Báo cáo khoa học: The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase pptx

The role of evolutionarily conserved hydrophobic contacts
in the quaternary structure stability of Escherichia coli
serine hydroxymethyltransferase
Rita Florio
1
, Roberta Chiaraluce
1
, Valerio Consalvi
1
, Alessandro Paiardini
1
, Bruno Catacchio
1,2
,
Francesco Bossa
1,3
and Roberto Contestabile
1
1 Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, ‘Sapienza’ Universita
`
di Roma, Italy
2 CNR, Istituto di Biologia e Patologia Molecolari, ‘Sapienza’ Universita
`
di Roma, Italy
3 Centro di Eccellenza di Biologia e Medicina Molecolare (BEMM), ‘Sapienza’ Universita
`
di Roma, Italy
Pyridoxal 5¢-phosphate (PLP)-dependent enzymes are a
large ensemble of biocatalysts that make use of the
same cofactor but have distinct evolutionary origins
and protein architectures [1–3]. According to their 3D

structure, PLP-dependent enzymes are grouped into
five evolutionarily unrelated superfamilies, correspond-
ing to as many different folds (fold types) [4]. The fold
type I group, also referred to as the aspartate amino-
transferase family [5], is the largest, functionally most
diverse and best characterized. Its members are catal-
ytically active as homodimers, although they may
assemble into higher order complexes. A single subunit
folds into two domains [6]. The central feature of the
N-terminal, larger domain is a seven-stranded b-sheet.
In some instances, the N-terminal tail does not partici-
pate as a part of the large domain but comprises a
separate structural element. The small, C-terminal
domain, comprises a three- or four-stranded b-sheet,
covered with helices on one side. The active site is
located at the interface of the domains and is delimited
by amino acid residues that are contributed by
both subunits of the catalytic dimer. Remarkably, the
Keywords
conserved hydrophobic contacts; fold type I
enzymes; pyridoxal phosphate; quaternary
structure; serine hydroxymethyltransferase
Correspondence
R. Contestabile, Dipartimento di Scienze
Biochimiche, ‘Sapienza’ Universita
`
di Roma,
Piazzale Aldo Moro 5, 00185 Rome, Italy
Fax: +39 0649 917566
Tel: +39 0649 917569

E-mail:
Website: />sito_biochimica/EN/index.html
(Received 18 September 2008, revised 23
October 2008, accepted 27 October 2008)
doi:10.1111/j.1742-4658.2008.06761.x
Pyridoxal 5¢-phosphate-dependent enzymes may be grouped into five struc-
tural superfamilies of proteins, corresponding to as many fold types. The
fold type I is by far the largest and most investigated group. An important
feature of this fold, which is characterized by the presence of two domains,
appears to be the existence of three clusters of evolutionarily conserved
hydrophobic contacts. Although two of these clusters are located in the
central cores of the domains and presumably stabilize their scaffold, allow-
ing the correct alignment of the residues involved in cofactor and substrate
binding, the role of the third cluster is much less evident. A site-directed
mutagenesis approach was used to carry out a model study on the impor-
tance of the third cluster in the structure of a well characterized member
of the fold type I group, serine hydroxymethyltransferase from
Escherichia coli. The experimental results obtained indicated that the clus-
ter plays a crucial role in the stabilization of the quaternary, native assem-
bly of the enzyme, although it is not located at the subunit interface. The
analysis of the crystal structure of serine hydromethyltransferase suggested
that this stabilizing effect may be due to the strict structural relation
between the cluster and two polypeptide loops, which, in fold type I
enzymes, mediate the interactions between the subunits and are involved in
cofactor binding, substrate binding and catalysis.
Abbreviations
CHC, conserved hydrophobic contact; eSHMT, Escherichia coli serine hydroxymethyltransferase; H
4
PteGlu, tetrahydropteroylglutamate; PLP,
pyridoxal 5¢-phosphate; SCR, structurally conserved region.

132 FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS
superimposition of fold type I enzymes reveals that the
location of the cofactor in the active site is virtually
identical in all members of the group [7].
Despite the high similarities of their 3D structures,
many fold type I enzymes show very little sequence
identity, highlighting the need to identify the structural
features that determine the common fold. Accordingly,
a computational analysis that made use of 23 nonre-
dundant crystal structures and 921 sequences of fold
type I enzymes identified 17 structurally conserved
regions (SCRs), which form the common cores of the
large and small domains. Within these SCRs, there are
three clusters of evolutionarily conserved hydrophobic
contacts (CHCs) [8]. The first and second cluster are
located in the cores of the large and small domains,
respectively, and appear to stabilize their protein scaf-
folds, allowing the proper positioning of the residues
involved in PLP binding, substrate binding and modu-
lation of the cofactor’s catalytic properties. The third
cluster forms a hinge between two conserved a-helices
(which correspond to two SCRs), located at the begin-
ning and at the end, respectively, of the large domain
(Fig. 1). Examination of the contact network shows
that the CHCs lie along one side of each helix, forming
a buried spine at positions i, i + 4, and i +7. By
apparent contrast to the two previously described clus-
ters, the third cluster does not appear to be directly
involved in the proper positioning of any active site
residue, suggesting that its high degree of evolutionary

conservation could be due to a merely structural,
rather than functional role.
In the present study, the importance of the third
hydrophobic cluster as a structural determinant of the
Escherichia coli serine hydroxymethyltransferase
(eSHMT) overall native fold was investigated by
decreasing the hydrophobic contact area of the cluster,
using a site-directed mutagenesis approach. The effects
of L85A, L276A and L85A⁄ L276A mutations on the
native structure of the enzyme were analyzed (Fig. 1).
Results
The consequences of the mutations on the native struc-
ture of eSHMT were evaluated by analyzing and
comparing the ultracentrifuge sedimentation, cofactor
binding, catalytic and spectral properties of wild-type
and mutant apo- and holoenzymes.
Quaternary structure analysis
The subunit assembly of apo- and holo-forms of wild-
type and mutant eSHMTs was characterized by analyt-
ical ultracentrifugation. Table 1 shows the values of
sedimentation coefficient and dissociation constant
(K
d
) calculated from combined sedimentation velocity
and equilibrium approaches. As established in the
available literature [9,10], wild-type eSHMT exists as a
dimer in both apo- and holo-forms, with a molecular
mass of approximately 91 kDa [9]. The ultracentrifuga-
tion experiments confirmed that the depletion of the
cofactor does not have any effect on the dimeric

assembly of the enzyme. The sedimentation velocity
Fig. 1. Schematic representation of the monomeric structure of eSHMT. Cartoon representation of a single subunit of the eSHMT ternary
complex with glycine and 5-formyl H
4
PteGlu (Protein Data Bank: 1dfo) [14], showing the N-terminal tail (residues 1–61) colored in orange,
the large domain (residues 62–211) in salmon, the interdomain segment (residues 212–279) in green and the small domain in blue. The PLP-
Gly complex is shown as yellow sticks, with the phosphorus atom in orange, the oxygen atoms in red and the nitrogen atoms in blue. The
two a-helices involved in the formation of the third cluster of CHCs are enclosed in a circle. A magnified view of these helices shows the
residues that form the CHCs represented both as sticks and as transparent spheres. L85 and L276 are indicated by arrows.
R. Florio et al. Role of hydrophobic contacts in serine hydroxymethyltransferase
FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS 133
patterns of apo- and holo-forms are indeed almost
superimposable, with a single symmetrical peak char-
acterized by a sedimentation coefficient, S
20,w
, of 5.5S
(Fig. 2), which is the value expected for a hydrated
eSHMT dimer endowed with an approximately spheri-
cal shape. The sedimentation equilibrium experiments
showed that, in the 2.5–25 lm subunit concentration
range, the wild-type eSHMT is a dimer, either in the
presence or absence of cofactor. In the same concen-
tration range, the L85A and L276A mutant holoen-
zymes are also dimers, with sedimentation coefficients
of 5.5S. Interestingly, the frictional ratio (f ⁄ f
0
, the ratio
between the experimentally calculated friction coeffi-
cient and the minimum friction coefficient of an anhy-
drous sphere) of dimeric wild-type, L85A and L276A

eSHMT holoenzymes is close to that of a spherical
protein, namely 1.2–1.3, suggesting that the single
mutations did not result in significant changes in the
shape of the dimeric protein.
Depletion of the cofactor affected the quaternary
structure of both single mutants, which showed an
extra sedimentation peak, at approximately 2.7S in the
case of L85A and at 3.1S with the L276A mutant
(Fig. 2). The smaller sedimentation coefficient corre-
sponds to that of monomeric eSHMT. Therefore, the
single mutant apoenzymes exist as an equilibrium mix-
ture of dimers and monomers, which interconvert
slowly with respect to the period of elapsed time in the
sedimentation velocity experiments. A comparison of
the dissociation constants of the single mutant apo-
forms, obtained by a fitting of the sedimentation equi-
librium curves to a monomer-dimer model, indicates
that the destabilizing effect of PLP depletion is greater
in L276A (K
d
= 2.7 · 10
)6
m
)1
) than in L85A
(K
d
= 4.0 · 10
)9
m

)1
). When both mutations are pres-
ent, as in the L85A ⁄ L276A double mutant, the apoen-
zyme exists as a monomer in the range of
concentrations tested (2.5–25 lm). The frictional ratio
of this monomeric species was calculated to be approx-
imately 1.2, suggesting that the dissociation determined
by the double mutation was not accompanied by large
structural changes.
Compared to that observed with the single L85A
and L276A mutants, cofactor binding to the
L85A ⁄ L276A double mutant apoenzyme did not shift
the equilibrium completely in favor of the dimer. In
the double mutant holoenzyme, obtained by adding
PLP to 98% of saturation (as calculated from the dis-
sociation constant of the related cofactor binding equi-
librium; see below), a residual 35% fraction of
monomer is in equilibrium with the dimer (Table 1
and Fig. 2). A dissociation constant of 1.7 · 10
)6
m
)1
was calculated for this equilibrium. Because it is
known that PLP bound to eSHMT through a Schiff
base linkage to the active site lysine residue absorbs
light maximally at 420 nm [9], a sedimentation velocity
experiment was performed on a double mutant holoen-
zyme sample (33 lm), measuring absorbance at this
wavelength. The presence of a 3.1S peak in the sedi-
mentation pattern indicated that the cofactor was

covalently bound to the monomeric form of the
enzyme (Fig. 2). The lower percentage of monomer
present in this sedimentation profile (12% instead of
35%; Fig. 2 and Table 1) is accounted for by the
higher concentration of enzyme employed in the exper-
iment, and as calculated by using the equation describ-
Table 1. Sedimentation and dissociation constants calculated from
ultracentrifuge experiments on apo- and holo-forms of wild-type
and mutant eSHMTs. Values are shown of the S
20,w
sedimentation
coefficient calculated in sedimentation velocity experiments on
enzyme samples at 2.5 l
M subunit concentration, in 50 mM
NaHepes buffer (pH 7.2), containing 200 lM dithiothreitol and
100 l
M EDTA, at 20 °C. Percentages in parenthesis correspond to
the fraction of enzyme subunits that sediment with the related
coefficient and were calculated from an integration of the sedimen-
tation profiles shown in Fig. 2. The dissociation constants of dimer–
monomer equilibria (K
d
) were determined from sedimentation
equilibrium experiments carried out on enzyme samples in the
2.5–25 l
M subunit concentration range.
S
20, w
(S) K
d

(M
)1
)
a
Holoenzyme forms
WT 5.5 ND
L85A 5.5 ND
L276A 5.5 ND
L85A ⁄ L276A 5.5 (66%) 3.3 (34%)
5.5 (88%)
b
3.1 (12%)
b
1.7 · 10
)6
Apoenzyme forms
WT 5.5 ND
L85A 5.5 (90%)
c
2.7 (8%)
c
4.0 · 10
)9
L276A 5.5 (65%) 3.1 (35%) 2.7 · 10
)6
L85A ⁄ L276A 3.1 ND
a
Dissociation constants could not be calculated for wild-type and
single mutant holoenzymes and for the double mutant apoenzyme
because these were completely either in the dimeric or monomeric

state in the range of protein concentration used (ND, not deter-
mined). However, the detection limit of the instrumentation
employed, which may be estimated to be approximately 1% (per-
centage of detectable monomer in a dimeric sample or vice versa),
restricts the K
d
for the dimeric holo-forms to values £ 8 · 10
)9
M
)1
and the K
d
for the monomeric double mutant apoenzyme to values
‡ 5 · 10
)4
M
)1
(calculated on the basis of Eqn (1), assuming that
1% of undetected dimer or monomer were present in the sedimen-
tation velocity experiments carried out at a subunit concentration of
2.5 l
M).
b
Calculated on data collected at 420 nm with an enzyme
sample at a subunit concentration of 33 l
M. Data of all other exper-
iments were collected at 277 nm.
c
In the sedimentation velocity
experiments on the L85A apoenzyme, approximately 2% of sub-

units sedimented very slowly in the form of aggregates.
Role of hydrophobic contacts in serine hydroxymethyltransferase R. Florio et al.
134 FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS
ing the dissociation equilibrium (Eqn 1). A complete
shift of the equilibrium in favor of the dimeric form
was obtained when l-serine (1 mm) was added to the
double mutant holoenzyme (Fig. 2).
PLP binding properties
The affinity of wild-type and mutant forms for the
cofactor was measured to evaluate the impact of the
mutations on the structure of the PLP binding site.
Because PLP binding to apo-eSHMT is known to
quench the intrinsic fluorescence emission of the
enzyme [10], without changing the wavelength of maxi-
mum emission, the dissociation constant of the binding
equilibrium was calculated from saturation curves
obtained by measuring the fluorescence emission of
apoenzyme samples (26 nm subunit concentration) at
increasing PLP concentrations (Fig. 3). Table 2 shows
that the apparent K
d
values calculated from least
square fitting of experimental data points to Eqn (2)
are essentially the same for all enzyme forms. The cal-
culated relative fluorescence intensities in the absence
of PLP (F
0
) or in the presence of saturating concentra-
tions of cofactor (F
inf

) were: F
0
= 125 ± 1 and
F
inf
=65 ± 1 for the wild-type enzyme; F
0
= 139 ± 1
and F
inf
= 66 ± 1 for L85A; F
0
= 122 ± 1 and
F
inf
= 63 ± 1 for L276; and F
0
= 103 ± 1 and
F
inf
= 73 ± 1 for L85A ⁄ L276A. The higher fluores-
cence with respect to wild-type observed with the L85A
apoenzyme may be explained by the presence of a small
percentage of subunits present as aggregates (Table 1).
A remarkable difference is noted with respect to the
intrinsic fluorescence emission intensities of apo- and
holo-forms of the L85A ⁄ L276A double mutant: the
relative fluorescence emission of the double mutant
apoenzyme is considerably lower than that of the other
apo-forms and PLP binding does not quench fluores-

cence to the same extent it does with the other holo-
forms, although the wavelength of maximum emission
is the same for all enzyme forms (Fig. 3, insets).
In the light of the results obtained from the ultra-
centrifuge experiments, it should be noted that, at a
concentration of 26 nm, the association state of subun-
its is expected to vary among wild-type and mutant
enzymes. Indeed, it may be calculated, using the disso-
ciation constants showed in Table 1 and according to
Eqn (1), that, at this concentration, the apoenzyme
subunits of wild-type eSHMT are mostly in the
dimeric state (for a fraction ‡ 90%), the apo-L85A is
approximately 75% dimeric, whereas the apo-L276A
and the apo-L85A ⁄ L85A mutants are in the mono-
meric state. The association state of the holoenzymes
can be estimated to be ‡ 90% dimeric for all enzyme
forms, except for the L85A ⁄ L276A double mutant,
Fig. 2. Sedimentation velocity distributions obtained with apo- and holo-forms of wild-type and mutant eSHMTs. Sedimentation velocity
measurements were performed at 116 480 g on 2.5 l
M (subunit concentration) holoenzyme (—) and apoenzyme (- - -) samples kept at
20 °Cin50m
M NaHepes buffer (pH 7.2), containing 200 lM dithiothreitol and 100 lM EDTA. L-serine (1 mM) was added to a sample of the
L85A ⁄ L276A double mutant holoenzyme (ÆÆÆÆÆ). Absorbance data were all collected at 277 nm, except in the case of a sedimentation experi-
ment carried out on the double mutant holoenzyme at a subunit concentration of 33 l
M, when the absorbance of protein-bound cofactor
was measured at 420 nm (Æ-Æ-Æ).
R. Florio et al. Role of hydrophobic contacts in serine hydroxymethyltransferase
FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS 135
which is expected to be mostly in the monomeric state.
Therefore, the similar values of dissociation constant

obtained with wild-type and mutant enzymes suggest
that the cofactor binds to monomeric and dimeric
forms with similar affinities.
Catalytic properties
SHMT catalyses the reversible transfer of the C
b
of
l-serine to tetrahydropteroylglutamate (H
4
PteGlu),
with formation of glycine and 5,10-methylene-H
4
Pte-
Glu. However, in the absence of H
4
PteGlu, it also
accelerates the cleavage of several different l-3-hydr-
oxyamino acids to glycine and the corresponding
aldehyde [11]. Both erithro and threo forms of l-3-
phenylserine are rapidly cleaved to glycine and benzal-
dehyde [12,13]. The serine hydroxymethyltransferase
and l-threo-phenylserine aldolase activities of all
eSHMT forms were assayed using enzyme samples (at
0.05 and 3 lm subunit concentrations, respectively)
saturated with PLP. The calculated kinetic parameters
of both reactions are shown in Table 2. The L85A
mutation had virtually no effect on the catalytic prop-
erties of the enzyme. Minor differences with respect to
Table 2. Dissociation constants of PLP binding equilibrium and kinetic parameters determined with wild-type and mutant eSHMTs. Para-
meters are expressed as the mean ± SD determined by nonlinear least squares fitting of data to the related equation (see Experimental

procedures).
K
d
PLP
a
(nM)
aK
m
b
Ser
(m
M)
aK
m
b
H
4
PteGlu
(l
M)
k
cat
SHMT
c
(min
)1
)
K
m
/-Ser

d
(mM)
k
cat
/-Ser
d
(min
)1
)
Wild-type 5.11 ± 1.14 0.14 ± 0.01 7.03 ± 0.88 686.6 ± 21.7 36.3 ± 1.4 202.1 ± 4.2
L85A 5.88 ± 0.73 0.15 ± 0.01 7.16 ± 1.57 647.4 ± 36.3 37.4 ± 2.9 257.8 ± 13.7
L276A 5.01 ± 0.87 0.20 ± 0.01 4.35 ± 0.52 400.5 ± 10.8 42.6 ± 1.8 132.3 ± 3.2
L85A ⁄ L276A 6.50 ± 1.69 0.20 ± 0.01 11.20 ± 1.34 400.0 ± 15.3 42.1 ± 4.5 173.9 ± 10.7
a
Dissociation constant of PLP binding equilibrium.
b
Apparent K
m
of either L-serine or H
4
PteGlu in serine hydroxymethyltransferase reaction
when the other substrate is at saturating concentration.
c
Catalitic constant of the serine hydroxymethyltransferase reaction.
d
Kinetic param-
eters of the
L-threo-3-phenylserine cleavage reaction.
Fig. 3. Comparison of PLP-binding saturation curves obtained with wild-type and mutant enzymes. Apoenzyme samples (26 nM) were mixed
with different concentrations of PLP (1–400 n

M)in50mM NaHepes (pH 7.2), containing 200 lM dithiothreitol and 100 lM EDTA, at 20 °C.
Fluorescence emission spectra were measured in a 1-cm quartz cuvette with excitation at 280 nm. The graphs report the relative fluores-
cence intensity at 336 nm (F
r
) as a function of the total PLP concentration (i.e. the concentration of free and enzyme-bound PLP). The contin-
uous lines are those calculated from the least square fitting of experimental data to Eqn (2). Insets show comparisons between the intrinsic
fluorescence emission spectra of holoenzyme (—) and apoenzyme (- - -) forms of wild-type (thick lines) and mutant (thin lines) eSHMT.
Role of hydrophobic contacts in serine hydroxymethyltransferase R. Florio et al.
136 FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS
wild-type were observed with the L276A and the
L85A ⁄ L276A mutants, which yielded similar kinetic
parameters overall: with both reactions tested, k
cat
values were decreased by up to two-thirds and the
K
m
for amino acid substrates were slightly increased.
Given the dimerization effect observed in the ultracen-
trifuge experiments upon addition of l-serine to the
double mutant holoenzyme, it may be assumed that
the slightly different kinetic parameters of L276A and
L85A ⁄ L276A mutants do not depend on the oligomer-
ization states of the enzymes. It may be inferred that
the minor changes of catalytic properties observed with
the double mutant enzyme are determined by the
L276A mutation.
Far and near-UV circular dichroism spectra
The far-UV CD spectra of wild-type and mutant apo-
and holoenzymes at a subunit concentration of 2.5 lm
were virtually identical (data not shown), indicating

that the mutations did not alter the secondary structure
of the enzyme. The tertiary structure of all apo- and
holo-forms was analyzed measuring and comparing
their near-UV CD spectra at a subunit concentration
of 35 lm. A substantial similarity was observed among
the aromatic CD spectra of wild-type, L85A and
L276A apo-eSHMTs, with respect to fine structure and
representative bands (Fig. 4). It may be estimated that
the L276A mutant at this concentration is approxi-
mately 80% dimeric, whereas the other apo-forms are
completely dimeric. In the case of the L85A ⁄ L276A
double mutant apoenzyme, the fine structure of the
spectrum is markedly less resolved than it is with all
other apo-forms, despite the similarity in overall ellipt-
icty (Fig. 4). The loss of fine structure may be
accounted for by the fact that, at a concentration of
35 lm, the enzyme exists mostly as a monomer (for a
fraction ‡ 90%). Because PLP binds to the monomeric
and dimeric apoenzyme with similar affinities, it may
be deduced that the structure of the monomer is analo-
gous to that of the subunits in the dimeric enzyme.
The protein-bound cofactor contributes to the CD
spectra of all the holoenzymes with a broad positive
band centered at 420 nm and a negative band at
approximately 340 nm, which are similar in all enzyme
forms (data not shown). The presence of the cofactor
negative band determines a significant increase of the
overall negative ellipticity below 320 nm (Fig. 4). PLP
binding to the wild-type apoenzyme also determines an
increase of the aromatic 285 nm band contribution. A

very similar spectral change is observed with the single
mutants that, at a concentration of 35 lm and in the
holo-form, are fully dimeric. PLP binding to the dou-
ble mutant apoenzyme definitely improves the resolu-
Fig. 4. Comparison of the near-UV CD spec-
tra of apo- and holo-forms of mutant (contin-
uous lines) and wild-type (dashed lines)
eSHMTs. Enzymes samples (35 l
M) were
dissolved in 50 m
M NaHepes (pH 7.2), con-
taining 200 l
M dithiothreitol and 100 lM
EDTA, at 20 °C. CD spectra were measured
using a 1 cm quartz cuvette.
R. Florio et al. Role of hydrophobic contacts in serine hydroxymethyltransferase
FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS 137
tion of the fine structure of the CD spectrum. How-
ever, the CD spectrum of the double mutant
holoenzyme (85% dimeric at a concentration of
35 lm) shows lower negative ellipticity in the aromatic
region and a less pronounced 285 nm band (Fig. 4),
indicating that the formation of the dimer induced by
PLP binding is not accompanied by a complete recov-
ery of the native tertiary structure.
Discussion
The decrease of hydrophobic contact area determined
by the mutations in the third cluster of CHCs caused a
shift of the equilibrium between dimeric and monomeric
forms of eSHMT in favor of the latter. The extent to

which this happened was maximum for the
L85A ⁄ L276A double mutation and minimum for the
L85A mutation, whereas the L276A had an intermedi-
ate effect. The alteration of the dimer–monomer equi-
librium determined by the single L85A and L276A
mutations was not accompanied by any significant
change of tertiary structure, as determined by analysis
of the fluorescence and near-UV CD properties. On the
other hand, the L85A ⁄ L276A double mutation had visi-
ble effects on both the intrinsic fluorescence and near-
UV CD properties of the enzyme. Both L276A and
L85A ⁄ L276A apoenzymes are in the monomeric state
at the concentration used in the intrinsic fluorescence
measurements. However, the fluorescence emission of
the double mutant apoenzyme is much less intense than
that of the L276A apoenzyme, for which the emission
spectrum, in turn, is very similar to that of the wild-type
apoenzyme (Fig. 3). This indicates that the presence of
both mutations slightly perturbed the native structure
of the monomer. When PLP binds to the monomeric
double mutant, the intrinsic fluorescence is quenched to
a lesser extent than with the other enzyme forms. This
difference may be attributed to the fact that, upon PLP
binding, the double mutant holoenzyme stays in the
monomeric state, whereas all the other forms become
dimeric, as revealed by analytical ultracentrifugation.
Nevertheless, at a higher enzyme concentration, when
the subunits of the double mutant associate into a
dimer, the aromatic CD spectrum of the double mutant
indicates that minor structural changes with respect to

the wild-type enzyme are present.
None of the introduced mutations had large conse-
quences on the catalytic properties of the enzyme. In
this respect, it should be noted that substrate binding
to the double mutant eSHMT was observed to stabi-
lize the dimeric, catalytically competent form of the
enzyme (Fig. 2). Therefore, it is expected that all
enzyme forms were dimeric in the conditions used to
assay their catalytic activity. It may be deduced that
the structural alterations determined by the mutations
had a modest impact on the overall tertiary structure
of the enzyme and were largely confined to the stabil-
ity of the native quaternary assembly. Initially, the
observed effects of the mutations on the quaternary
structure may appear to be rather surprising because
the residues replaced by site-directed mutagenesis are
far away from the subunit interface. Nevertheless, in
SHMT, an important interaction between the subunits
is established between the a-helices of the third cluster
of CHCs and the N-terminal a-helix of the adjacent
subunit (Fig. 5). This observation may be sufficient to
explain the increase of the dissociation constant of the
monomer–dimer equilibrium determined by the muta-
tions. The results obtained in the present study also
show that the monomeric form of the enzyme is able
to bind PLP and that this binding event counteracts
the effect of the mutations, stabilizing the dimeric
form. The stabilizing effect is even more pronounced if
l-serine binds to the cofactor at the active site, as is
evident in the case of the double mutant enzyme. This

is monomeric in the apo-form, exists as an equilibrium
mixture of monomers and dimers even when all active
sites are occupied by PLP and is fully converted into a
dimer by the addition of l-serine. Evidently, the muta-
tions caused a slight and indirect alteration of crucial
interactions at the subunit interface. The stabilizing
effect of PLP and l-serine on the dimeric assembly
suggests that these alterations involve regions at the
subunit interface that are contacted by cofactor and
substrates, when these are bound to the active site of
the adjacent subunit. Scrutiny of the eSHMT crystal
structure [14] reveals that two polypeptide loops, at
the N-terminal ends of the a-helices that form the third
cluster of CHCs, are likely to be the relevant structural
regions (Fig. 5). One is the polypeptide section made
of residues 55¢–67¢ (where the primes indicate that the
residues are contributed by the other subunit). In
eSHMT, Y55¢ interacts with the phosphate moiety of
PLP; E57¢ is crucial in the binding of the l-serine
hydroxyl group [15] and in the catalysis of the hydrox-
ymethyltransferase reaction [16]; and Y65¢ interacts
with the carboxylate group of substrates and plays a
key role in substrate binding [17]. An alignment among
63 amino acid sequences of the enzyme from several
different sources showed that all three residues are
invariant in SHMT [18]. The second polypeptide loop,
comprising residues 258¢–264¢, is a very conserved
region which interacts with the phosphate moiety of
PLP. It is delimited by two invariant proline residues,
P258¢ (which is in a cis configuration in all five known

structures from mouse [19], human [20], rabbit [21],
Role of hydrophobic contacts in serine hydroxymethyltransferase R. Florio et al.
138 FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS
E. coli [14] and Bacillus stearothermophilus [15]) and
P264¢. The importance of this structural region is testi-
fied by site-directed mutagenesis studies on P258¢ and
P264¢, which showed how the native conformation of
the loop is pivotal to PLP binding and catalysis [22].
Given the above considerations, a series of possible,
correlated outcomes of the mutations may be envis-
aged. The decrease of the hydrophobic contact area in
the third cluster of CHCs is expected to alter the asso-
ciation of the a-helices that form the cluster and,
consequently, to weaken their interaction with the
N-terminal a-helix. This can be imagined to be the
main cause of subunit dissociation in apo-eSHMT. At
the same time, the mutations may indirectly alter the
structure of the 55¢–67¢ and 258¢–264¢ loops. In holo-
eSHMT, this alteration may compromise the interac-
tions between the cofactor bound at the active site and
the loops of the adjoining subunit, promoting subunit
dissociation. In the holoenzyme, effects on both the
N-terminal helix and on the 55¢–67¢ and 258¢–264¢
loops would be present. On the other hand, PLP bind-
ing to the apoenzyme is expected to stabilize the struc-
ture of the loops and, indirectly, the association
between the helices of the cluster. This, as a conse-
quence, would reinforce the interaction among the
helices and the N-terminal helix of the other subunit,
promoting dimerization. At this point, the observation

that the monomeric eSHMT binds PLP (Fig. 2) and
that all mutant forms show similar affinity for the
cofactor (Table 2), although they exist as different
mixtures of monomers and dimers at equilibrium, is
rather puzzling. One possible explanation is that the
affinity of monomeric eSHMT for PLP is lower than
that of the dimer, although not drastically different.
This difference may not be detectable in the experi-
ments performed in the present study.
The high degree of sequence and structural conserva-
tion of the third cluster of CHCs, observed for the
majority of fold type I enzymes, suggests that its stabi-
lizing function, hypothesized for eSHMT, could be
extended to the whole superfamily. The extent to which
the third hydrophobic cluster is involved in the stabil-
ization of the dimer assembly might be different
amongst fold type I enzymes, depending on the size
and orientation of the N-terminal arm. However,
although the 55¢–67¢ loop of eSHMT is a highly diversi-
fied region in fold type I enzymes, it invariably contains
residues involved in PLP and substrate binding and
catalysis [8,23]. Moreover, the second polypeptide loop
(residues 258¢–264¢ in eSHMT) represents a very con-
served region in this group of enzymes, interacting with
the phosphate moiety of PLP. Therefore, the capability
of the third cluster of CHCs to confer stability to the
polypeptide loops of the active site is presumably
important in all members of the fold type I family.
An additional clue on the structural importance of the
third cluster of CHCs in fold type I enzymes comes from

folding studies on eSHMT. The folding mechanism of
eSHMT may be divided into two phases [10,22,24,25].
In the first, relatively rapid phase, the two domains fold
into a native-like intermediate that has virtually all of
the native secondary and tertiary structure, but is unable
Fig. 5. Schematic representation of the
dimeric structure of eSHMT. Enzyme subun-
its of the eSHMT•Gly•5-formyl H
4
PteGlu
ternary complex (Protein Data Bank: 1dfo)
[14] are shown in salmon and cyan,
whereas the PLP-gly complex is shown as
in Fig. 1. The a-helices involved in the for-
mation of the third cluster of CHCs belong
to the subunit shown in salmon. The first
a-helix and the 55¢–67¢ loop are colored in
red; the second a-helix and the 258¢–264¢
loop are shown in magenta. The N-terminal
helix of the cyan subunit is shown in blue.
Residues mentioned in the text are labeled.
R. Florio et al. Role of hydrophobic contacts in serine hydroxymethyltransferase
FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS 139
to bind PLP. In this intermediate, the N-terminus and
an inter-domain segment remain exposed to solvent. In
the following, slow phase, these structural elements
assume their native conformation, completing the active
site that acquires the capability to bind PLP. Notice-
ably, one helix of the third cluster of CHCs is part of the
inter-domain segment and the other helix lines the

boundary between the major domain and the N-termi-
nus (Fig. 1). Therefore, the formation of the third
cluster may represent a key event of the final phase
of folding, determining the assembly of eSHMT active
site and promoting dimerization.
Experimental procedures
Materials
Ingredients for bacterial growth were obtained from Sigma-
Aldrich (St Louis, MO, USA). Chemicals for the purifica-
tion of the enzymes were obtained from BDH ⁄ Merck
(Whitehouse Station, NJ, USA); DEAE-Sepharose and
Phenyl-Sepharose were obtained from GE Healthcare (Mil-
waukee, WI, USA). Wild-type and mutant forms of
eSHMT and methylenetetrahydrofolate dehydrogenase were
purified as previously described [26,27]. PLP was added to
protein samples during the purification procedure, but it
was left out in the final dialysis step. (6S)H
4
PteGlu, was a
gift from Eprova AG (Schaffhausen, Switzerland). PLP was
obtained from Sigma-Aldrich (98% pure). All other
reagents were obtained from Sigma-Aldrich.
Preparation of apoenzyme and holoenzyme
samples
Apo-eSHMT was prepared using l-cysteine as previously
described [10]. The apoenzyme, for which the subunit con-
centration was calculated according to a molar absorptivity
value of e
280
= 42790 cm

)1
Æm
)1
[28], was stored in 10%
glycerol at )20 °C for no more than 3 days before use. A
small, residual fraction (less than 5%) of holoenzyme, esti-
mated with activity assays, was present in the apoenzyme
samples. We noticed that different batches of purified
eSHMT samples contained variable holoenzyme ⁄ apo-
enzyme ratios. This observation was made with either
wild-type or mutant forms of the enzyme. To carry out
comparable experiments with the holo-forms, it was man-
datory to prepare enzyme samples containing the same
fraction of protein-bound PLP (possibly close to saturation)
and, at the same time, devoid of any excess of cofactor.
Holoenzymes were prepared from apoenzyme samples, by
addition of PLP at the concentration needed to obtain a
98% saturation, calculated on the basis of the related disso-
ciation constant of PLP binding equilibrium (Table 2),
using Eqn (2A). The subunit concentration of the holo-
enzyme was calculated according to a molar absorptivity
value of e
280
= 44884 cm
)1
Æm
)1
[28].
Site-directed mutagenesis
Site-directed mutagenesis of E. coli glyA (SHMT encoding

gene) coding region was performed with the Quick-
ChangeÔ kit from Stratagene (La Jolla, CA, USA), using
the pBS::glyA plasmid as template [26] and two comple-
mentary oligonucleotide primers containing the mutations,
which were synthesized by MWG-Biotech AG (Anzinger,
Germany). The L85A and L276A mutants were produced
using as primers 5¢-CGTGCAAAGAA
GCGTTCGGCGC-
3¢ and 5¢-GCGGTTGCT
GCGAAAGAAGCG-3¢, respec-
tively, and their complementary oligonucleotides (the
mutated bases are underlined). The L85 ⁄ L276A double
mutant was obtained introducing the L276A mutation into
a template pS::glyA plasmid that already contained the
L85A mutation. E. coli DH5a cells were used to amplify
the mutated plasmids . Both strands of the coding region of
the mutated genes were sequenced. The only differences
with respect to wild-type nucleotide sequence were those
that were intended. Enzyme expression was performed
using the GS1993 recA) strain of E. coli [26].
Analytical ultracentrifugation analyses
Sedimentation velocity and equilibrium experiments were
carried out at 20 °Cin50mm NaHepes buffer (pH 7.2),
containing 200 lm dithiothreitol, 100 lm EDTA, on a
Beckman XL-I analytical ultracentrifuge equipped with
absorbance optics and an An60-Ti rotor (Beckman Coul-
ter, Fullerton, CA, USA). In the sedimentation velocity
experiments, performed at 116 480 g the protein concentra-
tion was 2.5 lm for both apo- and holo-forms. Data were
collected at 277 nm and at a spacing of 0.003 cm with

three averages in a continuous scan mode. In the case of
the double mutant L85A ⁄ L276A holoenzyme the sedimen-
tation velocity experiments were also performed at
420 nm, aiming to evaluate the presence of PLP covalently
bound to the monomeric species as a Schiff base with the
active site lysine residue [9]. Sedimentation coefficients and
integration of data were obtained using the software sed-
fit (provided by P. Schuck, National Institutes of Health,
Bethesda, MD, USA). The values were reduced to water
(S
20,w
) using standard procedures. The buffer density and
viscosity were calculated by the software Sednterp. The
ratio f ⁄ f
0
was calculated from the diffusion coefficient,
which, in turn, is related to the spreading of the bound-
ary, using the software sedfit. Sedimentation equilibrium
experiments were performed at 7280, 10 483 and 14 270 g
on enzyme samples at subunit concentrations of 2.5 and
25 lm. Data were collected every 3 h at a spacing of
Role of hydrophobic contacts in serine hydroxymethyltransferase R. Florio et al.
140 FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS
0.001 cm with ten averages in a step scan mode. Equilib-
rium was checked by comparing scans for up to 24 h
using the software winmatch (J. Lary, National Analyti-
cal Ultracentrifugation Center, Storrs, CT, USA). Data
sets were edited by reedit (J. Lary, National Analytical
Ultracentrifugation Center, Storrs, CT, USA) and ana-
lyzed using the software sedphat (National Institutes of

Health). Data from different speeds were combined for
global fitting. When a monomer–dimer association model
was required for data fitting, the monomer molecular mass
was fixed at the value determined from the amino acid
sequence (45 320 Da).
Measurement of the K
d
of PLP binding
equilibrium
PLP binding equilibria were analyzed taking advantage of
the protein intrinsic fluorescence quenching observed upon
the binding event [10]. Dissociation constants of binding
equilibria were then calculated from saturation curves
obtained measuring the protein fluorescence emission inten-
sity as a function of increasing PLP concentration. The
cofactor (range 1–400 nm) was added to apoenzyme sam-
ples (26 nm)at20°Cin50mm NaHepes (pH 7.2), contain-
ing 200 lm dithithreitol and 100 lm EDTA. Preliminary
experiments demonstrated that, with all enzyme forms, the
binding equilibrium was established within the mixing time.
Fluorescence emission spectra (300–450 nm; 7 nm emission
slit) were recorded immediately after mixing PLP and apo-
enzyme with a LS50B spectrofluorimeter (Perkin-Elmer,
Waltham, MA, USA), with excitation wavelength set at
280 nm (5 nm excitation slit), at the same temperature and
with a 1 cm path length quartz cell. Data were analyzed
according to Eqn (2).
Activity assays
All assays were carried out at 20 °Cin50mm NaHepes
(pH 7.2), containing 200 lm dithiothreitol and 100 lm

EDTA. The serine hydroxymethyltransferase activity was
measured with 0.05 lm enzyme samples with l-serine and
H
4
PteGlu as substrates, as previously described [9]. To
determine the K
m
for l-serine, H
4
PteGlu was maintained at
0.23 mm and the l-serine concentration was varied in the
range 0.06–6 mm. For K
m
determinations of H
4
PteGlu,
l-serine concentrations were held constant at 30 mm and
H
4
PteGlu concentrations varied in the range 3–500 lm. The
rate of l-threo-phenylserine cleavage (3 lm enzyme samples)
was obtained from spectroscopic measurement of benzalde-
hyde production at 279 nm, employing a molar absorptivity
value of e
279
= 1400 cm
)1
Æm
)1
[12,13]. The K

m
for l-threo-
phenylserine was determined by varying the substrate
concentration in the range 4–200 mm.
Spectroscopic measurements
Fluorescence emission measurements were carried out at
20 °C with a LS50B spectrofluorimeter (Perkin-Elmer)
using a 1 cm path length quartz cuvette. Fluorescence emis-
sion spectra were recorded from 300–450 nm (1 nm sam-
pling interval), with the excitation wavelength set at
280 nm. Far- (190–250 nm), near-UV (250–310 nm) and
visible (310–500 nm) CD spectra were measured using 0.2
and 1 cm path length quartz cuvettes and the results
obtained were expressed as the mean residue ellipticity (Q),
assuming a mean residue molecular mass of 110 per amino
acid residue. UV–visible spectra were recorded with a dou-
ble-beam Lambda 16 (Perkin-Elmer). Kinetic measurements
in the activity assays were performed on a HP 8453 diode-
array spectrophotometer (Hewlett-Packard, Palo Alto, CA,
USA). All spectroscopic measurements were carried out at
20 °Cin50mm NaHepes (pH 7.2), containing 200 lm dith-
iothreitol and 100 lm EDTA.
Data analysis
Kinetic parameters were determined by nonlinear least
squares fitting of initial velocity data to the Michaelis–
Menten equation using the software prism (GraphPad Soft-
ware Inc., San Diego, CA, USA). The concentrations of
monomeric and dimeric species at equilibrium were calcu-
lated from Eqn (1) [29], in which [D
eq

] and [E
t
] are the
equilibrium concentrations of the dimer and the total con-
centration of the enzyme, respectively, expressed as dimer
equivalents, and K
d
is the dissociation constant of the
related equilibrium.
D
eq
ÂÃ
¼
8 Â E
t
½þK
d
À
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
16 Â E
t
½ÂK
d
þ K
2
d
p
8
ð1Þ
Fluorescence data obtained in PLP binding equilibrium

experiments were analyzed according to Eqn (2), in which
F
rel
is the measured relative fluorescence, F
0
is fluorescence
in the absence of PLP, F
inf
is fluorescence at infinite PLP
concentration, [E] is the total enzyme subunit concentration,
[PLP] is the total cofactor concentration and K
d
is the disso-
ciation constant of the equilibrium HOLO ¢ APO þ PLP:
F
rel
¼ F
0
À F
0
À F
inf
ðÞÂ
PLP½þE½þK
d
À
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
À K
d
þ PLP½þE½ðÞ

2
þ4 Â PLP½þK
d
ðÞÀ4 Â E½ÂPLP½
q
2
E½
ð2Þ
R. Florio et al. Role of hydrophobic contacts in serine hydroxymethyltransferase
FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS 141
The fraction present in Eqn (1) corresponds to the
fraction of subunits that binds PLP at equilibrium
( HOLO
eq
ÂÃ
= E½
). The concentration of holoenzyme at equi-
librium ([HOLO
eq
]) was derived from the equation for the
dissociation constant of the binding equilibrium:
K
d
¼
E½ÀHOLO
eq
ÂÃÀÁ
 PLP½ÀHOLO
eq
ÂÃÀÁ

HOLO
eq
ÂÃ
; ð2aÞ
as one of the two solutions of the quadratic equation:
HOLO
eq
ÂÃ
2
À K
d
þ PLP½þE½ðÞÂHOLO
eq
ÂÃ
þ E½ÂPLP½¼0:
ð2bÞ
Acknowledgements
We thank Professor Verne Schirch for helpful discus-
sions. We are grateful to Eprova AG (Schaffhausen,
Switzerland) for providing pure (6S)H
4
PteGlu. This
work was supported by grants from the Italian Minis-
tero dell’Universita
`
e della Ricerca. Rita Florio is the
recipient of a fellowship from the Facolta
`
di Scienze
Matematiche, Fisiche e Naturali of ‘Sapienza’ Univer-

sita
`
di Roma, Italy.
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