Conformational analysis of putative regulatory
subunit D of the toluene/o-xylene-monooxygenase
complex from Pseudomonas stutzeri OX1
ROBERTA SCOGNAMIGLIO,1EUGENIO NOTOMISTA,1PAOLA BARBIERI,2
PIERO PUCCI,1,3FABRIZIO DAL PIAZ,1,3ANNA TRAMONTANO,4
AND ALBERTO DI DONATO1
1Dipartimento di Chimica Organica e Biologica, Universita ´ di Napoli Federico II, 80134 Napoli, Italy
2Dipartimento di Genetica e di Biologia dei Microrganismi, Universita ´ di Milano, 20133 Milano, Italy
3Centro Internazionale di Servizi di Spettrometria di Massa, 80131 Napoli, Italy
4IRBM P. Angeletti, Via Pontina Km 30.600—00040 Pomezia (RM), Italy
(RECEIVED August 22, 2000; FINAL REVISION October 26, 2000; ACCEPTED November 23, 2000)
A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for
toluene/o-xylene-monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and
purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the
sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was
probed by limited proteolysis carried out under strictly controlled conditions using several proteases as
proteolytic probes. The same experiments were carried out on the homologous P2 component of the
multicomponent phenol hydroxylase from Pseudomonas putida CF600. The proteolytic fragments released
from both proteins in their native state were analyzed by electrospray mass spectrometry, and the prefer-
ential cleavage sites were assessed.
The results indicated that despite the relatively high similarity between the sequences of the two proteins,
some differences in the distribution of preferential proteolytic cleavages were detected, and a much higher
conformational flexibility of subunit D was inferred. Moreover, automatic modeling of subunit D was
attempted, based on the known three-dimensional structure of P2. Our results indicate that, at least in this
case, standard modeling procedures based on automatic alignment on the structure of P2 fail to produce a
model consistent with limited proteolysis experimental data. Thus, it is our opinion that reliable techniques
such as limited proteolysis can be employed to test three-dimensional models and highlight problems in
automatic model building.
Keywords: Monooxygenase; expression; Pseudomonas; recombinant; limited proteolysis; mass spectrom-
A gene cluster has been isolated by screening a library of
Pseudomonas stutzeri OX1 genomic DNA, and cloned
(Bertoni et al. 1996). Sequence analysis of the cluster re-
vealed the presence of six open reading frames (ORFs) with
a high degree of identity with components of monooxygen-
ase systems (Bertoni et al. 1998) found in benzene- and
toluene-degradative pathways. These similarities were con-
firmed by individual ORF subcloning, expression, and elec-
trophoretic analyses of the expressed proteins (Bertoni et al.
1998). The results of these studies strongly suggest that the
gene cluster encodes a novel oxygenase system named tolu-
ene/o-xylene-monooxygenase (Tomo). It has been shown
(Bertoni et al. 1996) that the Tomo system is able to cata-
Reprint requests to: Alberto Di Donato, Dipartimento di Chimica Or-
ganica e Biologica, Universita ´ di Napoli Federico II, Via Mezzocannone,
16. 80134 Napoli, Italy; e-mail: email@example.com; fax (39) (081) 552-
Abbreviations: Tomo, toluene/o-xylene-monooxygenase; Tomo D, sub-
unit D of the complex Tomo; DmpM (protein P2), regulatory component
of phenol hydroxylase; ORF, open reading frame; ES/MS, electrospray
mass spectrometry; MALDI, matrix assisted laser desorption ionization;
LB, Luria-Bertani; EDTA, ethylenediaminetetraacetic acid; PAGE, poly-
acrylamide gel electrophoresis; SDS, sodium dodecylsulfate; Tris, tris(hy-
droxymethyl)aminomethane; DTT, dithiothreitol; HPLC, high pressure liq-
uid chromatography; IPTG, isopropyl-?-D-thiogalacto-pyranoside; PVDF,
polyvinyl difluoro; PDB, Protein Data Bank; V-8, V-8 endoproteinase;
Asp-N, endoproteinase Asp-N.
Article and publication are at www.proteinscience.org/cgi/doi/10.1110/
Protein Science (2001), 10:482–490. Published by Cold Spring Harbor Laboratory Press. Copyright © 2001 The Protein Society
lyze two consecutive hydroxylation reactions of toluene and
o-xylene, via the intermediate production of a mixture of o-,
m-, and p-cresol, and 2–3-dimethylphenol and 3–4-dimeth-
ylphenol, respectively. Moreover, Tomo is also able to de-
grade highly chlorinated compounds (Ryoo et al. 2000).
Homology studies (Bertoni et al. 1998) suggest that P.
stutzeri OX1 toluene/o-xylene-monooxygenase is a multi-
component enzyme, made up of six different subunits, simi-
lar to the composition of monooxygenases from B. cepacia
AA1 (Newman and Wackett 1995), B. picketii PKO1
(Byrne et al. 1995), and P. mendocina KR1 (Yen et al.
1991; Yen and Karl 1992). In fact, each polypeptide com-
ponent of Tomo shows similarities with components of sev-
eral enzymatic complexes involved in the oxygenation of
aromatic compounds (Bertoni et al. 1998).
The similarity between subunit D of the Tomo complex
and the low molecular weight proteins TmoD from P. men-
docina KR1 (Pikus et al. 1996), MmoB from M. capsulatus
(bath) (Stainthorpe et al. 1990), and P2 from P. putida sp.
strain CF600 (Nordlund et al. 1990) appears particularly
interesting. It has been demonstrated that these proteins are
involved in the stimulation of their respective hydroxylase
components (Froland et al. 1992; Pikus et al. 1996; Qian et
al. 1996) and do not play a direct role in the electron trans-
This paper reports the conformational analysis of the sub-
unit D component of the toluene/o-xylene-monooxygenase
system from P. stutzeri OX1 by the limited proteolysis/mass
spectrometry approach (Zappacosta et al. 1996; Scaloni et
al. 1998; Orru ` et al. 1999) using several proteases as con-
formational probes. The same experiments were also per-
formed on the homologous (65% similarity) P2 protein
whose three-dimensional structure has been solved by
NMR. The analysis indicates that P2 is very flexible, and
well suited to bind and interact with the hydroxylase com-
ponent of the complex.
Molecular models of subunit D were constructed by au-
tomatic modeling using the P2 protein as a template (Qian
et al. 1996), and used to interpret the results of limited
proteolysis/mass spectrometry. The analysis indicated that
the automatically derived model is somewhat inadequate to
explain all the selective cleavages found in subunit D. Our
results suggest that, whereas automatic model building is a
valuable system for obtaining approximate structural mod-
els, experimental validation methodologies, such as limited
proteolysis, are also needed to evaluate and validate its re-
Amino acid sequence of subunit D of Tomo
The primary structure of the recombinant subunit D of
Tomo as derived from the translation of the coding gene
sequence (Bertoni et al. 1996; Bertoni et al. 1998) was
verified by the peptide mapping strategy. Aliquots of the
HPLC-purified protein were digested with either cyanogen
bromide or Asp-N endoprotease, and the resulting peptide
mixtures were analyzed by MALDI/MS. The mass signals
recorded in the spectra were mapped onto the anticipated
sequence of subunit D on the basis of their mass value and
the specificity of the enzyme, leading to the complete veri-
fication of the amino acid sequence of subunit D, shown in
Topological studies of subunit D of Tomo and dmpM
The surface topology of subunit D in solution was investi-
gated by a strategy that combines limited proteolysis ex-
periments with mass spectrometric procedures (Zappacosta
et al. 1996; Scaloni et al. 1998; Orru ` et al. 1999). The
overall strategy is based on the evidence that the amino acid
residues located within exposed and flexible regions of the
protein can be recognized by proteases, leading to a reason-
ably good indication of the conformation of the protein in
Limited proteolysis experiments were performed by us-
ing trypsin, V-8 endoprotease, Asp-N endoprotease, chy-
motrypsin, elastase, and subtilisin as proteolytic probes. The
protein was incubated with each protease under strictly con-
trolled conditions to ensure the maintenance of its native
conformation, and to increase the selectivity of proteases
(Zappacosta et al. 1996; Orru ` et al. 1999). The extent of the
enzymatic hydrolysis was monitored on a time-course basis
by sampling the incubation mixtures at different interval
times, followed by HPLC fractionation. The fragments re-
leased from the proteins were identified by ES/MS, leading
to the assignment of the cleavage sites. Preferential proteo-
lytic sites were assigned from the identification of the two
complementary peptides released from the intact protein
following a single proteolytic event. When two or more sites
were identified in the same experiment, these cleavages
were always due to a single proteolytic event on the intact
protein molecule, apparently occurring with the same kinet-
ics of hydrolysis.
Peptides generated by subsequent digestion of larger
fragments and not released from the intact protein were not
considered in the interpretation of proteolysis data. As an
example, Figure 2 shows the HPLC chromatograms of the
aliquots withdrawn after 10 min, 30 min, and 60 min of
trypsin digestion of subunit D. Under the controlled condi-
tions used (1 : 1000 (w : w) trypsin : Tomo D), most of the
protein remained undigested and only a few specific frag-
ments were released (Fig. 2C). The ES/MS analysis of in-
dividual fractions identified these fragments as the peptides
1–25, 1–56, 1–79, 80–109, and 57–109, respectively. The
major component contained a mixture of the intact protein
and the peptide 26–109. The presence of the complementary
Conformational analysis of subunit D of the Tomo complex
peptide pairs, that is, 1–25 and 26–109, 1–56 and 57–109,
and 1–79 and 80–109, clearly indicates that these fragments
originated from a single proteolytic event that occurred on
the native protein. These data demonstrate that Arg25,
Arg56 and Arg79 were the preferential tryptic cleavage sites
in subunit D. Similar results were obtained when the spe-
cific proteolytic enzymes V-8 protease and endoprotease
Asp-N were used.
Experiments were also carried out by incubating subunit
D with broader specificity proteases such as chymotrypsin,
elastase, and subtilisin. As an example, chymotrypsin di-
gested subunit D preferentially at Leu8, Phe78, Phe103, and
Tyr104 (Fig. 1 and Table 1) as inferred by the identification
of the peptide pairs 1–8 and 9–109, 1–78 and 79–104, and
the fragments 1–103 and 1–104.
The overall data from the limited proteolysis experiments
performed on subunit D are summarized in Table 1 and
The data indicate that the preferential proteolytic sites are
scattered along the entire sequence of the protein. Besides
few isolated cleavages essentially occurring within the N-
terminal portion (Leu8, Asp12, and Glu35), the preferential
proteolytic sites are gathered within a few well-defined re-
gions of the subunit D sequence. Four short segments,
Arg25-Asp28, Tyr54-Arg56, Asp61-Glu62 and Phe103-
Tyr104, and the longer region spanning residues 78–88
were identified as accessible areas. Thus, the four short
sequences might constitute mobile loops connecting ordered
secondary structure elements, whereas the segment 78–88
might represent a very flexible and unstructured region.
A further consideration suggested by the limited prote-
olysis results concerns the three very hydrophilic regions in
the sequence of subunit D (segments 38–51, 66–72, and
95–102). These segments, while containing a large number
of putative cleavage sites, were resistant to proteases.
These regions were thus suspected to be either highly
structured or not exposed to the solvent, although it should
be noted that unpaired charged residues are rarely found
buried inside proteins.
The same limited proteolysis experiments were carried
out on the homologous DmpM (P2) protein for comparison.
The data collected are reported in Figure 1 and Table 1,
indicating that the proteolytic pattern of P2 is similar but not
identical to that defined for subunit D of the Tomo complex.
However, it should be emphasized that for all proteases, a
higher enzyme-to-substrate ratio had to be used in the P2
Modeling of subunit D of Tomo
The NMR structures of three different regulatory compo-
nents of multicomponent oxygenases are currently avail-
able: the P2 protein, the regulatory component of a phenol
hydroxylase (PDB code 1HQI), and the components of two
different methane monooxygenases (PDB codes 1CKV and
2MOB). The structure of P2, the one sharing the higher
Fig. 1. Alignment of the P2 protein and Tomo D used to generate models. (#) Identical residues; (*) conservative substitutions. The
sites of proteolytic cleavages (see Results section) are highlighted in red. Residues of Tomo D not aligned with the P2 sequence are
shown in bold. The secondary structure of the P2 protein, determined by means of the DSSP program (Kabsch and Sander 1983), is
Scognamiglio et al.
Protein Science, vol. 10
sequence similarity with Tomo D, was selected as the tem-
plate for the model building of Tomo D. This choice is also
supported by the observation that their respective protein
complexes catalyze the transformation of the same type of
substrate, and it has been proposed that Tomo D and P2 act
as regulatory components and are involved in substrate
binding (Qian et al. 1996; Bertoni et al. 1998).
The amino acid sequences of Tomo D and P2 share
36.9% identity and 65.5% similarity in a 90-residue overlap
segment. In addition, the sequence of Tomo D shows a
15-residue N-terminal extension which was not considered
in the modeling procedure. The two sequences were aligned
using a standard algorithm and the BLOSUM62 matrix
The structure of P2 has been solved by NMR (Qian et al.
1996), and the coordinates for 12 models are present in the
Protein Data Bank entry 1HQI. P2 turned out to be ex-
tremely flexible in the experimental conditions used: the
RMS deviation from the average structure of the secondary
structure segments is around 2.5 Å, but the RMS deviation
of these segments among the 12 different submitted models
can be as high as 4.5 Å with an average value of about
The NMRCLUST algorithm (Kelley et al. 1996) was
used to cluster the 12 structures into three groups. Two
clusters contained only two models each, whereas eight
structures gathered within the largest cluster which was se-
lected for the modeling procedure. The remaining models
were discarded, as they appeared quite different from the
main group of structures.
The set of atoms whose positions are well defined
throughout this reduced set of structures were then extracted
and the conserved core of the selected eight models defined
using the NMRCore algorithm (Kelley et al. 1997). The
main portion which is consistently structurally conserved
among the eight structures is composed by residues 4–11,
29–32, 34–44, and 71–73 and accounts for 34.2% of the
total core. Finally, the structure closest to the centroid of the
largest cluster (model 3) was selected for modeling, as it is
more representative of the ensemble than the commonly
used minimized average structure.
Thus, one Swiss-PDB-Viewer-project file (Guex and
Peitsch 1997), containing the sequence alignment and the
set of coordinates of the selected structure 3 of P2 was
submitted to the automated modeling server. The resulting
model was visually examined with Swiss-PDB-Viewer. As
expected, given the high similarity between the template
and the query sequence, the model was almost superimpos-
able on the corresponding template structure (data not
Knowledge of a protein’s tertiary structure is a prerequisite
for the proper understanding of and for engineering its func-
tion. This makes the successful prediction of a protein ter-
tiary structure a central issue in proteomics, in cases in
which the experimental three-dimensional structure is not
yet available. However, although prediction and modeling
techniques have greatly improved in the past few years
(Moult et al. 1999), simple and effective tools are needed
Fig. 2. HPLC chromatograms of the tryptic digests of subunit D of Tomo
after 10 (A), 30 (B), and 60 (C) min of incubation.
Table 1. Limited proteolysis cleavage sites in subunit Tomo D
ratio Sites in Tomo D
ratioSites in DmpM
R25, R56, R79
Y54, F78, F103,
D12, D28, D61
E35, E62, E82
L8, Q81, S88
L8, F78, F103
Conformational analysis of subunit D of the Tomo complex
for experimentally testing tertiary structure models. This
would give confidence in the prediction results, especially
when automated procedures are used to generate the mod-
Limited proteolysis studies of protein molecules coupled
with fast and sensitive mass spectrometry analyses of pep-
tide fragments is one such tool. Its potency lies in the fact
that proteolytic enzymes are amenable probes of protein
secondary and tertiary structures (Fontana et al. 1997). Fur-
thermore, such studies can be used both to describe surface
topology and to monitor conformational changes of proteins
in solution (Orru ` et al. 1999)
The primary structure of Tomo D, as determined in the
present study, is largely similar to that of P2 protein from P.
putida, except for its 15 N-terminal amino acids (Fig. 1).
The Tomo D polypeptide chain contains 109 residues,
whereas P2 has only 90 residues. Both proteins were sub-
mitted to conformational analysis using the limited prote-
olysis/mass spectrometry approach. When the results ob-
tained on Tomo D and P2 were compared, similar but not
identical proteolytic patterns could be defined. Both pro-
teins showed short flexible segments accessible to proteases
and very likely connecting secondary structure elements and
a larger loop encompassing residues 76–83 (numbers refer
to Tomo D sequence). Moreover, the three hydrophilic re-
gions resistant to proteases present in Tomo D find their
homologous counterparts in P2 structure, although a low
degree of sequence similarity occurs in the segment 38–51
On the other hand, Tomo D appeared to be more flexible
and/or less structured with respect to its homolog P2 as
demonstrated by the lower enzyme-to-substrate ratios used
in all the proteolytic experiments,. Moreover, the region
encompassing residues 25–28 and 61–62 and the Glu resi-
due at position 35, accessible to proteases in Tomo D, were
not recognized in P2, although putative cleavage sites oc-
curred at corresponding sequence positions.
Thus, despite their high degree of similarity, a number of
topological differences could be mapped on the surface of
the two proteins.
The results of the limited proteolysis experiments carried
out on P2 were then mapped onto the three-dimensional
structure of the protein (Qian et al. 1996) as a test of the
reliability of our structure validation procedure. Figure 3A
shows the location of the preferential proteolytic sites
within model 3 of P2. The accessible segment Met54-Arg56
(numbering refers to Tomo D sequence) occurs within a
flexible portion of the protein at the junction between a loop
region and strand ?2. Analogously, the region encompass-
ing residues Arg76 to Met83, readily cleaved by proteases,
corresponds to the long segment closing the hydrophobic
cavity where the substrate is thought to bind to the protein
and interact with the hydroxylase complex. It should be
stressed that this segment belongs to the region showing the
largest deviation of atomic coordinates among the 12 NMR
structures of P2, and it has been described as highly flexible
(Qian et al. 1996). Accordingly, the more representative
models, as defined by the clustering algorithm, display a
nonstructured region spanning residues 76–86. This find-
ing demonstrates that sensitive conformational probes such
as proteolytic enzymes were able to correctly discriminate
among the ensemble of NMR structures depicted for P2.
The two hydrophilic portions 66–72 and 95–100 not rec-
ognized by proteases were identified as located within two
Fig. 3. Structure 3 of the P2 template (A) and one of the modeled struc-
tures (model 3) of Tomo D (B) are shown, respectively, with the residue
side chains at the limited proteolysis sites. Color codes in (C) and (D)
correspond to secondary structure: ?-helix (red), ?-strands (blue), loops
(white). The same color code for P2 structure in (A) has been used for the
corresponding model in (B).
Scognamiglio et al.
Protein Science, vol. 10
highly structured regions corresponding to the helix ?2 and
the strand ?5, respectively, thus explaining their resistance
to protease action. The third protease-resistant region 40–51
is located within a large loop connecting helix ?1 and strand
?2 and was therefore expected to be proteolytically acces-
However, the specific amino acid residues occurring in
this segment are poorly amenable to proteolysis, causing
this portion to be left untouched. It should be noted that the
amino acid sequence of this segment shows the lowest de-
gree of similarity with Tomo D.
Finally, unexpected cleavages were observed at Phe101,
Leu103, and Trp105 within strand ?6, indicating that this
region might be endowed with considerable conformational
flexibility. It should be noted that the C-terminal portion of
P2 does not belong to the regions structurally conserved
among the eight clustered NMR structures.
When the data collected from the limited proteolysis ex-
periments carried out on Tomo D were located on the pro-
tein model obtained from the Swiss Prot Server (Fig. 3B),
an intriguing picture emerged. Most of the preferential
cleavage sites identified on P2 found their homologous
counterparts in Tomo D (Fig. 1), whereas few discrepancies
could be observed. In particular, the regions Arg25-Asp28
and Asp61-Glu62 occur in exposed loops connecting the
end of strand ?1 with the first ?-helix and strand ?4 with
the following helix ?2, respectively. Accordingly, these
portions were found accessible to proteases. The sequences
containing these segments are well aligned with P2 protein,
and there are no insertions or deletions between the two
proteins. Thus, it is surprising that Tomo D is cut by the
proteolytic enzymes, whereas P2 is not. Moreover, the flex-
ible loop connecting ?1 with strand ?2 in Tomo D (region
40–51) contains a number of putative cleavage sites, and yet
it was not recognized by proteases.
Given these discrepancies, we also verified whether al-
ternative models of Tomo D structure based on the struc-
tures of methane monooxygenase regulatory components,
PDB codes 1CKV and 2MOB, would be more consistent
with the proteolytic patterns, but this was not the case (data
Thus, it is evident that there is a discrepancy between the
model of the three-dimensional structure of Tomo D and the
results of the partial proteolysis experiments. This cannot be
due to a difference in flexibility or underdetermination of
the NMR structure of P2, since these regions belong to the
conserved core. It is then possible that the automatically
derived sequence alignment used to build up the model does
not correspond to the optimal structural alignment.
Several attempts to obtain an alignment more consistent
with the data using different algorithms and scoring systems
and including other members of the family did not provide
a satisfactory answer. It is obviously possible to manually
modify the alignment according to the experimental data.
Unfortunately, the constraints given by the proteolytic
cleavages do not allow discrimination between a number of
alternative alignments. As an example, we show one of
them in Figure 4. This alignment is not optimal in terms of
sequence identity (25% with respect to 37% of the auto-
matic alignment) and would imply an insertion immediately
after the first ?-strand of P2 and a deletion of the irregular
region following helix ?1. However, the inserted region
25–35 would be exposed to the solvent and would not have
any structurally equivalent counterpart in P2, consistent
with the proteolysis data.
The discrepancy observed at the level of Asp61 and
Glu62 might in principle be explained on the basis of the
different character of the amino acid residues occurring at
homologous positions in P2, that is, Lys and Arg, which can
be involved in different interactions. However, neither the
Fig. 4. Suboptimal sequence alignment obtained by manual modification of the alignment shown in Fig.1. The sites of proteolytic
cleavages are highlighted in red.
Conformational analysis of subunit D of the Tomo complex
structure of P2 nor the Tomo D model supports this hypoth-
esis, as these two residues are exposed and not involved in
hydrogen bonds or salt bridges. A more convincing expla-
nation might be given by considering that in P2, the Lys and
Arg residues lie quite close to the N-terminal tail, the C-?
distance between Lys and residue 4 being about 5 Å. As
judged from the NMR data, this part of the protein is flex-
ible and not anchored to the core of the structure, and might
hinder the Lys and Arg residues, thus preventing proteolytic
cleavages. On the other hand, the long N-terminal extension
(16 residues) occurring in Tomo D is most likely structured
as being not accessible to proteases (cleavages were only
observed at Leu8 and Asp12) and should then not be able to
impair cleavages at residues 61 and 62.
Thus, the analysis of the new proposed sequence align-
ment and the experimental data suggest that the structural
similarity between Tomo D and P2 is higher in the C-ter-
minal portion and that the N-terminal region including the
end of the first strand, the first ? helix and the loop con-
necting this helix to the second ? strand is differently folded
in the two proteins.
It is well known that a major problem in predicting pro-
tein structure by homology modeling is that the sequence
alignment from which the model is built may not be the best
with respect to the correct equivalence of residues assessed
by structural or functional criteria. It has been proposed
(Saqi et al. 1992) that a number of suboptimal alignments
should be generated and examined before selecting the ap-
propriate one for model building. In this case, the problem
is to find a meaningful procedure to rapidly filter the dif-
ferent possible alignments. This still represents the major
obstacle in large-scale automatic modeling, where manual
inspection and/or careful analysis of all possible suboptimal
sequence alignments is prohibitive.
The results presented in this paper indicate that in this
case, standard modeling procedures based on automatic
alignment on the structure of P2 fail to produce a model
consistent with experimental data. Moreover, this example
clearly shows that fast and reliable techniques such as lim-
ited proteolysis can be employed to highlight problems in
automatic model building, and algorithms should be devised
to use this type of experimental data to select among
A further conclusion can be drawn from these data. In all
the experiments, a higher enzyme-to-substrate ratio was al-
ways needed for P2 with respect to that required for Tomo
D, under the same conditions (Table 1). This indicates that,
despite the high structural flexibility of P2, Tomo D is even
more flexible, and adaptive to its hydroxylase partner. This
is in line with the wider substrate specificity of the complex
Tomo with respect to other multicomponent monooxygen-
ases (Bertoni et al. 1996). In fact, it has been proposed (Qian
et al. 1996) that the flexible cavity defined by helix 2 and
strands 3 and 5 is the site where the substrate enters the
complex of phenol hydroxylase. If the same functional role
is fulfilled by the cavity present in all the models derived for
the subunit D of Tomo, then its greater flexibility with
respect to that of P2 would allow a different variety of
aromatic substrates to entry into the core of the complex.
Materials and methods
The DNA coding for DmpM inserted into vector pET3a (pET3a-
dmpM) was provided by Dr. Justin Powlowski. E. coli strain
JM109 was purchased from Boehringer. Labeled oligonucleotides
were from Amersham Italia. The Wizard DNA purification kit for
elution of DNA fragments from agarose gel was obtained from
Promega Italia. Enzymes and other reagents for DNA manipula-
tion were from Promega Italia. PVDF membranes were from Per-
kin Elmer. Cyanogen bromide, acetic anhydride, trypsin, endopro-
teinase V-8, chymotrypsin, elastase, and subtilisin were purchased
from Sigma. Endoproteinase Asp-N was from Boehringer. Sol-
vents were HPLC-grade from Baker.
Bacterial cultures, plasmid purifications, and transformations were
performed according to Sambrook et al. (1989). Double-strand
DNA was sequenced with the dideoxy method of Sanger et al.
(1977), carried out with a Sequenase version II Kit (Amersham
Italia) with deoxynucleotide triphosphates purchased from Phar-
macia Italia (Italy).
Expression and purification of subunit D of Tomo
Aliquots of 20 ng of each plasmid (pMZ1204) (Bertoni et al. 1998)
and pET3a-DmpM (Cadieux and Powlowski 1999) were used to
transform competent E. coli JM109 cells or BL21(DE3), respec-
tively, plated onto LB/ampicillin plates. One recombinant clone
from each transformation was grown in 10 mL of LB-medium,
supplemented with ampicillin, at 37°C up to O.D.600 nm? 0.7.
These cultures were used to inoculate 1 L of LB supplemented
with 50 ?g/mL ampicillin, and grown at 37°C until A600ranged
from 0.6 to 0.7. Expression of the recombinant protein was in-
duced by IPTG at a final concentration of 0.5 mM, and the culture
was kept for another 3 h at 37 °C under vigorous shaking. Cells
were then collected by centrifugation for 10 min at 5,520 × g, at
4°C, and the cell paste stored at −80°C. SDS-PAGE analysis of an
aliquot of induced and noninduced cells extracted in electropho-
resis loading buffer showed that in each culture, a protein with a
molecular mass of about 12 kDa, the expected molecular size of
recombinant subunit D and of DmpM, was produced only in the
The identity of the expressed proteins was further checked by
N-terminal sequence determination, run on samples directly blot-
ted on PVDF membranes from the electrophoretic gel.
Cells from 1-L cultures were suspended in 10 mL of buffer A
(25 mM MOPS at pH 6.9, containing 5% glycerol, 1 M DTT) and
disrupted by sonication (6 × 1 min cycle, on ice). The soluble
fraction was separated by the insoluble fraction by centrifugation
at 17,400 × g for 60 min at 4°C. SDS-PAGE analysis of the soluble
and insoluble fractions (data not shown) revealed that the proteins
Scognamiglio et al.
Protein Science, vol. 10
of interest were present only in the soluble fraction. Yields were of
∼20–30 mg of protein/L of bacterial culture, on the basis of a
densitometric scanning of the electrophoretic profile.
The crude extracts were loaded onto a Fast Flow Q-Sepharose
column (10 × 200 mm), equilibrated in buffer A. Elution was car-
ried out at 4°C, at a flow rate of 7 mL/h, with a linear gradient (0
to 0.5 M, 160 mL) of NaCl. The proteins of interest were identified
in the eluate by running SDS-PAGE of alternate fractions, and then
pooled. Protein samples were concentrated by ultrafiltration in an
Amicon apparatus equipped with YM3 membrane, then loaded on
a Sephadex G75 superfine column (28 × 350 mm), and eluted at 12
mL/h with buffer A containing 0.3 M NaCl. The analysis of the
eluate carried out by SDS-PAGE of alternate fractions revealed
that either in the case of DmpM or of subunit D, two distinct peaks,
both containing a protein of the expected molecular weight (about
10 kDa in the case of DmpM, and about 12 kDa in the case of
subunit D), can be separated (data not shown). It has already been
shown (Cadieux and Powlowski 1999) that DmpM exists in two
forms: an active monomeric form, and an inactive dimer, which
can convert one into the other. Thus, we discarded in both cases
the fast-eluting peak and collected the fractions eluting at the cor-
rect (monomeric) elution volume.
Samples of purified subunit D and DmpM were desalted by
RP-HPLC on a Phenomenex C18 column (see Protein sequence
determination section below) and subjected to electrospray mass
spectrometry.The average molecular
12,142.2 ± 0.6 Da, and 10,360.1 ± 0.9 Da, for Tomo D and
DmpM, respectively. These values are in agreement with the theo-
retical values calculated on the basis of the deduced amino acid
sequence of subunit D (12,141.8 Da), and that of DmpM (10,359.7
Da), respectively, and confirm that the protein species purified
through the procedure above are in their monomeric state. How-
ever, given the possibility of the conversion of these species to
“oligomeric” forms (Cadieux and Powlowski 1999), we always
stored the proteins in glycerol containing buffers, which have been
shown (Cadieux and Powlowski 1999) to prevent interconversion
of the two forms. Finally, we routinely checked the aggregation
state of the samples prior to their use by running analytical gel
filtration chromatography on a HiLoad 10/30 Superdex 75 column,
eluted with 50 mM ammonium acetate at pH 5.0, containing 0.3 M
NaCl, at a flow rate of 0.3 ml/min.
Protein sequence determination
Proteins were desalted on Phenomenex Jupiter C18 reverse-phase
columns (250 × 2.1 mm, 100 Å pore size) eluted at 0.2 mL/min
with a linear gradient of a two-solvent system. Solvent A was 0.1%
TFA in water, solvent B was acetonitrile containing 0.1% TFA.
The gradient was constructed increasing the concentration of sol-
vent B from 20% to 60% in 50 min. Desalted proteins (2 nmol)
were incubated in 100 ?L of a 10-fold molar excess of CNBr
dissolved in 70% TFA, for 12 h under nitrogen in the dark. Re-
action was stopped by dilution with 1 mL of cold water.
Digestion with endoprotease Asp-N was carried out in 50 mM
NaHCO3at pH 8.0, overnight at 37°C, using a 1 : 50 w : w, en-
Peptide mixtures were directly analyzed by MALDI mass spec-
Limited proteolysis experiments
Limited proteolysis experiments were carried out on 3 nmol of
proteins not subjected to the desalting procedure described in the
previous paragraph, with trypsin, V-8 protease, endoproteinase
Asp-N, chymotrypsin, elastase, and subtilisin. Enzymatic diges-
tions were all performed in 0.4 % ammonium bicarbonate at pH
7.0, at 37°C with enzyme-to-substrate ratios ranging from 1 : 1500
to 1 : 30 w : w (see Table 1). The extent of digestion was moni-
tored on a time-course basis by sampling the reaction mixture at
different time intervals from 10 to 60 min. Digested protein
samples were acidified by adding TFA to lower the pH to 2.5, and
analyzed by RP-HPLC as described in the section on protein se-
quence determination. Elution was monitored at 220 and 280 nm.
Individual fractions were collected and identified by ES/MS.
Protein samples or proteolytic fragments were analyzed by elec-
trospray mass spectrometry using either a BIO-Q triple quadrupole
mass spectrometer (Micromass) or an API-100 single quadrupole
instrument (Perkin Elmer). Samples were directly injected into the
ion source via a loop injection at a flow rate of 5 ?l/min. Data were
acquired and elaborated using either the MASS-LINX (Micro-
mass) or the Biomultiviewer (Perkin Elmer) program.
Mass calibration was performed by means of the multiply
charged ions from a separate injection of horse heart myoglobin
(average molecular mass 16,951.5 Da). All masses are reported as
average mass. Peptide mixtures were analyzed by MALDI mass
spectrometry (MALDI/MS) using a Voyager DE instrument (Per-
kin Elmer). Typically, 1 ?L of analyte solution was mixed with 1
?L of ?-cyano-4-hydroxycinammic acid 10 mg/mL in acetonitrile/
0.2% TFA, 70 : 30 v/v, containing 250 fmoL of insulin. The mix-
ture was applied onto the metallic sample plate and air dried. Mass
calibration was performed with the mass signals of insulin at m/z
5734.5 and a matrix peak at m/z 379.1. Raw data were analyzed by
using a computer program provided by the manufacturer. All mass
values are reported as average masses.
Sequence alignment and modeling
Protein sequences were aligned using the Fasta3 program (Pearson
and Lipman 1988) available on-line at http://www2.ebi.ac.uk/
Protein modeling was carried out with the automated compara-
tive protein server freely available at http://www.expasy.ch/
swissmod/SWISS-MODEL.html (Peitsch 1996; Guex and Peitsch
1997). Models were energy-minimized with Gromos96.
N-terminal protein sequence determinations were performed on an
Applied Biosystems sequenator (model 473A), connected on-line
with an HPLC apparatus for identification of phenylthiohydanto-
ins. SDS-PAGE was carried out according to Laemmli (1970).
Protein concentrations were determined by a colorimetric assay
(BCA Protein Assay, Pierce).
The authors are indebted to Dr. Justin Powlowski, Dept. of Chem-
istry and Biochemistry, Concordia University, Canada for having
kindly provided the DNA coding for DmpM, Dr. Antimo Di Maro
for the determination of the N-terminal sequence of the proteins,
Dr. Alessia Palmieri for contributing to the preparation and char-
acterization of Tomo D, and Mrs. Aida Milano for her contribution
Conformational analysis of subunit D of the Tomo complex
in the purification of DmpM. Special thanks go to Prof. Giuseppe
D’Alessio, Dept. of Organic and Biological Chemistry, University
of Naples Federico II, Italy, for critically reading the manuscript.
This work was supported by grants from the Ministry of University
and Research (PRIN/98, CSEB:O&O, and PRIN/97, Biologia
The publication costs of this article were defrayed in part by
payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 USC section 1734
solely to indicate this fact.
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