McGuire2 1 Department of Biological Chemistry and2Center for Enzyme Research, Institute of Molecular Biology, University of Copenhagen, Denmark The amino acid sequence of 5-phospho-a-D-r
Trang 1Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase
Bjarne Hove-Jensen1and James N McGuire2
1
Department of Biological Chemistry and2Center 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
caldolyticusis 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 60–65C
and a half-life of 26 min at 65C, compared to values of
46C and 60 s at 65 C, respectively, for the B subtilis
enzyme Chemical cross-linking shows that both enzymes
are hexamers Vmax is determined as 440 lmolÆmin)1Æmg
protein)1and Kmvalues for ATP and ribose 5-phosphate
are determined as 310 and 530 lM, respectively, for the
B caldolyticusenzyme The enzyme requires 50 mMPias
well as free Mg2+ for maximal activity Manganese ion
substitutes for Mg2+, but only at 30% of the activity obtained with Mg2+ ADP and GDP inhibit the B caldo-lyticusenzyme in a cooperative fashion with Hill coefficients
of 2.9 for ADP and 2.6 for GDP Kivalues are determined as
113 and 490 lMfor ADP and GDP, respectively At low concentrations ADP inhibition is linearly competitive with respect to ATP A predicted structure of the B caldolyticus enzyme based on homology modelling with the structure 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 intermediate [3]
PRibPP is the substrate 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 species like Bacillus subtilis and Escherichia coli
contain 10 enzymes, which utilize PRibPP as a substrate [5]
The synthesis 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 reaction proceeds by attack of the b-phosphate by O-1 of Rib5P [7,8] PRibPP synthase from
E coli [9,10], Salmonella enterica serovar Typhimurium [11,12] and B subtilis [13] requires two Mg2+per subunit and a Pi concentration of 50 mM S enterica and E coli PRibPP synthases bind ATP (as MgÆATP) before Rib5P The E coli enzyme furthermore binds free Mg2+ before binding MgÆATP in the catalytic cycle [14] Regulation of the activity of PRibPP synthase is achieved primarily through the inhibition by ADP or GDP It has been shown that ADP inhibits the enzyme by binding to the allosteric site in competition with Pias well as by competing with ATP for the active site [9,15,16] GDP also inhibits PRibPP synthases from Gram-negative bacteria and mammals, but to a lesser extent and by binding at the allosteric site [13,17] PRibPP synthase is active as a homomultimer with oligomerization states ranging from hexamer to higher states of aggregation depending on the detection method and the source of organism [18] In the present work we describe the charac-terization of PRibPP synthase, which is encoded by the prs gene, from the thermophile Bacillus caldolyticus and compare it with the enzyme from the mesophile B subtilis
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-mail: hove@mermaid.molbio.ku.dk
Abbreviations: PRibPP, 5-phospho-a- D -ribosyl 1-diphosphate;
Rib5P, ribose 5-phosphate.
Enzyme: 5-phospho-a- D -ribosyl 1-diphosphate synthase or ATP:
D -ribose-5-phosphate pyrophosphotransferase (EC 2.7.6.1).
Note: A department website is available at http://www.molbio.ku.dk
Note: Dedicated to the memory of the late Professor Agnete
Munch-Petersen, a fine colleague and a great mentor.
(Received 4 August 2004, revised 17 September 2004,
accepted 4 October 2004)
Trang 2heim, 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˚rhus, Denmark) or Hobolth DNA
Syntese (Hillerød, Denmark) FPLC was performed using a
Bio-Rad Bio Logic system with UV detection at 280 nm
Polyethyleneimine-cellulose coated TLC sheets were from
Baker-flex (J T Baker, Phillipsburg, NJ, USA)
Cloning and expression of theB caldolyticus prs gene
The prs gene was synthesized by PCR with pHO219 DNA
[19] as the template, the oligodeoxyribonucleotides 5¢-AA
GAAAGAATTC-TAGCGGAGGTCTATCATG-3¢ and
5¢-ATGTTTAAGCTTA-TTAGTCGAACAGGACGCT-3¢
as primers and DNA polymerase from Pyrococcus furiosus
in the presence of the four deoxyribonucleoside
triphos-phates The nucleotides preceding hyphens indicate
non-complementary extensions and recognition sites for the
restriction endonuclease EcoRI and HindIII are underlined
Standard procedures were used for thermocycling in a
Trio-Thermoblock (Biometra, Go¨ttingen, Germany) The PCR
product was digested by EcoRI and HindIII, and ligated to
EcoRI and HindIII digested DNA of the expression vector
pUHE23-2 [H Bujard, University of Heidelberg, Germany,
personal communication] The nucleotide sequence of the
insert of the resulting plasmid, pJM1, was determined in an
Abi Prism Genetic Analyser (model 310) with the Bigdye
Terminator Cycle Sequencing Ready Reaction Kit as
recommended by the supplier (PE Applied Biosystems,
Foster City, CA, USA)
Purification of recombinantP RibPP synthase
ofB caldolyticus and B subtilis
The plasmid pJM1 was transformed into the
PRibPP-less E coli strain HO1986 (Dprs-4::KanR araCam araD
D(lac)U169 trpammalamrpsL relA thi deoD gsk-3 udp supF
FR/F lacIq zzf::Tn10), which contains no endogenous
PRibPP synthase activity HO1986 is a derivative of strain
HO1088 [20] and was kindly provided by B N Krath (this
institute) It is resistant to an unspecified nonlambdoid,
non-P type bacteriophage Cultures of strain HO1986/pJM1 were
grown at 37C to an attenuance at 436 nm of 1.2–1.5
( 3 · 1011 cellsÆL)1), measured in an Eppendorf 6121
spectrophotometer At this time isopropyl thio-b-D
-galacto-side was added to a final concentration of 50 lM, and
incubation continued for 16 h Unless otherwise stated the
following steps were performed at 4C Cells were harvested
by centrifugation at 20 000 g for 20 min Collected cells were
resuspended in five volumes of 50 mMpotassium phosphate
buffer (pH 7.5), and sonicated for 20 min (60 s bursts with
60 s pauses) followed by centrifugation at 20 000 g for
15 min The supernatant fluid was 40% saturated with ammonium sulphate The precipitate was removed by centrifugation, and the supernatant fluid was 60% saturated with ammonium sulphate The precipitate, collected by centrifugation, was redissolved in 50 mMpotassium phos-phate buffer (pH 7.5) in half the original volume and dialyzed for 16 h against 2 L of 50 mMpotassium 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 mMpotassium phosphate buffer (pH 8.2) Protein was eluted by using a linear gradient over six column volumes from 50 mMpotassium phosphate buffer (pH 8.2) to 50 mM potassium phosphate, 300 mMpotassium chloride (pH 8.2) PRibPP synthase activity eluted as two major peaks, which were pooled, dialyzed against 50 mMpotassium 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 mMpotassium 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 PRibPP synthase activity eluted as three or four peaks The largest was chosen for further study The final enzyme fraction was greater than 95% pure as determined by SDS/ PAGE and staining in Coomassie Brilliant Blue The enzyme was stored in 50% glycerol in aliquots at)80 C
Recombinant B subtilis PRibPP synthase was isolated from cells overexpressing the prs gene essentially as described previously [21] with a modification of the final anion exchange step as follows The enzyme, dissolved in
50 mMpotassium phosphate buffer (pH 7.5) was applied to
a 20 mL anion exchange Hiload Q-Sepharose column (Pharmacia), previously equilibrated with the same buffer PRibPP synthase was eluted by applying a salt gradient of 0% Salt Buffer [50 mM potassium phosphate buffer (pH 7.5)] to 100% Salt Buffer [1M sodium chloride in
50 mMpotassium phosphate buffer (pH 7.5)] at a rate of
2 mLÆmin)1over 60 min The gradient was an initial linear increase from 0 to 20% Salt Buffer, followed by a hold for
40 mL and an increase to 35% Salt Buffer over approxi-mately 120 mL and finally a raise to 100% Salt Buffer PRibPP synthase eluted at a sodium chloride concentration
of approximately 0.30M The fractions with highest purity evaluated by assay of PRibPP synthase activity and by SDS/PAGE were pooled and dialyzed against 50 mM 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 catalysed by PRibPP.
Trang 3USA Amino acid sequencing by automated Edman
degradation was performed by the Department of Protein
Chemistry, Institute of Molecular Biology, University of
Copenhagen, Denmark
Assay ofP RibPP synthase activity
The standard reaction buffer consisted of 50 mMTris/HCl,
50 mM potassium phosphate, 2.0 mM EGTA (pH 8.5,
adjusted at 65C) The standard reaction contained
2.0 mM (10 GBqÆmol)1) [32P]ATP[cP] (prepared as
des-cribed previously [24]), 5.0 mMRib5P, 5.0 mMmagnesium
chloride Unless otherwise indicated the Mg2+
concentra-tion was 3.0 mM in excess of the ribonucleoside
triphos-phate concentration In analyses of inhibition by ADP and
in determination of Km for ATP and Rib5P, a buffer
without EGTA was used For the Pior sulphate dependence
analysis, the enzyme was diluted in 50 mMTris/HCl buffer
(pH 8.2) containing BSA (2 gÆL)1) without prior dialysis
The reaction buffer for these studies was 50 mMTris/HCl
(pH 8.5, adjusted at 65C) In all cases, the assay buffer
with ATP, Rib5P and magnesium chloride present was
prewarmed for 2 min at the desired temperature and
reaction initiated by the addition of enzyme The enzyme
had been previously diluted in 50 mMpotassium phosphate
buffer (pH 8.5, adjusted at 20C) containing BSA
(2 mgÆmL)1) and prewarmed for 2 min at 20C Reaction
was performed for 3 min at three different enzyme dilutions
The reaction was terminated by mixing the sample (10 lL)
with 0.33Mformic acid (5 lL) and applying the 15 lL to a
polyethyleneimine-cellulose coated TLC sheet The
chro-matogram was developed in 0.85Mpotassium phosphate,
which had been previously titrated to pH 3.4 with 0.85M
phosphoric acid The radioactive content in individual spots
was determined in a Packard Instant Imager (model 2024)
B subtilis PRibPP synthase activity was assayed by
the same procedure Enzyme activity is expressed as
lmolÆmin)1Æmg protein)1
Kinetic analysis
Results of initial velocity determinations, which were
averages of at least three determinations, were fitted to the
following equations using the programULTRAFIT(version
3.0.5, Biosoft, Cambridge, UK) Equation 1 is the
Micha-elis–Menten equation for hyperbolic substrate saturation
kinetics, whereas Eqn 2 is the rate equation for a sequential
mechanism For competitive and noncompetitive inhibition
the initial velocities were fitted to Eqn 3 and 4, respectively
[25] Equation 5 was used to estimate the Hill coefficient in
inhibition studies
v¼ VappS
KATP½Rib5PþKRib5P½ATPþKiATPKRib5Pþ½ATP½Rib5P
ð2Þ
Km 1þ IK
is
Km 1þ IK
is
þ S 1 þ IK
ii
v¼ Vmax
1þ IK i
where v is the initial velocity, Vappis the apparent maximal velocity, Kmis the apparent Michaelis–Menten constant for the varied substrate S, Vmaxis the maximal velocity, KATP and KRib5Pare the Michaelis–Menten constants for ATP and Rib5P, respectively KiATPis the dissociation constant for ATP, Kisand Kiiare inhibitor constants for the inhibitor
I obtained from the effect on slopes and intercept, respect-ively, Kiis 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 mM in 20 mM potassium phosphate buffer (pH 8.3) with a protein concentration range of 91–910 lgÆmL)1 (equivalent to 3–30 lMPRibPP synthase subunit) The reaction (10 lL) was incubated at room temperature for 30 min followed
by quenching with an equal volume of 100 mMTris/HCl (pH 8.5) Samples were analysed by SDS/PAGE (10% acrylamide)
Molecular modelling Molecular modelling was based on the coordinates of the crystal form of B subtilis PRibPP synthase with sulphate present [26] An unresolved loop, RPKPNVAEVM(199– 208), was added to this structure usingHOMOLOGYsoftware (Biosym/Msi, San Diego, CA, USA) and minimized using the manufacturer’s suggested settings The resulting struc-ture was used as a template to build a model of B caldo-lyticus PRibPP synthase by using the programHOMOLOGY The residues that deviated from the B subtilis sequence were minimized to remove any gross errors The whole structure was subjected to repeated rounds of minimization and molecular dynamics using the DISCOVER module (Biosym/Msi) again using the manufacturer’s suggested settings The final root-mean-square deviation between the two backbones was 0.005 Analysis of the structure with PROSTATin HOMOLOGYand VERIFY3-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 is flexible anyway, so small errors were of little consequence Graphics were made by using the programINSIGHT(Biosym/Msi)
Results
Purification and characterization
B caldolyticus PRibPP synthase was purified to homo-geneity by ammonium sulphate precipitation, triazyl dye
Trang 4chromatography 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 from the deduced amino acid
sequence N-terminal sequencing revealed 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 that
the initial methionine has been removed Comparison of the
nucleotide sequences of the insert of pJM1 and the original
insert of pHO219 (GenBank and EBI Data Bank accession
number X83708) revealed three discrepancies Lys289 and
Arg294 were found to be glutamic acid and alanine,
respectively The codon for Val292 was found to be GUG
and not GUC as published originally [19]
Temperature and pH dependency
Temperature dependency of the enzymatic activity of
B caldolyticus PRibPP synthase was determined in the
range 40–75C using the standard reaction buffer A bell
shaped profile was obtained with maximal activity at 60C
(data not shown) In all of the experiments reported here,
the reactions were initiated with enzyme that had been
prewarmed at room temperature Initiating the reaction
with Rib5P gave an optimum at 60–65C This suggests
that the presence of the substrate ATP prior to initiating
the reaction may stabilize the enzyme The optimal
temperature appeared to vary between 60 and 65C
among enzyme preparations For comparison the
tem-perature dependency of the enzymatic activity of B subtilis
PRibPP synthase was determined as well and revealed an
optimal temperature of 46C The stability of the two
enzymes at 65C was determined A dramatic difference
was observed The half-life of the B caldolyticus enzyme
was 26 min, whereas that of the B subtilis enzyme was 60 s
(data not shown)
The optimal pH of B caldolyticus PRibPP synthase was
8.25–8.75 when the activity was assayed at 65C The
activity dropped to 80% of maximal at pH 9.5 and to only
about 25% at pH 6.5 compared to the activity at pH 8.50
At least in part this reduction in enzyme activity at higher
pH may be caused by the formation of magnesium–
phosphate complexes, and, thus, cause a depletion of
Mg2+ An identical pH optimum was obtained with
B subtilis PRibPP synthase when activity was assayed at
37C
Piand metal ion requirements
In the absence of added Pi, which corresponds to a minimal
Piconcentration of 12.5 lMin the assay, the enzyme was
weakly active (4.8% of maximum) As the Piconcentration
was raised, the enzyme gained activity and reached a
maximum at 50 mM, whereas it was slowly reduced to 58%
at 120 mM and 17% at 200 mM The enzyme could use
sulphate ion in place of Pi but only at about 30% of
maximal activity at a concentration of 0.50M At 50 mM,
the optimal concentration for Pi, sulphate was hardly
activating (5% of maximal activity), whereas 1Msulphate
was strongly inhibitory (5% of maximal activity) The
enzyme clearly preferred Mg2+as the metal ion, but could
use Mn2+, Zn2+, Cd2+ or Cu2+ The activity in the
presence of Mn2+was about 30% of the activity determined
in the presence of Mg2+, while the activity in the presence
of Zn2+, Cd2+or Cu2+was only 5–10% of the activity determined in the presence of Mg2+ It is likely that two
Mg2+were bound per subunit, one in complex with ATP and one bound at the active site, because activity increased
as the Mg2+ concentration was raised above the ribo-nucleoside triphosphate concentration No activity was observed in the presence of Ca2+, Fe2+, Co2+or Ni2+ Kinetic analysis
It was necessary to use an excess of Mg2+over ATP, similar
to what has been observed for other PRibPP synthases Even under these conditions ATP exerted substrate inhibi-tion at concentrainhibi-tions above 1 mM However, results of initial velocity vs the concentration of ATP or Rib5P were found to follow Michaelis–Menten kinetics at ATP con-centrations below 0.8 mM In double reciprocal plots of the data, intersecting lines indicated that the reaction followed a sequential mechanism (Fig 1) The data were fitted to Eqn 2 and the following values were obtained: KATP
310 ± 110 lM, KRib5P 530 ± 140 lM and Vmax
440 ± 69 lmolÆmin)1Æmg 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 mM, had little or no
Fig 1 Reaction mechanism of PRibPP synthase and determination of kinetic constants Activity was determined as described in Experimental procedures The magnesium chloride concentration was 3.0 m M over the ATP concentration 1/v is expressed as lmol)1ÆminÆmg 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, 0.1 m M ; n, 0.2 m M ; h, 0.4 m M ; ·, 0.6 m M ; or s, 0.8 m M Lines represent fitting of the data to Eqn 2.
Trang 5effect on the enzyme activity, as activity varied from 92
to 109% of the activity obtained in the absence of these
nucleotides The activity in the presence of 5.0 mM UTP
was only 20% of that in the absence of UTP, indicating
significant inhibition Only ADP and GDP showed
significant inhibition at physiologically relevant
concen-trations, less than 1% residual activity in the presence of
1 mM ADP or 5 mM GDP As expected from these
results, GDP was a less efficient inhibitor (Fig 2)
Inhibition by ADP as well as by GDP was strongly
cooperative, with Hill coefficients for ADP and GDP
determined as 2.9 ± 0.1 and 2.6 ± 0.1, respectively The
apparent Ki values determined under these assay
condi-tions (3.0 mM ATP) were 113 ± 1 lM for ADP and
490 ± 9 lM for GDP
Inhibition with ADP at various ATP concentrations was
analysed In the inhibitor concentration range employed
here, 0.06–0.18 mM, ADP was a linear competitive inhibitor
of ATP saturation (Fig 3) Analysis of the data with respect
to noncompetitive inhibition (Eqn 4) failed to give a
satisfying fit
Quaternary structure
Chemical cross-linking of PRibPP synthase followed by
SDS/PAGE revealed two major bands of Mr220 000 and
100 000 (Fig 4) The monomer behaved as a 36 000 Mr
polypeptide This result indicates the formation of
hexa-mers and trihexa-mers In addition some higher order oligohexa-mers
were seen Interestingly, no or very little dimer was
observed Higher order oligomers of B caldolyticus
PRibPP synthase were consistently seen by gel filtration,
and they possessed significant activity 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 r 208 000; II, M r 115 000; III, M r
79 500; IV, M r 49 500; V, M r 34 800 Lane 2 contains untreated enzyme (0.9 lg applied in gel) Lanes 3–6 contain cross-linked enzyme The amount of protein loaded in each lane of the gel: lane 3, 4.5 lg applied in gel; lane 4, 2.3 lg; lane 5, 1.1 lg; lane 6, 0.5 lg.
Fig 3 Inhibition of B caldolyticus PRibPP synthase activity by ADP Activity was determined as described in Experimental procedures The magnesium chloride concentration exceeded total nucleotide concen-tration by 3.0 m M 1/v is expressed as lmol)1ÆminÆmg protein Double reciprocal plots of initial velocity vs ATP at six concentrations of ADP are shown The concentration of ATP was varied from 0.05 to 0.80 m M in the presence of different concentrations of ADP: ,, 0 m M ;
s, 0.06 m M ; h, 0.09 m M ; n, 0.12 m M ; e, 0.15 m M , or · , 0.18 m M Lines represent fitting of the data to Eqn 3.
Fig 2 Inhibition by ADP and GDP of B caldolyticus PRibPP
syn-thase activity Activity was determined as described in Experimental
procedures with ATP and Rib5P concentrations of 3.0 and 5.0 m M ,
respectively, and Mg 2+ exceeding the total ribonucleotide
concentra-tion by 3.0 m M The specific activity of the enzyme was 400 lmolÆ
min)1Æmg 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 Eqn 5 h, ADP; s, GDP.
Trang 6chemical cross-linking products with Mr of 220 000 and
100 000 were obtained with B subtilis PRibPP synthase
as well
Model structure
An alignment of B caldolyticus and B subtilis PRibPP
synthases is shown in Fig 5 The amino acid sequences of
the two polypeptides are 81% identical The crystal
structure of B subtilis PRibPP synthase has been solved
with two ADP molecules per monomer, one bound at the
active site and one bound in an allosteric cleft The
structure has also been solved with sulphate bound in the
allosteric cleft and in place of the phosphate group of
Rib5P in the active site [26] A model based on the
sulphate structure was constructed using a
homology-based method (Fig 6) All of the amino acids of the
active sites as well as those of the monomer–monomer
contact surfaces were identical in the two proteins The
only exceptions were Leu70 and Lys199, which are
isoleucine and arginine, respectively, in the B subtilis
enzyme In addition, all of the amino acids involved in
allosteric regulation by ADP were conserved [28]
Inter-estingly, of the 59 altered amino acid residues, 54
(i.e 92%) were solvent exposed in the hexameric
struc-ture The five buried residues of B caldolyticus PRibPP
synthase were as follows, with the corresponding amino
acid of B subtilis PRibPP synthase given in parenthesis:
Ile43 (Val), Val56 (Cys), Leu70 (Ile), Asn108 (Glu) and
Val115 (Phe) Consistent with the surface location of the
altered amino acids were hydrophobicity surface maps of
monomers from the two Bacillus PRibPP synthases
These revealed an 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 same basic structure as its mesophilic
relative and that both enzymes function by the same
mechanism In particular all of the residues identified as
important in catalysis and allosteric regulation as well as in monomer–monomer contact of the B subtilis PRibPP synthase were retained in the B caldolyticus enzyme with the two exceptions of conservative replacements mentioned above [22,26,28–30] Thus, the mechanism of catalysis and regulation appears to be similar for the two enzymes The two enzymes differed primarily in their thermal properties The origin of this difference is at present unknown In general, the number of individual amino acids varied little among the two enzymes Exceptions were asparagine, alanine, glycine and methionine Analysis of the number of 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 compared 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 caldolyticusenzyme Thus, the Asn + Gln content may
Fig 5 Alignment of B caldolyticus and B subtilis PRibPP synthase amino acid sequences Bc, B caldolyticus; Bs, B subtilis b-Sheets are shown as yellow letters, a-helices as blue letters Residues that are different among the two sequences, are shown as red letters in the B caldolyticus sequence.
Fig 6 Model structure of hexameric B caldolyticus PRibPP synthase One dimer is shown with grey shading, a second dimer with green and purple shading and a third dimer with blue and yellow shading Red atoms indicate amino acids that differ among B caldolyticus and
B subtilis PRibPP synthases (detailed in Fig 5).
Trang 7be of significance for the enhanced thermostability of
B caldolyticus PRibPP synthase, similar to what has been
shown for certain enzymes from hyperthermophilic
organ-isms [31] Furthermore, the B caldolyticus enzyme
con-tained 33 alanines compared to 28 in the B subtilis enzyme
as well as one additional change to alanine The amino acids
of the B subtilis enzyme at positions corresponding to these
six alanines were serine, glutamate, valine, lysine and two
glycines It is possible therefore that these alanines contribute
compactness to the thermophilic enzyme The glycine
content of the B caldolyticus enzyme was three less than
that of the B subtilis enzyme In the former enzyme the
corresponding amino acids were cysteine, alanine and serine
Therefore, it is possible that the thermophilic enzyme is more
rigid in structure than the mesophilic enzyme Finally, the
B caldolyticusenzyme contains four more methionines than
the B subtilis enzyme, corresponding to proline, valine,
isoleucine and glutamine in the latter enzyme The
signifi-cance of this difference, if any, remains unknown It is
possible that subtle changes along the primary structure
together contribute to the increased thermostability [32]
Altogether the modelling of B caldolyticus PRibPP
syn-thase indicated that the altered amino acids were primarily
located on the surface of the hexameric protein
Apart from the thermal properties, the two enzymes also
differ widely in their regulation We determined Kivalues
for ADP and GDP, in the presence of 3.0 mMATP and
5.0 mM Rib5P, as 113 and 490 lM, respectively, for the
B caldolyticusenzyme In comparison, the concentration of
ADP and GDP resulting in 50% inhibition, and determined
at identical substrate concentrations as before, were greater
than 1 mM and greater than 5 mM, 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 the two enzymes is given in
Table 1
Acknowledgements
We are grateful to M Willemoe¨s for discussions and for carefully
reading the manuscript, to B N Krath for providing strain HO1986
and for assistance with analysis of kinetic data We wish to thank T D.
Hansen for excellent technical assistance Financial support was
obtained from the Danish Natural Science Research Council.
References
1 Hove-Jensen, B (1988) Mutation in the
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Source of PRibPP
synthase
No of amino acids
Optimal K m
V max (lmolÆ min)1Æmg protein)1) KADP
i (m M )
Predominant oligomer Temp (C) pH ATP (m M ) Rib5P (m M )
B caldolyticus 314 60–65 8.5 0.31 0.53 440 0.113 Hexamer
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