sMMO, which catalyses the NADPH-depen-dent and O2-dependent oxygenation of methane and numerous adventitious substrates, is an enzyme complex consisting of three components: a multisubun
Trang 1Residues near the N-terminus of protein B control autocatalytic
proteolysis and the activity of soluble methane mono-oxygenase
Anastasia J Callaghan*, Thomas J Smith†, Susan E Slade and Howard Dalton
Department of Biological Sciences, University of Warwick, Coventry, UK
Soluble methane mono-oxygenase (sMMO) of
Methylo-coccus capsulatus (Bath) catalyses the O2-dependent and
NAD(P)H-dependent oxygenation of methane and
numer-ous other substrates During purification, the sMMO
enzyme complex, which comprises three components and
has a molecular mass in excess of 300 kDa, becomes
inac-tivated because of cleavage of just 12 amino acids from the
N-terminus of protein B, which is the smallest component of
sMMO and the only one without prosthetic groups Here we
have shown that cleavage of protein B, to form the inactive
truncated protein B¢, continued to occur when intact protein
B was repeatedly separated from protein B¢ and all
detect-able contaminants, giving compelling evidence that the
protein was cleaved autocatalytically The rate of
autocata-lytic cleavage decreased when the residues flanking the
cleavage site were mutated, but the position of cleavage was
unaltered Analysis of a series of incremental truncates
showed that residue(s) essential for the activity of sMMO, and important in determining the stability of protein B, lay in the region Ser4–Tyr7 Protein B was shown to possess intrinsic nucleophilic activity, which we propose initiates the cleavage reaction via a novel mechanism Proteins B and B¢ were detected in approximately equal amounts in the cell, showing that truncation of protein B is biologically relevant Increasing the growth-medium copper concentration, which inactivates sMMO, did not alter the extent of in vivo cleav-age, therefore the conditions under which cleavage of protein
B may fulfil its proposed role as a regulator of sMMO remain to be identified
Keywords: autocatalytic inactivation; methane mono-oxy-genase; methanotroph; N-terminal autoprocessing; regula-tory protein
Methane mono-oxygenase (MMO) catalyses the oxidation
of methane to methanol and is essential for the growth of
methanotrophic bacteria using methane as the growth
substrate [1] Methanotrophic bacteria such as
Methylococ-cus capsulatus (Bath) possesses two forms of MMO, the
copper-requiring particulate form (pMMO) and the
iron-containing soluble form (sMMO), the expression of which is
regulated by the concentration of available copper in the
medium [2] sMMO, which catalyses the
NAD(P)H-depen-dent and O2-dependent oxygenation of methane and
numerous adventitious substrates, is an enzyme complex
consisting of three components: a multisubunit hydroxylase,
a reductase, and a regulatory component known as protein
B [3]
The hydroxylase (250.1 kDa) has an (abc)2quaternary structure [4] in which each a subunit contains a l-(hydr) oxo-bridged di-iron centre that is the presumed site of substrate oxygenation [5,6] The reductase (38.5 kDa) contains FAD and Fe2S2 centres and supplies electrons from NADH to the hydroxylase [7] Protein B (16 kDa), which is devoid of prosthetic groups and metal cofactors, is essential for natural, O2-dependent substrate oxygenation
by the sMMO complex [3] Owing to its diverse effects on the catalytic properties of sMMO, protein B is potentially a powerful regulator of sMMO activity Protein B has been shown to (a) couple electron transfer to substrate oxygen-ation thus converting sMMO from an oxidase into an oxygenase [8]; (b) reduce the redox potentials of the di-iron site [9,10] and hence increase the reactivity of the diferrous di-iron site to oxygen; (c) accelerate formation of the high-valent intermediate Q, which appears to be responsible for oxygenation of methane [11–13]; (d) alter product distri-bution with complex substrates [14,15]; and (e) inhibit oxygenation reactions when the hydroxylase is artificially activated by hydrogen peroxide via the peroxide shunt reaction [14]
Protein B binds to the hydroxylase [16] but not directly to the reductase [17] There are currently no high-resolution structural data for the complex formed between protein B and the hydroxylase; however, a cross-linking study using the homologous sMMO of Methylosinus trichosporium OB3b showed that protein B bound to the a subunit [18] A variety of spectroscopic techniques have demonstrated that protein B perturbs the environment of the di-iron site, presumably by altering the conformation of the hydroxylase [19–21] Consistent with this, small-angle X-ray scattering
Correspondence to H Dalton, Department of Biological Sciences,
University of Warwick, Coventry CV4 7AL, UK.
Fax: + 44 24 7652 3568, Tel.: + 44 24 7652 3552,
E-mail: hdalton@bio.warwick.ac.uk
Abbreviations: ESI, electrospray ionization; GST, glutathione
S-transferase; SAXS, small angle X-ray scattering; s/pMMO, soluble/
particulate methane mono-oxygenase.
Enzyme: methane monooxygenase (EC 1.14.13.25).
*Present address: Department of Biochemistry, University of
Cambridge, Old Addenbrookes Site, Cambridge , UK.
Present address: Biomedical Research Centre, Sheffield Hallam
University, Howard Street, Sheffield, UK.
Note: a web page is available at http://www.bio.warwick.ac.uk/dalton/
(Received 8 November 2001, revised 6 February 2002, accepted 8
February 2002)
Trang 2(SAXS) has given direct, though low-resolution, structural
evidence for a large conformational change in the
hydrox-ylase that protein B and the reductase together induce [22]
Thus, current evidence suggests that protein B influences
sMMO activity through the conformational change that it
causes in the hydroxylase, although a direct role in
transferring electrons from the reductase to the hydroxylase
is also possible
The NMR structure of protein B from Mc capsulatus
(Bath) [23] shows that it has a folded central,
two-domain core region, while the N-terminal region until
Val31 and the C-terminus (Met130–Ala140) are mobile
and largely unstructured NMR measurements in the
presence of the hydroyxlase showed exchange broadening
of specific nuclear Overhauser effect cross-peaks that
grouped around the so-called ÔnorthernÕ domain of the
core of protein B [23] These results were interpreted as
showing that hydroxylase-bound and unbound protein
B were in dynamic equilibrium and that the hydrophobic
ÔnorthernÕ half of the core of protein B interacted with
the hydroxylase Docking studies showed that protein
B could bind in the hydrophobic cleft formed by two
of the four iron-co-ordinating a helices that lie in a
canyon formed between the ab pairs of the hydroxylase
[6,23–25]
Protein B from Mc capsulatus (Bath) is unusually
sensitive to inactivation because of truncation reactions
During and after purification, protein B degrades by
cleavage, principally between Met12 and Gly13, to give
protein B¢, which is completely inactive in the sMMO
whole-complex reaction [16,26] Cleavage is also observed
between Gln29 and Val30, giving protein B¢¢, which is also
inactive [16] Mutagenesis studies have shown that the
residues around the cleavage site influence the rate of
inactivation Mutation of the Met12–Gly13 cleavage site in
protein B of the Mc capsulatus (Bath) site to Met12–Gln13,
equivalent to the site found in Ms trichosporium OB3b
protein B (in which truncation had not been reported),
enhanced the stability of the protein [16] The triple mutant
G10A/G13Q/G16A was also resistant to truncation but had
diminished activity [27] Protease inhibitors did not prevent
cleavage of protein B, and recombinant protein B expressed
in a protease-deficient strain of Escherichia coli was cleaved
to protein B¢ [16] despite the absence of Mc
capsulatus-specific proteases and the major intracellular proteases of
E coli
It is remarkable that the sMMO complex, the
compo-nents of which total 300 kDa, is exquisitely sensitive to
inactivation by removal of just 12 amino acids from the
unstructured terminus of its smallest component The fact
that those 12 amino acids are lost spontaneously under a
range of conditions raises important questions about the
mechanism of cleavage and suggests that cleavage may
occur in vivo If it is an in vivo phenomenon, truncation of
protein B offers a possible mechanism to control the
amount of active protein B within the cell and thus regulate
the rates of methane oxidation and NADH consumption by
sMMO, e.g in response to intracellular or extracellular
conditions To address these questions, we conducted a
detailed characterization of the mechanism of cleavage, the
roles of specific amino acids near to the N-terminus in
catalytic activity, and the significance of the cleavage
reaction in vivo
M A T E R I A L S A N D M E T H O D S Bacterial growth
sMMO-expressing Mc capsulatus (Bath) cells were grown
in nitrate minimal salts medium using methane as the growth substrate, as described previously [7] The switch from sMMO to pMMO expression was effected in fermen-tor cultures by increasing the CuSO4.5H2O concentration of the medium from 0.1 to 1.0 mgÆL)1 E coli strains were grown at 37°C in Luria–Bertani broth [28], with ampicillin (100 lgÆmL)1) added for selection of plasmids as required
Purification of the sMMO components fromMc capsulatus
The hydroxylase, reductase and protein B components of sMMO were purified from Mc capsulatus (Bath) as described previously [22,29] As protein B underwent truncation during purification, protein prepared by this method contained a mixture of proteins B and B¢ The relative abundance of proteins B and B¢ was assessed by using SDS/PAGE and electrospray ionization (ESI)-MS Incubation of the purified protein B/B¢ mix at 20 °C for 1–2 days enabled complete conversion of protein B to B¢
Separation of proteins B and B¢ by chromatofocusing chromatography
Chromatofocusing chromatography was achieved using a Mono P FPLC column (HR 5/20) (Amersham Pharmacia) The column was equilibrated with buffer A (25 mM
methylpiperizine, pH 5.64 or 5.7) before loading of the protein in the same buffer Elution using buffer B [1 : 10 dilution of PolyBuffer 74TM (Amersham Pharmacia)] at either pH 3.5 or 4, with a flow rate of 0.3–1.0 mLÆmin)1 over 15 col vol., separated proteins B and B¢ according to the difference in their isoelectric pH
Genetic manipulations The construct for expression of the M12A/G13Q double mutant of protein B was made by amplification of mmoB (which encodes protein B) from pGEX-WTB [16] by PCR with primers mmoB-M12A/G13Q-1 (5¢-CGCGGATCC ACGATGAGCGTAAACAGCAACGCATACGACGCC GGCATCGCGCAGCTGAAAGGCAAG-3¢; M12A and G13Q mutations shown in bold, start codon in italics and BamHI site underlined) and primer mmoB-2 (5¢-GGCGAA
underlined) and cloning into the glutathione S-transferase (GST)-fusion expression vector pGEX-2T (Amersham-Pharmacia) using BamHI and EcoRI
The plasmids for expression of the C-terminally 6-His-tagged G13Q mutant of protein B and N-terminal trunca-tions thereof were constructed by PCR amplification of the appropriate section of mmoB using pGEX-mtB [16] as the template and cloning into pET3a (Novagen) using NdeI and BamHI The truncated constructs and the proteins they encoded were numbered according to the first amino acid after the start codon The forward PCR primers for the various constructs were as follows: G13Q-tag (full-length construct), 5¢-GGGAATTCCATATGAGCGTAAACAG
Trang 3CAACGCATAC-3¢; truncate 4, 5¢-GGGAATTCCATAT
GAGCAACGCATACGACGCCGGCATC-3¢; truncate 5,
5¢-GGGAATCCATATGAACGCATACGACGCCGGCA
TCATGCAGCTGAAA-3¢; truncate 6, 5¢-GGGAATTCC
ATATGGCATACGACGCCGGCATCATGCAGCTGAA
A-3¢; truncate 7, 5¢-GGGAATTCCATATGTACGACGC
CGGCATCATGCAGCTGAAA-3¢; truncate 8, 5¢-GGGA
ATTCCATATGGACGCCGGCATCATGCAGCTGAA
A-3¢; truncate 9, 5¢-GGGAATTCCATATGGCCGGCAT
CATGCAGCTGAAAGGCAAG-3¢; truncate 10, 5¢-GGG
AATTCCATATGGGCATCATGCAGCTGAAAGGCA
AG-3¢; truncate 13, 5¢-GGGAATTCCATATGCAGCTG
AAAGGCAAGGACTTC-3¢ (NdeI sites underlined and
start codons italicized) The PCR reverse primer was the
same in each case (5¢-TGTATAGGATCCTCAGTGATGG
tag shown in bold, stop codon italicized, and BamHI
restriction site underlined) The absence of unwanted
mutations from all cloned PCR products was confirmed
by DNA sequencing
Purification of recombinant protein B derivatives
The GST-tagged wild-type, G13Q and M12A/G13Q
deriv-atives of protein B were purified from strains of E coli
AD202 containing the appropriate plasmids by affinity
chromatography [16] The GST affinity tag was removed by
the addition of thrombin [2 ng thrombinÆ(lg fusion
protein))1] for 5–10 min at room temperature, after which
the recombinant protein B derivative was separated by gel
filtration with a Superdex 75 FPLC column (2.6 cm
· 61 cm; Amersham Pharmacia), eluted with 25 mMMops
buffer, pH 7
Plasmids for expression of the His6-tagged protein B
derivatives were transformed into E coli BL21(DE3)
(Novagen) Cells were grown, induced with isopropyl
thio-b-D-galactopyranoside, and soluble extracts were prepared
as described previously [16], except that the cells were
broken in 20 mM sodium phosphate buffer, pH 7.4–7.6,
containing 0.5MNaCl and 10 mMimidazole Purification
of the His6-tagged protein B derivative was accomplished
using the HisTrapTMkit (Amersham Pharmacia) according
to the manufacturer’s instructions The purified fusion
protein was then exchanged into 25 mMMops buffer, pH 7,
by gel filtration as described above
Determination of protein concentration
Concentrations of protein B and protein B¢ samples
were determined spectrophotometrically at 280 nm
using the absorption coefficients 16 839M )1Æcm)1 and
16 032 M )1Æcm)1, respectively, which were determined
experimentally by established methods [30,31]
Concentra-tions of the hydroxylase and reductase were determined by
the method of Bradford [32] using BSA as the protein
standard and commercially available reagent (Bio-Rad)
Enzyme assays
The semiquantitative naphthalene oxidation test to detect
sMMO activity in liquid culture samples was performed as
previously described [33] Quantitative propylene oxidation
assays using the whole sMMO complex (hydroxylase,
reductase and protein B) were performed by the method
of Pilkington & Dalton [29] The effect of protein B derivatives on the propylene oxidation activity of the hydroxylase via the peroxide shunt reaction was measured
in the presence of 24 lMhydroxylase and 100 mMhydrogen peroxide as published [14], except that the epoxypropane product was quantified by GC of 0.5-mL gas-phase samples
Circular dichroism Protein samples in 25 mMsodium phosphate buffer were scanned 10 times in a Jasco J715 spectropolarimeter in a 1-mm path-length quartz cuvette between 190 nm and
250 nm (for far-UV CD analysis) or in a 1-cm path-length quartz cuvette between 260 nm and 300 nm (for near-UV CD) In all cases the response time was 0.25 s and the scan speed was 100 nmÆmin)1 Scans were blanked against fresh buffer recorded under the same conditions
Fluorescence Protein samples in 25 mMsodium phosphate buffer, pH 7, were placed in a 3-mL quartz cuvette with a 10-mm path length Fluorescence measurements were made using a PerkinElmer LS-50 fluorimeter at room temperature with a scan speed of 500 nmÆmin)1and an excitation wavelength of
280 nm, and scanned over the range 300–450 nm An accumulation of eight scans was taken for each sample, and scans were blanked against buffer data collected under the same conditions
Other methods Protein B-associated nucleophile activity was measured at
20°C by the protein B-dependent conversion of p-nitro-phenylacetate to p-nitrophenol, monitored spectrophoto-metrically at 400 nm [34] SDS/PAGE [35] was performed using 12% (w/v) polyacrylamide gels SDS cell extracts of
Mc capsulatuscells were prepared from cells harvested by centrifugation (14 000 g, 5 min, room temperature), which were immediately resuspended in SDS/PAGE loading buffer [65 mMTris/HCl (pH 8.8), 1 mMEDTA, 1% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 0.0025% (w/v) brom-ophenol blue, 10% (v/v) glycerol] and boiled (100°C,
5 min) before centrifugation (10 000 g, 10 min) to remove particulate material Preparation of anti-(protein B) serum, Western blotting, and ESI-MS were as described previously [16]
R E S U L T S Cleavage of protein B did not require detectable extrinsic proteases
To determine whether the proteolytic activity responsible for cleavage was intrinsic or extrinsic to protein B, proteins
B and B¢ were separated from one another by chomatofo-cusing chromatography on a Mono P column The protein
B used for this separation was a highly purified sample [prepared from Mc capsulatus (Bath), with a specific activity of 3500 nmolÆmin)1Æmg)1] that had undergone partial cleavage SDS/PAGE analysis of this sample showed
Trang 4that it contained proteins B, B¢ and B¢¢ in the proportions
10 : 6 : 1, and MS analysis confirmed the presence of
proteins with masses of 15 852, 14 629 and 12 718 Da,
which corresponded to the calculated masses of proteins B,
B¢ and B¢¢ On chromatofocusing the sample could be
readily resolved into proteins B, B¢ and B¢¢ Immediate SDS/
PAGE analysis of the protein B fraction at this stage
showed it to be pure by Coomassie Blue staining, and it was
active (6380 nmolÆmin)1Æmg)1) by the propene oxidation
assay In contrast, no activity could be detected from the
fraction eluted as protein B¢ The active protein B was then
subjected to further rounds of chromatofocusing, and on
each occasion the protein was resolved into B and B¢
During these operations, the highly purified protein B was
observed to degrade by 50% to B¢ over a 3-h period at
20°C This spontaneous truncation of highly purified
samples of protein B upon repeated repurification, despite
the absence of detectable contaminating proteins, strongly
suggested that truncation was autocatalytic
Effect of the structure of the cleavage site
on protein B activity and the cleavage reaction
We were interested to investigate the roles of the residues
near to the cleavage site in the rate and position of cleavage
because it seemed likely that the side chains near to the
cleavage site were important in the autocatalytic cleavage
process We had already shown that the G13Q mutation,
which changed the amino acid immediately C-terminal to
the cleavage site, reduced the rate of truncation of protein B
[16], but the precise position of cleavage in this mutant had
not been determined After a preparation of the G13Q
mutant protein had been incubated at 20°C for 48 h,
ESI-MS analysis revealed the presence of two major molecular
ions, of 16 300 Da (corresponding to the mass of the intact
G13Q mutant) and 14 700 Da (corresponding to the mass
of a truncate beginning at Gln13) This illustrated that
replacement of the small Gly13 with the bulkier, hydrophilic
Gln did not affect the principal site of cleavage of protein B,
which remained immediately N-terminal to residue 13
To investigate the role of the residue immediately
N-terminal to the cleavage site and to see whether the
G13Q mutant could be further stabilized by removal of
side-chain functionality at this position, the M12A/G13Q
mutant was constructed The activity of this double mutant
was indistinguishable from that of the wild-type (data not
shown), and ESI-MS analysis of the freshly prepared
mutant protein confirmed the predicted molecular mass of
16 240 Da Analysis of a sample that had been incubated at
20°C for 48 h showed that the major new molecular ion
had a mass of 14 701 Da, which corresponded to the mass
of the truncate produced by cleavage between amino acids
12 and 13 Thus, despite radically changing the amino acids
on both sides of the cleavage site, the position of cleavage
was unchanged and the protein remained active
A surprising difference between the wild-type and mutant
forms of protein B was observed during the peroxide shunt
reaction The peroxide shunt, which allows the hydroxylase
to be activated by hydrogen peroxide to perform
oxygen-ation reactions in the absence of the reductase and NADH,
is inhibited by intact wild-type protein B [14] Just as the
stimulatory effect of protein B¢ in the whole-complex
sMMO reaction was much lower than that of intact protein
B, the inhibitory effect of protein B¢ during the peroxide shunt was markedly lower than that of protein B (Fig 1) However, the intact G13Q and M12A/G13Q mutant forms
of protein B, both of which had wild-type activity in the whole-complex reaction, were significantly poorer inhibitors
of the peroxide shunt reaction than wild-type protein B (Fig 1) Thus the inhibitory effect of protein B was more sensitive to structural changes near to the N-terminus than its better-documented stimulatory effect
As previous SAXS studies had indicated that protein B elongated on truncation [22], we studied the effect of truncation on the overall conformation of protein B so as to assess whether the inactivity of protein B¢ is associated with
a conformational change However, far-UV CD spectra of proteins B and B¢ were identical (data not shown), showing that truncation caused no detectable change in the second-ary-structure content of the protein Likewise, near-UV CD and fluorescence spectra were scarcely different for proteins
B and B¢, showing little difference in the environments of aromatic side chains between the full-length and truncated proteins (data not shown) These data, taken together with the SAXS study, are consistent with a relatively minor change in conformation on truncation of free protein B Similar structural studies were performed with the two mutant forms of protein B and their respective truncates, and the results were indistinguishable from those obtained with the wild-type protein B/B¢ system (data not shown) Incremental truncation of protein B
To investigate more thoroughly the role of the N-terminal region in the truncation reaction and in the activity of protein B, a series of N-terminal truncates was constructed genetically These all contained the stability-enhancing G13Q mutation to minimize loss of additional amino acids
by spontaneous cleavage and had a C-terminal 6-His tag to
Fig 1 Inhibition of the peroxide shunt reaction by protein B and its derivatives Oxygenation of propylene was measured in the presence of the hydroxylase and hydrogen peroxide as described in Materials and methods at various concentrations of intact wild-type protein B (solid line), intact G13Q (broken line) or M12A/G13Q (dashed line) mutant protein B or protein B¢ derived from wild-type protein B (dotted line) Enzyme activity is shown as a percentage of the activity [9.2 nmolÆmin)1Æ(mg of hydroxylase))1] with no added protein B The activities presented are the mean of three or four separate experiments Standard error bars are shown.
Trang 5permit affinity purification while allowing free manipulation
of the N-terminus ESI-MS of the full-length and truncated
proteins confirmed their integrity and showed whether the
initial methionine residue was preserved (Fig 2) The
C-terminal His6 tag had no effect on activity because the
activity of the full-length fusion (G13Q–His6) was
approx-imately the same as that observed for the original G13Q
mutant with no C-terminal tag Full activity was retained on
truncation as far as Asn5 (truncate 5), but truncation
beyond this led to progressive loss of activity until truncate
Asp8 (truncate 8), when activity was lost completely
(Fig 3)
The cleavage of the truncates was analysed to investigate
the role of the N-terminal region in the cleavage process
ESI-MS analysis was performed at 24-h intervals during
incubation at 20°C over 6 days The full-length construct
G13Q-tag was detectable for 3 days Truncate 4 was less
stable, having completely degraded to truncates within 24 h, whereas the shorter truncates 5, 6 and 7 were still observed
in uncleaved form after 6 days Further truncation beyond this, to truncates 8 and 9, which abolished activity, also enhanced the cleavage reaction, which was complete within
24 h General destabilization or reorganization of the secondary structure of the truncated forms was unlikely to
be the cause of their destabilization of the truncates because the far-UV CD spectra for truncates 7 (stable) and 8 (rapidly cleaving) were indistinguishable and very similar to that of native, intact protein B (data not shown) Truncates
10 and 13, both of which retained the scissile Met12–Gln13 peptide bond (Fig 2), were as stable for at least 6 days, showing that at least four of the amino acids N-terminal to the cleavage site are required for rapid autocatalytic cleavage
Mechanism of autocatalytic cleavage The probable autocatalytic mechanism of cleavage of protein B posed the question of which intrinsic groups on protein B were responsible for the reaction Recent research has identified autoprocessing reactions in other systems, such as aminohydrolase and aspartate decarboxylase, which rely on the formation and resolution of internal (thio) esters [36,37] In these examples, a nucleophilic amino acid (cysteine, serine or threonine) rearranges within the protein, thus replacing the amide peptide bond between itself and the preceding amino acid with a more reactive thioester or ester linkage Such bonds then hydrolyse spontaneously and thus effect cleavage [38–40]
In wild-type protein B, the amino acid on the C-terminal side of the cleavage site is glycine and so nucleophilic attack from this site is impossible Nevertheless, it was possible that cleavage of protein B occured via a similar chemical mechanism, initiated by attack from a nucleophile elsewhere
in the protein This would transfer the N-terminal region of the protein on to a (thio)ester linkage on the nucleophile, which would then spontaneously hydrolyse to yield the truncated protein (Fig 4)
To investigate the feasibility of such a mechanism, the protein was tested for the presence of accessible nucleo-phile(s) by reaction with p-nitrophenylacetate, which reacts with nucleophilic groups to form p-nitrophenol [34] The results (Fig 5) indicated that a nucleophilic group was indeed present, because an increased reaction rate of p-nitrophenol formation was observed in the presence of increasing concentrations of protein B Control reactions (Fig 5) confirmed that the rate of p-nitrophenol production was significantly higher than the background rate that was observed in the absence of protein or when denatured (boiled) protein B or the hydroxylase were used
Detection of protein B¢in vivo
To investigate the in vivo significance of truncation of protein B, sMMO-expressing Mc capsulatus (Bath) whole cells were analysed for the presence of proteins B and B¢
Mc capsulatus (Bath) was cultivated under low-copper, oxygen-limiting conditions as described in Materials and methods A positive naphthalene oxidation test confirmed the expression of sMMO because pMMO is inactive with this substrate [33] The cells were rapidly harvested and
Fig 2 Deduced N-terminal sequences of the incremental truncates of
protein B, each of which was constructed in the G13Q background and
had the C-terminal 6-His tag The presence of the initial methionine
residues (shown in bold) was determined experimentally by ESI-MS.
The site of cleavage for formation of protein B¢ is indicated.
Fig 3 Effect of incremental truncation on the activity of protein B.
Activity was measured as the rate of propene oxygenation in the
presence of excess hydroxylase and reductase and is shown as a
per-centage of the activity [1956 nmolÆmin)1Æ(mg protein B))1] observed
with the G13Q mutant prepared using the GST-tag system (G13Q).
The activities presented are the mean of three or four separate
exper-iments Standard error bars are shown.
Trang 6immediately boiled in SDS-containing sample-loading
buf-fer, thus capturing the cellular proteins with minimal
opportunity for degradation before exposure to the
dena-turant SDS/PAGE and Western-blotting analysis, using
anti-(protein B) sera (that cross-reacted with protein B¢), clearly showed that protein B¢ was present in vivo in sMMO-expressing cells of Mc capsulatus (Bath) (Fig 6) Control samples from pMMO-expressing cells (grown
Fig 4 Proposed mechanism for autocatalytic cleavage of protein B A nucleophilic side chain, probably on the surface of the folded core region of protein B, is proposed to attack the carbonyl group of the scissile peptide bond Cleavage then follows via cyclic zwitte-rion and ester intermediates as indicated.
Fig 5 Nucleophilic activity of protein B Formation of p-nitrophenol
from p-nitrophenyl acetate was monitored spectrophotometrically as
described in Materials and Methods The reaction mixtures contained
protein B at 1 mgÆmL)1 (dotted line), protein B at 0.5 mgÆmL)1
(broken line), boiled protein B at 1 mgÆmL)1(dashed line),
hydroxy-lase at 1 mgÆmL)1(broken/dotted line) and no protein (solid line).
Fig 6 Detection of protein B in vivo Western blot probed with anti-(protein B) sera showing SDS cell extracts of Mc capsulatus (Bath) from fermentor cultures expressing (lane 1) sMMO and (lane 2) pMMO (negative control containing no protein B or B¢) Molecular masses of standards are indicated in kDa.
Trang 7under high-copper conditions), which did not exhibit
sMMO activity, confirmed that the proteins that reacted
with the antisera corresponded to proteins B and B¢
These results strongly suggested that protein B degraded
to B¢ in vivo and were consistent with the hypothesis that the
cleavage reaction may serve to control the in vivo activity of
sMMO One possibility was that conversion of protein B to
B¢ may account, at least in part, for the observed
inactiva-tion of sMMO when the growth-medium copper
concen-tration was increased, which also causes cessation of sMMO
expression and induces the copper-dependent pMMO, via
the so-called copper switch [2,41] Experiments were
there-fore conducted to determine whether addition of copper to a
Mc capsulatus(Bath) culture altered the cellular levels of
proteins B and B¢ during the switch from sMMO to pMMO
expression The abundances of proteins B and B¢ remained
constant throughout the time course, even until the cells
began to express pMMO, and so formation of protein B¢
did not appear to control the activity of sMMO during the
copper switch
D I S C U S S I O N
Mechanism of truncation
Previous observations that protein B underwent cleavage
during purification regardless of whether it had been
expressed in Mc capsulatus or E coli, even when the
protein had been purified to apparent homogeneity [16], had
shown that either truncation was autocatalytic or the
peptide bond between amino acids 12 and 13 was unusually
susceptible to digestion by very small amounts of extrinsic
proteases Here, by demonstrating that repeatedly repurified
protein B continued to undergo cleavage, we have provided
strong evidence that the cleavage reaction is independent of
the presence of contaminating proteins and so is almost
certainly autocatalytic
The insensitivity of the position of cleavage to the
amino-acid sequence around the cleavage site implies the
involve-ment of other parts of the structure in determining the
sensitivity of the scissile peptide bond to cleavage Intrinsic
nucleophilic activity within protein B is consistent with a
cleavage mechanism that proceeds via an intramolecular
rearrangement beginning with attack on the scissile peptide
bond by a nucleophilic amino-acid side chain elsewhere in
the protein (Fig 4) Thus we are able to propose the first
credible mechanism for autocatalytic cleavage of protein B
This could explain both the occurrence of cleavage and its
position, determined by distance constraints when the
flexible N-terminal region approaches the nucleophile,
which we suspect resides on the core of the protein The
core of protein B [23] has 12 exposed potential nucleophiles
(serines 34, 44, 92, 109, 110, 126; threonines 36, 49, 57, 68,
111, 117, 123, 125; cysteine 88), all of which are exposed to
the solvent, and so the precise residue(s) involved cannot
currently be assigned The residues flanking the cleavage site
do affect the rate of cleavage, perhaps by altering the steric
accessibility of the scissile peptide linkage to the nucleophile
It was also interesting to note that, as the protein was
progressively shortened from the N-terminus, a marked
decrease in stability was observed concomitant with loss of
activity, but stability was restored after removal of a further
two amino acids If binding of protein B to the hydroxylase
prevented the N-terminal region from approaching the nucleophilic group (e.g by the nucleophile being occluded
by binding to the hydroxylase) the presence of the hydroxylase may also serve to stabilize protein B
Role of the N-terminus in catalysis
By constructing a series of incremental truncates, we have shown that the amino acids from the N-terminus to Ser4 are not required for catalysis As truncation beyond Ser4 led to progressive loss of activity until the truncate that began with Asp8 (truncate 8), which was inactive, the critically impor-tant N-terminal region appears to correspond to Ser4-Asn5-Ala6-Tyr7 We presume therefore that the presence of these residues is essential for protein B to induce the conforma-tional change in the hydroxylase whereby it exerts its effect
on catalysis It is also possible that one or more of these residues is directly involved in the pathway for electron transfer between the reductase and the hydroxylase Precise assignment of the essential N-terminal residues is problem-atic because not all the truncates retained the initiating methionine For instance, it is difficult to assess the role of Tyr7 because truncate 7 had the initial methionine but truncate 8 did not
NMR analysis of protein B from Mc capsulatus (Bath)
in the presence of the hydroxylase indicated that the structured core region of protein B interacted with the hydroxylase but gave no indication of the involvement of the N-terminus [23] This was difficult to reconcile with the observed critical importance of the N-terminal region in the functional [26] and physical [16] interaction of protein B with the hydroxylase One possibility was that the structure
of the core region was different in proteins B and B¢ The available structural data, however, lend little weight to this theory SAXS data suggested elongation of the overall structure of protein B on truncation [22], but CD and fluorescence spectroscopy showed that any change in conformation on truncation must be extremely slight Recent NMR results with the sMMO system from
Ms trichosporium OB3b [42], however, have shown that the N-terminal region of protein B does indeed interact with the hydroxylase In the presence of the hydroxylase, NMR signals due to His4 (equivalent to Ser4 in the Mc capsulatus system) and Tyr7 (which is conserved in both Ms trichos-poriumand Mc capsulatus) broadened, indicating interac-tion with the hydroxylase These assignments are consistent with our incremental truncation studies, which showed that the functionally important residues lay in the region Ser4– Tyr7 Signals due to His32, which is toward the inner end of the flexible N-terminal region, were also perturbed by the presence of the hydroxylase, again indicative of binding [42], although analysis of the function of this residue is not accessible via the incremental truncation method used here The NMR study indicated that the isolated 29 N-terminal residues of protein B bound to the hydroxylase in a manner that was competitive with the full-length protein B [42] Conversely, an artificially synthesized dodecapeptide corresponding to amino acids 1–12 of protein B (SVNSNAYDAGIM, which was purified to homogeneity and confirmed by ESI-MS to have a molecular mass of 1241.3 Da), did not restore function to protein B¢ [43] Thus
it is possible that the N-terminus and core of protein B can bind independently to the hydroxylase, but the covalent
Trang 8connection between them is needed to induce the required
conformational change in the hydroxylase
It is also intriguing that the capacity of protein B to
activate the hydroxylase during the whole-complex sMMO
reaction was not affected by changes to the amino acids
flanking the site of cleavage for protein B¢ formation, but
the inhibitory effect of intact protein B in the peroxide shunt
reaction was diminished by such mutations This suggests a
fundamental difference in the nature of the interactions
required for the stimulatory and inhibitory effects of protein
B Either the two types of effect result from binding at
different sites on the hydroxylase, or the conformational
change that the mutant forms elicit in the hydroxylase is
different from that elicited by the wild-type such that the
inhibitory effect alone is diminished
Biological significance of truncation
All known sMMOs require a protein B component for full
activity, and other homologous binuclear iron active-centre
mono-oxygenases also have an essential regulatory protein
that is, like protein B of sMMO, a small protein without
prosthetic groups [44] The susceptibility of protein B and
other regulatory proteins (such as the regulatory protein of
alkene monooxygenase from Rhodococcus rhodochrous
B-276 (S C Gallagher & H Dalton, unpublished
observa-tions) to inactivation by proteolytic degradation offers a
mechanism by which their activity, and hence the activity of
the whole enzyme complex, could be controlled It also
offers an explanation for the persistence of the regulatory
components during evolution
In the case of sMMO, autocatalytic cleavage may ensure
that the half-life of active protein B is short, and so the
activity of protein B could be controlled at the
transcrip-tional or translatranscrip-tional levels with minimal lag time
Alternatively, other factors in the cell may control the rate
of autoproteolysis of protein B, in response to
environ-mental factors or the metabolic state of the cell It is
interesting to note that the protein B of Ms trichosporium
OB3b is much less susceptible to cleavage than that of
Mc capsulatus(Bath) and that the truncated form of the
Ms trichosporium protein is not observed in vivo during
growth using sMMO (A J Callaghan, S E Slade & H
Dalton, unpublished observations) Also, although protein
B from Methylocystis sp strain M does undergo N-terminal
truncation, this is prevented by protease inhibitors [45]
Thus, it may be that the importance of truncation of protein
B in determining sMMO activity differs among the
sMMO-expressing methanotrophs
As proteins B and B¢ were observed in vivo at comparable
levels during steady-state growth of Mc capsulatus (Bath),
truncation of protein B is evidently a significant factor in
determining the amount of active protein B in the cell It was
suspected that truncation of protein B played a role in the
observed rapid inactivation of sMMO when the copper
concentration of the medium was increased, in advance of
the induction of the copper-dependent pMMO via the
copper switch However, the abundance of proteins B and
B¢ was unchanged even after detectable sMMO activity had
been lost and pMMO induced (data not shown), so it
appears that truncation of protein B does not play a role in
controlling sMMO activity during the copper switch It
remains to be demonstrated whether regulation of sMMO
by other factors, such as starvation and other metabolic stresses, is effected in vivo via truncation of protein B
A C K N O W L E D G E M E N T
This work was funded through a Biotechnology and Biological Sciences Research Council (BBSRC) Studentship to A J C.
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