Surface exposed amino acid differences between mesophilic and
thermophilic phosphoribosyl diphosphate synthase
Bjarne Hove-Jensen
1
and James N. McGuire
2
1
Department of Biological Chemistry and
2
Center for Enzyme Research, Institute of Molecular Biology, University of Copenhagen,
Denmark
The amino acid sequence of 5-phospho-a-
D
-ribosyl
1-diphosphate synthase from the thermophile Bacillus
caldolyticus is 81% identical to the amino acid sequence of
5-phospho-a-
D
-ribosyl 1-diphosphate synthase from the
mesophile Bacillus subtilis. Nevertheless the enzyme from the
two organisms possesses very different thermal properties.
The B. caldolyticus enzyme has optimal activity at 60–65 °C
and a half-life of 26 min at 65 °C, compared to values of
46 °Cand60sat65°C, respectively, for the B. subtilis
enzyme. Chemical cross-linking shows that both enzymes
are hexamers. V
max
is determined as 440 lmolÆmin
)1
Æmg
protein

enzyme based on homology m odelling with the structu re of
B. subtilis 5-phospho-a-
D
-ribosyl 1-diphosphate synthase
shows 92% of the amino acid differences to be on solvent
exposed surfaces in the hexameric structure.
Keywords: kinetics; mesophile; nucleotide metabolism;
PRPP; thermophile.
The compound 5-phospho-a-
D
-ribosyl 1-diphosphate
(PRibPP) is a central intermediate in the de novo and salvage
biosynthesis of pyrimidine, purine and pyridine nucleotides
as well as in the biosynthesis of the amino acids histidine
and tryptophan [1,2]. In addition, methanopterin, a folate
analogue involved in C1 metabolism of methanogenic
archaea, is synthesized with PRibPP as an inte rmediate [3].
PRib PP is the s ubstrate for a number of phosphoribosyl-
transferases which catalyse the phosphoribosylation of a
variety of nucleobases to the corresponding ribonucleoside
monophosphates, i.e. the formation of N-glycosidic bonds.
In methanopterin biosynthesis, a carbon–carbon bond is
formed to C1 of the phosphoribosyl moiety of PRibPP [3,4].
Bacterial s pecies like Bacillus subtilis and Escherichia coli
contain 10 enzymes, which utilize PRibPP as a substrate [5].
The s ynthesis of PRibPP is catalysed by PRibPP synthase,
which transfers the b,c-diphosphoryl group of ATP to ribose
5-phosphate (Rib5P) to produce PRibPP and 5¢-AMP [6,7]
(Scheme 1 ). The r eaction proceeds by a ttack of the b-
phosphate by O-1 of Rib5P [7,8]. PRibPP synthase from

Experimental procedures
Materials
Ribonucleotides were obtained from Pharmacia (Uppsala,
Sweden), Sigma (St. Louis, MO, USA) or Roche (Mann-
Correspondence to B. Hove-Jensen, Department of Biological Chem-
istry, Institute of Molecular Biology, University of Copenhagen, 83H
Sølvgade, DK-1307 Copenhagen K, Denmark. Fax: +45 3532 2040,
Tel.: +45 3532 2027, E- m ail: hov
Abbreviations: PRibPP, 5-phospho-a-
D
-ribosyl 1-diphosphate;
Rib5P, ribose 5-phosphate.
Enzyme: 5 -phospho-a-
D
-ribosyl 1-di phosphate synthase or A TP:
D
-ribose-5-phosphate p yrophosphotransferase ( EC 2. 7.6.1).
Note: A department website i s available at
Note: Dedicated to the memory of the late Professor Agnete M unch-
Petersen, a fine colleagu e and a great mentor.
(Received 4 August 2004, rev ised 17 September 2004,
accepted 4 October 2004)
Eur. J. Biochem. 271, 4526–4533 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04412.x
heim, Germany). Antibiotics, isopropyl thio-b-
D
-galactoside
and EGTA were obtained from Sigma. Restriction endo-
nucleases were obtained from Promega (Madison, WI,
USA). Oligodeoxyribonucleotides were purchased from
DNA Technology (A

Terminator Cycle S equencing Ready Reaction Kit as
recommended by the supplier (PE Applied Biosystems,
Foster City, CA, USA).
Purification of recombinant
P
Rib
PP
synthase
of
B. caldolyticus
and
B. subtilis
The plasmid pJM1 was t ransformed i nto the PRibPP-
less E. coli strain HO1986 (Dprs-4::Kan
R
araC
am
araD
D(lac)U169 trp
am
mal
am
rpsL relA thi deoD gsk-3 udp supF
ÔFÕ
R
/F lacI
q
zzf::Tn10), which contains no endogenous
PRib PP synthase activity. HO1986 is a deriva tive of strain
HO1088 [20] and was kindly p rovided b y B . N . K rath (t his

dialyzed for 16 h against 2 L of 50 m
M
potassium phosphate
buffer (pH 8.2). The dialysed enzyme preparation was
applied to a Dyematrex Gel Green A column (Millipore,
Bedford, MA, USA), and washed with five volumes of
50 m
M
potassium phosphate buffer (pH 8.2). Protein was
eluted by u sing a linear gradient o ver s ix column volumes
from 50 m
M
potassium phosphate buffer (pH 8.2) to 50 m
M
potassium phosphate, 300 m
M
potassium chloride (pH 8.2).
PRib PP synthase activity eluted as two major peaks, which
were pooled, dialyzed against 50 m
M
potassium phosphate
buffer (pH 8.2), reapplied to the same column, and eluted
under the same conditions as before. The larger of two
activity peaks (fraction A) was further dialysed against
50 m
M
potassium phosphate buffer (pH 8.2) eluted isocrat-
ically through a Pharmacia Superose 12 10/30 gel filtration
column using an FPLC instrument at room temperature.
PRib PP synthase activity eluted as three or four peaks. The

of approximately 0.30
M
. The fractions with highest purity
evaluatedbyassayofPRibPP synthase activity and by
SDS/PAGE were pooled and dialyzed against 50 m
M
potassium phosphate buffer (pH 7.5). The enzyme was
stored refrigerated [22].
Protein content was determined by the bicinchoninic acid
procedure (Pierce Chemical Company, Rockford, IL, USA)
as described previously with BSA as the standard [23].
MALDI-TOF mass spectrometry analysis was performed
by the School of Chemical Sciences Mass Spectrometry
Center, University of Illinois, Urbana-Champaign, IL,
Scheme 1. Reaction catal y sed by PRib PP.
Ó FEBS 2004 Bacillus caldolyticus PRibPP synthase (Eur. J. Biochem. 271) 4527
USA. Amino acid sequencing by automated Edman
degradation was performed by the Department of Protein
Chemistry, Institute of Molecular Biology, University of
Copenhagen, Denmark.
Assay of
P
Rib
PP
synthase activity
The standard reaction buffer consisted o f 50 m
M
Tris/HCl,
50 m
M

M
Tris/HCl buffer
(pH 8 .2) containing BSA (2 gÆL
)1
) without prior dialysis.
The reaction bu ffer for these studies was 50 m
M
Tris/HCl
(pH 8 .5, adjusted at 65 °C). In all cases, the assay buffer
with ATP, Rib5P and magnesium chloride present was
prewarmed for 2 m in at the desired temperature and
reaction initiated by the addition of enzyme. The enzyme
had b een previously diluted in 50 m
M
potassium phosphate
buffer (pH 8.5, adjusted at 20 °C) containing BSA
(2 mg ÆmL
)1
) and prewarmed for 2 min at 20 °C. Reaction
was performed for 3 min at three different enzyme dilutions.
The reaction was terminated by mixing the s ample ( 10 lL)
with 0.33
M
formic acid (5 lL) and applying the 1 5 lLtoa
polyethyleneimine-cellulose coated TLC sheet. The chro-
matogram was developed i n 0.85
M
potassium phosphate,
which had been previously titrated to pH 3.4 with 0.85
M

v¼
V
max
½ATP½Rib5P
K
ATP
½Rib5Pþ K
Rib5P
½ATPþ K
iATP
K
Rib5P
þ½ATP½Rib5P
ð2Þ
v ¼
V
app
S
K
m
1 þ
I
K
is

þ S
ð3Þ
v ¼
V
app

the varied substrate S, V
max
is the maximal velocity, K
ATP
and K
Rib5P
are the Michaelis–Menten constants for ATP
and R ib5P, respectively. K
iATP
is the dissociation constant
for ATP, K
is
and K
ii
are inhibitor constants f or the inhibitor
I obtained from t he effect on slopes a nd intercept, respect-
ively, K
i
is the inhibitor constant for the substrate S, and n is
the Hill coefficient.
Chemical cross-linking
Cross-linking was performed with bis(sulphosuccinimidyl)
suberate (Pierce) at a concentration of 1.8 m
M
in 20 m
M
potassium phosphate buffer (pH 8.3) with a protein
concentration range of 91–910 lgÆmL
)1
(equivalent to

two backbones was 0.005. Analysis of the structure with
PROSTAT
in
HOMOLOGY
and
VERIFY
3-
D
[27] revealed only
two problem areas. The first was the loop RQDRKAR-
SRN(99–108), which had some non-ideal torsion angles, but
they arose from the analogous loop in the original structure.
The other problem was the constructed loop (amino acids
residues 197–206), which i s flexible anyway , so s mall errors
were of little consequence. Graphics were made by using the
program
INSIGHT
(Biosym/Msi).
Results
Purification and characterization
B. caldolyticus PRibPP synthase was purified to homo-
geneity by ammonium sulphate precipitation, triazyl dye
4528 B. Hove-Jensen and J. N. McGuire (Eur. J. Biochem. 271) Ó FEBS 2004
chromatography and gel filtration. An approximate subunit
mass was determined by MALDI-TOF mass spectrometry
as 34 496.8 Da and agreed within 1% deviation with the
value, 34 296 Da, calculated f rom the deduced amino a cid
sequence. N-terminal sequencing r evealed the sequence Ser-
Asp-Xaa-Gln-His-Gln-Leu-Lys-Leu-Phe, which is in agree-
ment with the deduced amino acid sequence and shows t hat

about 25% at pH 6.5 compared to the activity at pH 8.50.
At least in part this reduction in enzyme activ ity at higher
pH may be caused by the formation of magnesium–
phosphate complexes, and, thus, cause a depletion of
Mg
2+
. An i dentical pH optimum was obtained w ith
B. subtilis PRibPP synthase when activity was assayed at
37 °C.
P
i
and metal ion requirements
In the a bsence of added P
i
, which corresponds to a minimal
P
i
concentration of 12.5 l
M
intheassay,theenzymewas
weakly ac tiv e (4 .8% of maximum). As the P
i
concentration
was raised, the enzyme gained activity and re ached a
maximum at 50 m
M
, whereas it was slowly reduced to 58%
at 120 m
M
and 17% at 200 m

2+
was about 30% of the activity determined
inthepresenceofMg
2+
, while the activity in the presence
of Zn
2+
,Cd
2+
or Cu
2+
was only 5–10% of the activity
determined in the presence of Mg
2+
.Itislikelythattwo
Mg
2+
were bound per subunit, one in complex with ATP
and one bound at the active site, because activity increased
as the Mg
2+
concentration w as raised above the ribo-
nucleoside triphosphate concentration. No activity was
observed in the presence of Ca
2+
,Fe
2+
,Co
2+
or Ni

Æmg protein
)1
.
Assay of enzyme activity in the presence of a variety
of nucleotides showed that 5¢-AMP, GTP, 5¢-GMP and
CTP, each at a concentration of 5.0 m
M
, had little or no
Fig. 1. Reaction mechanism o f PRibPP synthase and determination o f
kinetic constants. Activity was determined as described in Experimental
procedures. The magnesium ch loride c oncentration was 3.0 m
M
over
the ATP concentratio n. 1/v is expressed as lmol
)1
ÆminÆmg protein.
Double reciprocal plots of initial velocity vs. Rib5P at five concen-
trations of ATP are shown. The concentration of Rib5P was varied
from 0.2 to 0.8 m
M
in the presence of different concentrations of ATP:
e,0.1m
M
; n,0.2m
M
; h,0.4m
M
; ·,0.6m
M
;ors,0.8m

490 ± 9 l
M
for GDP.
Inhibition with ADP at various ATP c oncentrations was
analysed. In the inhibitor concentration range employed
here, 0.06–0.18 m
M
, ADP was a linear competitive inhibitor
of ATP saturation (Fig. 3). Analysis of the data with respect
to noncompetitive inhibition (Eqn 4) failed t o give a
satisfying fit.
Quaternary structure
Chemical cross-linking of PRibPP synthase followed by
SDS/PAGE revealed two major bands of M
r
220 000 and
100 000 (Fig. 4). The monomer behaved as a 36 000 M
r
polypeptide. This result indicates the formation of hexa-
mers and trimers. In addition some higher order oligomers
were seen. I nterestingly, n o o r very little dimer was
observed. Higher order oligomers o f B. caldolyticus
PRib PP synthase were consistently seen by gel fi ltration,
and they possessed significant a ctivity but not as high as
the hexamer (data not shown). Identical results, i.e.
Fig. 4. The quaternary structure of PRibPP synthase. Cross-linking
was performed as described in Experimental procedures. Lanes 1 and 7
contain M
r
standards (Bio-Rad): I, M

; h,0.09m
M
; n,0.12m
M
; e,0.15m
M
,or· ,0.18m
M
.
Lines represent fitting o f the data to Eqn 3.
Fig. 2. Inhibition by ADP and GDP of B. caldo lyticus PRibPP syn-
thase activity. A ctivity was determined as described in E xperimental
procedures wit h ATP a nd Rib 5P c onc entrations o f 3.0 and 5.0 m
M
,
respectively, a nd Mg
2+
exceeding the total ribon ucleotid e co ncentra-
tion by 3.0 m
M
. The specific activity of the enzyme was 400 lmolÆ
min
)1
Æmg protein
)1
(determined at 65 °C). Ribonucleoside diphos-
phate varied from 0 to 5 m
M
. Curves represent fitting of the entire data
sets to Eq n 5 . h,ADP;s,GDP.

Val115 (Phe). Consistent with the surface lo cation of the
altered amino acids were hydrophobicity surface maps of
monomers from the two Bacillus PRibPP synthases.
These revealed a n increase in polar surface area in the
B. caldolyticus enzyme compared to that of B. subtilis
(data not shown).
Discussion
It is apparent that the thermophilic version of the Bacillus
enzyme possesses the sam e basic s tructure as its m esophilic
relative and that both enzymes function by the same
mechanism. In particular all of t he residues identified as
important in c atalysis a nd allosteric regulation as well as in
monomer–monomer contact of the B. subtilis PRibPP
synthase were retained in the B. caldolyt icus enzyme with
the t wo exceptions of conservative replacements mentioned
above [22,26,28–30]. T hus, the mechanism of catalysis and
regulation appe ars to b e s imilar for the two enzymes. The
two enzymes differed primarily in their thermal properties.
The origin of this d ifference is at present unknown. In
general, the number of individual amino a cids varied little
among the two enzymes. Exceptions were asparagine,
alanine, glycine a nd methionine. Analysis of the number o f
asparagine and glutamine residues revealed a bias against
these thermolabile amino acids. Both enzymes contained
10 glutamine residues. B. subtilis PRibPP synthase con-
tained 17 asparagines c ompared to 11 of the B. caldolyticus
enzyme. Curiously, however, four of these 17 asparagines of
the B. subtilis enzyme were replaced by glutamines in the
B. caldolyticus enzyme. T hus, the A sn + G ln content may
Fig. 5. Alignment of B. ca ldolyticus and B. subtilis PRibPP synthase amino acid sequences. Bc, B. caldolyticus; Bs, B. subtilis. b-Sheets are shown as

located o n t he surface of t he hexameric protein.
Apart f rom t he thermal properties, the two enzymes also
differ widely in their regulation. We determined K
i
values
for ADP and GDP, in the presence of 3.0 m
M
ATP and
5.0 m
M
Rib5P, as 113 and 490 l
M
, respectively, for the
B. caldolyticus enzyme. In comparison, the concentration of
ADP and GDP resulting in 50% inhibition, and determined
at identical s ubstrate c oncentrations as before, w ere g reater
than 1 m
M
and greater than 5 m
M
, respectively, for the
B. subtilis enzyme [12]. Similarly, UTP inhibited the
B. caldolyticus to a higher extent, 20% residual activity,
than the B. subtilis enzyme, 80% residual activity. Again,
determined under identical assay conditions, other kinetic
values differed by approximately two-fold or less. A
summary of the properties of t he two enzymes is given in
Table 1.
Acknowledgements
We are grateful to M. Willem oe

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Ó FEBS 2004 Bacillus caldolyticus PRibPP synthase (Eur. J. Biochem. 271) 4533