.
During reduction from ‘E’ to ‘EH2’ the
hydride passes from the NAD(P)H through
the FAD to the nonflavin redox center An
additional possible species, EH2-NAD(P) + , is
not shown.
Trang 8the levels of O2present in ambient air Even if it were,
however, this apparent sulfenic acid state is freely
reducible, so that its production would not seem to
put the enzyme at a disadvantage
Physiological role of CoADR and NAD(P)H
in Pyrococcus
The maintenance of low intracellular levels of cysteine
in organisms has been attributed to the avoidance of
the hydrogen peroxide produced during the rapid O2
-dependent oxidation to cystine, necessitating the use of
other small molecular mass thiols such as glutathione
for the maintenance of internal redox levels The
results presented above are consistent with a role for
CoA in maintaining a reducing environment or serving
as a pool of reducing equivalents at the very high
tem-peratures and high concentrations of metals found in
the natural environment of Pyrococcus The observed
CoA concentration of 0.64 lmol CoAÆg dry weight)1
compares favorably with the result of 1.1 lmolÆg)1dry
weight obtained for S aureus [14], and the
correspond-ing intracellular concentration of 210 lm CoA is well
above the Km app of 30 lm for the CoA disulfide The
pfCoADR (nox A-2 in the annotation of the P
furio-sus genome) was found to be upregulated more than
fivefold by growth on elemental sulfur (S0) [21], a
result which suggests a role for this enzyme in the
eventual transfer of electrons to S0 Because CoA
lev-els were determined in cells which were grown in the
absence of S0, future studies will determine whether
CoA levels increase during growth in the presence of
S0 It is also worth noting that the low CoADR
activ-ity noted in crude extracts of P furiosus was obtained
from cells grown in the absence of elemental sulfur, so
it seems likely that greatly increased activity will be
seen in extracts of cells grown in the presence of S0
The NAD(P)H dependence of enzymes in the
disul-fide reductase family can be divided into two classes:
(a) enzymes whose function appears to be the
main-tenance of a high R-SH⁄ R-S-S-R ratio, such as
glutathione reductase, trypanathione reductase and
thi-oredoxin reductase, which tend to be NADPH
depend-ent; and (b) enzymes that appear to be involved more
directly in the reduction of oxidizing compounds and
in the regeneration of oxidized nucleotides for
glycoly-sis, which show a distinct preference for NADH, such
as the NADH oxidase, NADH peroxidase, the NOX1
of P furiosus, alkyl hydroperoxide reductase, and
lipo-amide dehydrogenase
The pyrococcal CoADR described in this work is
able to efficiently utilize both NADPH and NADH, a
result which is consistent with the unusual utilization
of reduced nucleotide coenzymes by Pyrococcus The central metabolism of this organism uses an unusual NADPH-dependent sulfide dehydrogenase which is capable of both the NADPH-dependent reduction of elemental sulfur and the NADP+-dependent oxida-tion of ferredoxin [22] A unique NADPH-dependent alcohol dehydrogenase with wide substrate specificity and a strong preference for the reduction of alde-hydes to alcohols is also found in this organism [23] This unusual mix of NADH and NADPH dependent reactions in catabolic processes may account for the finding that the pyrococcal CoADR, which would be expected to show a strong preference for NADPH, is able to efficiently utilize either reduced nucleotide substrate
Comparison of the phCoADR to the pyrococcal NADH oxidase
There are now two members of the disulfide reductase family that have been characterized from Pyrococcus, the NADH oxidase and the CoADR These two enzymes display unique biochemical and enzymatic properties that are not present in their mesophilic counterparts The P horikoshii CoADR and the
P furiosus NADH oxidase (NOX1) are 37% identical Each appears to play distinct roles in the fermentative metabolism of Pyrococcus and each is regulated differ-ently [1,21] The difference in their reactivity is reflec-ted in the behavior of these two enzymes in reductive anaerobic titrations As shown in Fig 2 and discussed above, upon addition of excess NAD(P)H the phCo-ADR forms what appears to be an EH2ÆNAD(P)H complex with little or no FADH2 character (Scheme 1) This can be contrasted to the behavior of the P furiosus NOX, which forms what appears to be
an EH4ÆNAD+ complex with a fully reduced FAD (Scheme 1) upon addition of two equivalents of NADH (data not shown) In-depth studies comparing the mechanisms of these two pyrococcal enzymes to each other and to their mesophilic counterparts will be presented in a future communication
Experimental procedures
Growth of microorganisms
as previously described [24] Cellobiose (30 mm), maltose
inclu-ded as primary carbon source E coli JM109(kDE3) and
Trang 9K2HPO4 (2.5 gÆL)1)] When required, the following
Cloning and expression of the gene encoding
the P horikoshii and P furiosus CoADRs
The structural gene encoding the CoADR [previously
referred to as NOX2 (PH0572)] was amplified from P
oligonucleo-tides TG100 (5¢-GGCCTCATGAAGAAAAAGGTCGTCA
TAATT-3¢), and TG101 (5¢-GGCCAAGCTTCTAGAAC
TTGAGAACCCTAGC-3¢) (for the P horikoshii CoADR),
and TG104 (5¢-CGCGCCATGGAAAAGAAAAAGGTA
GTCATAA-3¢) and TG105 (5¢-CGCGGTCGACCTAGAA
CTTCAAAACCCTGGC-3¢) for the P furiosus CoADR
The N terminus of the CoADRs were based on the
pres-ence and proper spacing of the ribosome binding site,
agreement with the NOX homologues from mesophilic
sources that had been characterized in detail PCR
amplifi-cation was carried out using Pfu polymerase (Stratagene,
La Jolla, CA), and the resulting 1.3 kb PCR product was
cloned in the NcoI and HindIII sites of pET-24d The
resulting plasmids, pSMC002 (P horikoshii) and pSMC004
(P furiosus) were transformed into E coli BL21(kDE3)
For production of the recombinant enzymes the strain
JM109 (kDE3) was used
Production and purification of recombinant
phCoADR
The phCoADR was overexpressed in E coli JM109(kDE3)
( 4.0 h) isopropyl thio-b-d-galactoside was added to a
final concentration of 1.0 mm to induce enzyme production
The culture was incubated for an additional 4.0 h and the
15 min The cells were washed with 50 mm Tris buffer
until purification
The thawed cells were resuspended in 50 mm Tris buffer
pH 7.5 (70 mL total volume) Eight-milliliter aliquots of
treat-ments Cell debris was removed by centrifugation at
and sonication treatments were repeated (20 mL total
vol-ume) and cell debris was removed by centrifugation
protein was removed by centrifugation at 15 000 g and
Chromatography was performed using an AKTA low-pressure chromatography system (FPLC) from Pharmacia Biotech (Piscataway, NJ) The supernatant was applied to a
25 mL Q-Sepharose Hi-Load column (Pharmacia Biotech) equilibrated with 50 mm Tris buffer pH 7.5 at 3.0 mLÆ
50 mm Tris pH 7.5 to remove excess protein phCoADR was eluted by a 120-mL linear gradient from 0.10 to 0.45 m KCl, with phCoADR eluting between 0.27 and 0.32 m KCl Fractions containing phCoADR were pooled and reconsti-tuted with a final concentration of 1 mm FAD by
concentrated and rinsed with 50 mm Tris buffer pH 7.50 by ultrafiltration with 30 k molecular mass limit filtration
The concentrate (total volume 1.7 mL) was applied to a 120-mL Sephacryl S-200 HR (Pharmacia Biotech) size-exclusion column equilibrated with 50 mm Tris buffer
were pooled (total volume 20 mL) The procedure yields
35 mg of enzyme with little loss between the steps following heat treatment ( 30% of activity is lost during the heat treatment, which is assumed to be due to association of the enzyme with the large pellet of heat denatured protein obtained at this stage) The enzyme shows a single band
Fig 5 Coomassie blue stained SDS ⁄ PAGE analysis of the purifica-tion of the recombinant P furiosus CoADR Lane 1, crude extract (15 lg); lane 2, heat-treated extract (5 lg); lane 3, pooled fractions from the Q-sepharose column (5 lg); lanes 4 and 5, pooled frac-tions from the size-exclusion column (1 and 2 lg, respectively) The left lane contains marker proteins with the indicated subunit molecular mass (top to bottom): myosin, 195 k; b-galactosidase,
112 k; BSA, 59 k; carbonic anhydrase, 30 k.
Trang 10gives a single peak by both conventional and HPLC
size-exclusion chromatography A summary of the purification
spec-trum of phCoADR, which is typical of a flavoprotein, with
Production and assay of the recombinant
pfCoADR
pfCoADR was overexpressed in E coli JM109(kDE3) A
2.8-L Fernbach flask with 325 mL TYP media and
inoculation of overnight culture Cells were grown, protein
production was induced, and cells were harvested as
des-cribed above Cells were disrupted by sonicating for
precipi-tated during the heat treatment were removed by
centrifu-ging in a microcentrifuge for 15 min at 14 000 g
Standard assays
tempera-ture) in 1.5-mL cuvettes All substrates, including oxidized
CoA and dephosphoCoA, were from Sigma (St Louis,
MO) Enzyme and substrate(s) were added to preheated
buffer for a total reaction volume of 840–950 lL Oxidation
of NAD(P)H was monitored at 340 nm The standard
oxid-ase assay used to monitor the purification of phCoADR
was conducted with 100 lm NADH and FAD as above A
unit of CoADR activity is defined as the amount of enzyme
potassium phosphate pH 7.5, 230 lm CoA disulfide and
Protein and enzyme-bound FAD extinction
coefficient determinations
Protein concentrations were determined using a modified
Bradford assay (Bio-Rad Protein Assay, Hercules, CA)
The extinction coefficient of the enzyme-bound FAD in
50 mm Tris pH 7.50 was determined using the following
Quaternary structure determination
The 120-mL Sephacryl S-200 HR (Pharmacia Biotech)
size-exclusion column was used to determine the quaternary
molecular mass of phCoADR The column was eluted with
molecular mass of phCoADR was determined by
(gamma globulin), 44 k (ovalbumin), and 17 k (myoglobin) This result was confirmed by size exclusion HPLC
column
Determination of enzyme free and released thiols
DTNB was used to detect both protein and small molecular mass thiols All determinations used a final concentration
of DTNB of 200 lm Small molecular mass thiols that might be in the form of mixed disulfide on the enzyme (Cys-S-S-R) were determined by reducing the enzyme (56 lm) in an anaerobic titration with NADPH, followed
by cooling the cuvette on ice, opening the cuvette to air, and immediately centrifuging the enzyme on a 30 k mole-cular mass cut-off filter to separate the enzyme from small molecular mass thiols The flow-through from the filtration experiment was assayed for thiol content (providing a measure of small molecular mass thiols), while the retentate was rinsed three times with 50 mm potassium phosphate, 0.5 mm EDTA pH 7.5 and also assayed for thiol content (providing a measure of air-stable, accessible protein thiols)
In order to detect protein thiols that were not stable in air (such as those that may be converted to sulfenic acids), the enzyme was reduced by 0.8 equivalents of NADPH in an anaerobic titration at room temperature (because of a slow turnover of the enzyme with DTNB, more than one equiv-alent of NADPH would result in a DTNB reductase activ-ity), followed by a tip to DTNB The enzyme as purified was assayed for reactive thiols simply by adding DTNB, as above
Thiol stability assays
Thiol assays were performed using DTNB as a thiol detec-tion reagent in a modificadetec-tion of a previously published pro-cedure [25] An aliquot of the thiol to be assayed (generally
100 lL) was removed and added to 900 lL of assay buffer containing (final concentrations) 5 mm DTNB, 0.25 m potassium phosphate, 0.5 mm EDTA pH 7.2 After a 5-min incubation the absorbance was read at 412 nm Thiol con-tent was determined by comparison to a standard curve determined under the same conditions with an extinction
Thermostability and thermoactivity assays
of enzyme was placed in a 2.0-mL microcentrifuge tube and sealed with Parafilm For lower temperatures, the tube was placed in the respective freezer and standard activity assays were performed after 1 month At higher temperatures, a