serine 948 and threonine 1042 are crucial residues for allosteric regulation of escherichia coli...

Article No. jmbi.1999.2561 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 286, 1217±1228

Serine 948 and Threonine 1042 are Crucial Residuesfor Allosteric Regulation of Escherichia coliCarbamoylphosphate Synthetase and IllustrateCoupling Effects of Activation and Inhibition Pathways

Sylviane Delannay1,2, Daniel Charlier3,4, Catherine Tricot5

5 1,5 1,5
Vincent Villeret , AndreÂ PieÂrard and Victor Stalon *
1Laboratoire de MicrobiologieUniversiteÂ Libre de Bruxelles1, Avenue E. GrysonB-1070 Brussels, Belgium2Haute Ecole Provinciale deCharleroi, Bd. Solvay, 31B-6000 Charleroi, Belgium3OnderzoekingsinstituutCOOVI, 1, E. GrysonlaanB-1070 Brussels, Belgium4Department of MicrobiologyFlanders InteruniversityInstitute for Biotechnology1, E. GrysonlaanB-1070 Brussels, Belgium5Institut de RecherchesMicrobiologiques Jean-MarieWiame, 1, Avenue E. GrysonB-1070 Brussels, Belgium

lyzes the complex synthesis

E-mail address of the [email protected]

Abbreviations used: CPSase, casynthetase, OTCase, ornithine carATCase, aspartate carbamoyltranscarbamoylphosphate; IMP, inosinars, arginine-sensitive; urs, uracil-pyruvate kinase/lactate dehydrog

0022-2836/99/091217±12 $30.00/0

Escherichia coli carbamoylphosphate synthetase (CPSase) is a key enzymein the pyrimidine nucleotides and arginine biosynthetic pathways. Theenzyme harbors a complex regulation, being activated by ornithine andinosine 50-monophosphate (IMP), and inhibited by UMP. CPSase mutantsobtained by in vivo mutagenesis and selected on the basis of particularphenotypes have been characterized kinetically. Two residues, serine 948and threonine 1042, appear crucial for allosteric regulation of CPSase.When threonine 1042 is replaced by an isoleucine residue, the enzymedisplays a greatly reduced activation by ornithine. The T1042I mutatedenzyme is still sensitive to UMP and IMP, although the effects of bothregulators are reduced. When serine 948 is replaced by phenylalanine,the enzyme becomes insensitive to UMP and IMP, but is still activatedby ornithine, although to a reduced extent. When correlating these obser-vations to the structural data recently reported, it becomes clear that bothmutations, which are located in spatially distinct regions correspondingrespectively to the ornithine and the UMP/IMP binding sites, havecoupled effects on the enzyme regulation. These results provide an illus-tration that coupling of regulatory pathways occurs within the allostericsubunit of E. coli CPSase.

In addition, other mutants have been characterized, which displayaltered af®nities for the different CPSase substrates and also slightlymodi®ed properties towards the allosteric effectors: P165S, P170L,A182V, P360L, S743N, T800F and G824D. Kinetic properties of thesemodi®ed enzymes are also presented here and correlated to the crystalstructure of E. coli CPSase and to the phenotype of the mutants.

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Keywords: CPSase; E. coli; in vivo mutagenesis; structure; allostery
*Corresponding author
Introduction

The single carbamoylphosphate synthetase(CPSase) of Escherichia coli [E.C. 6.3.5.5] (for areview, see Cunin et al., 1986; Meister, 1989) cata-

of carbamoylpho-

ding author:

rbamoylphosphatebamoyltransferase;ferase; CP,

e 50-monophosphate;sensitive; PK/LDH,enase.

sphate (CP) from bicarbonate, glutamine, and twomolecules of Mg-ATP, with the release of gluta-mate, phosphate and two Mg-ADP. The synthesisof CP is critical for two biosynthetic pathways,namely those of pyrimidine nucleotides and argi-nine residues. In the ®rst pathway, CP is coupledto aspartate via aspartate carbamoyltransferase(ATCase), initiating the biosynthesis of pyrimidinenucleotides such as UMP. In the second pathway,CP is coupled to ornithine via ornithine carbamoyl-transferase (OTCase), constituting the sixth step(starting from glutamate) in the arginine biosyn-thetic pathway.

E. coli CPSase is activated by ornithine and alsoby inosine 50-monophosphate (IMP, a precursor of

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and the UMP/IMP sites, have coupled effects on

1218 Allosteric Regulation of CPSase

purine nucleotides) and inhibited by UMP(Anderson & Meister, 1966a; PieÂrard, 1966; Trottaet al., 1971, 1974). In addition to the regulation ofactivity, CPSase production is also under strict con-trol. Transcription of the carAB operon, encodingthe two subunits of CPSase, is cumulativelyrepressed by the end-products of both pathways(arginine residues and pyrimidine nucleotides;PieÂrard & Wiame, 1964; PieÂrard et al., 1965). ThecarA gene is preceded by two promoters in tandem(Piette et al., 1984; Bouvier et al., 1984) controlledrespectively by arginine and the hexameric argi-nine repressor (Charlier et al., 1988, 1992; Wanget al., 1998) and by pyrimidine nucleotides(Glansdorff, 1996; Glansdorff et al., 1998).

The native E. coli CPSase is a heterodimer com-posed of a small subunit of 41,270 Da and a largesubunit of 117,710 Da. They are respectivelyencoded by the adjacent carA and carB genes, orga-nized in an operon polarized from carA to carB(Gigot et al., 1980; Crabeel et al., 1980). The smallsubunit catalyzes the hydrolysis of glutamine andis responsible for the transfer of NH3 to the largesubunit, where CP synthesis actually takes place.This latter subunit contains the binding sites forthe substrates bicarbonate, ammonia, two separatesites for Mg-ATP and a 18 kDa carboxyterminalregion which constitutes the regulatory domain(Rubio et al., 1991; Cervera et al., 1996).

The formation of CP has long since been pro-posed to be accomplished via a sequential mechan-ism (Anderson & Meister, 1966b) and appearscon®rmed by recent experimentations (Raushelet al., 1998; Rubio et al., 1998).

The high degree of sequence homology betweenthe amino and carboxy-terminal halves of the largesubunit (40 % amino acid sequence identitybetween regions 1-553 and 554-1073) implies asimilar folding of the two halves, and suggests theparticipation of both halves in a phosphorylationstep (Nyunoya & Lusty, 1983). The amino-terminalhalf (from residue 1 to 400) is primarily responsiblefor the phosphorylation of bicarbonate and for theformation of the reactive intermediate, carboxy-phosphate. The carboxy-terminal half (fromresidues 553 to 933) is responsible for the phos-phorylation of carbamate and for the ultimate for-mation of carbamoylphosphate (Post et al., 1990;Rubio, 1993). The large subunit can, therefore, beenvisioned as two halves designated as the carbox-yphosphate and carbamoylphosphate syntheticcomponents (Rubio et al., 1998). The crystal struc-ture of an allosterically activated form of CPSasehas recently been described (Thoden et al., 1997). Itindicates that each component contains fourdistinct domains labeled A, B, C, D. The ®rst threedomains in the two large subunit halves are verysimilar in terms of structure, but the fourth areentirely different. Earlier biochemical studies haveshown that the D domain of the carbamoylpho-sphate synthetic half (residues 937 to 1073) isresponsible for the binding and the allosteric regu-lation by effectors, IMP, UMP and ornithine. All

these ligands exert their effect essentially byaltering the enzyme af®nity for Mg-ATP.

Progress has been made in identifying and char-acterizing the E. coli CPSase, and in the molecularbasis of its allosteric regulation. Photoaf®nitylabeling techniques have shown that the inhibitorUMP binds in the carboxy-terminal domain of thelarge subunit, a domain of about 18 kDa (Rubioet al., 1991). Studies involving a variety of UMPand IMP analogs have shown that dialdehydeUMP can reduce the apparent inhibition inducedby UMP and activation by IMP, but has no effecton the extent of activation by ornithine. Theseresults suggest that UMP and IMP bind to thesame or overlapping allosteric sites of the enzyme,sites which are apparently distinct from the bind-ing site for ornithine (Boettcher & Meister, 1981;1982). Within this regulatory domain, conservedamino acid residues were mutated. Among thetargeted amino acid residues, lysine 992 is a resi-due which appears to be covalently attached toUMP (Cervera et al., 1996); the threonine 977 toalanine mutant is no longer regulated by UMP, itis yet still activated by ornithine (Czerwinski et al.,1995)

In order to gain additional insights into theorganization of the functional and regulatorydomains in CPSase, and since no X-ray structurewas available when this work was initiated, wehave chosen to study CPSase mutants selectedin vivo on the basis of particular phenotypes, e.g.mutants that grow in minimal medium unless themedium is supplemented with arginine (arginine-sensitive, ars) or, in other strains, by uracil (uracil-sensitive, urs), and, in one strain, by any of the two(ars and urs). This was done with the aim tofurther characterize the complex behaviour of theenzyme. We have analyzed in detail several carBmutants previously isolated, genetically mappedand partially characterized (Mergeay et al., 1974)and which display an altered allosteric behaviorwhen compared to that of the wild-type enzyme.The mutant carB alleles have been cloned andsequenced; whenever a mutant proved to bearmore than one substitution, the correspondingsingle substitution alleles were constructed inorder to precisely reveal the function of each of themutated residues. The corresponding mutantenzymes were puri®ed, studied for their catalyticand allosteric properties, and their response to thevarious effectors affecting CPSase activity ana-lyzed. The relationships between the enzymaticcharacteristics and the mutant phenotypes are dis-cussed and correlated to the recently reportedthree-dimensional structure of CPSase (Thodenet al., 1997).

This approach has allowed the identi®cation, inthe C-terminal part, of two residues crucial for theallosteric regulation of CPSase, S948 and T1042.Our results suggest that both mutations, S948F andT1042I, which are located in spatially distinct bind-ing sites corresponding to the ornithine-binding

decreasing the maximal velocity and increasing the

Allosteric Regulation of CPSase 1219

the enzyme regulation. This provides an illus-tration that coupling of regulation pathways occurswithin the allosteric subunit of E. coli CPSase. Inaddition, a more detailed view of the catalytic siteshas emerged. Finally, our results add to the struc-tural description of CPSase recently reported(Thoden et al., 1997).

Results

Phenotypes, cloning and sequencedetermination of carB mutants

We have analyzed six mutants: their phenotypeand their CPSase speci®c activities determined incultures grown in minimal medium and in the pre-sence of arginine, uracil, or both are presented inTable 1. Growth in the presence of uracil of four ofthese mutants was stimulated by addition of CO2

to the gas phase (Mergeay et al., 1974).The carB mutations, transferred to an expression

vector as described in Materials and Methods,were characterized by sequencing both strands ofthe entire carB gene.

Three mutants (carB678, carB801, carB1001)proved to bear a single base-pair substitution,whereas carB36, carB80 and carB803 harbored twosubstitutions in different regions of the large sub-unit (Table 1). For each of these, the correspond-ing single base-pair substitution mutants wereconstructed by subcloning carB fragments (seebelow).

Purification of CPSases

Wild-type CPSase and 11 mutant enzymeswere puri®ed using the procedure described byWeyens (1987) with modi®cations, including a®nal hydrophobic chromatography step andreplacing the heat step by precipitation in ethanolwhen the enzymes were unstable (P165S, P360L,T800F, G824D, T1042I and S743N � G824D; fordetails see Materials and Methods). Remarkably,the single substitution of glycine 824 by an aspar-tate residue induces a complete loss of activityfollowing a 15 minute incubation at 55 �C. Thewild-type CPSase and some of the mutants(S743N, and A182V � S948F) were puri®ed using

Table 1. Phenotype, amino acid substitutions and speci®cmutants

Strains Amino acid substitutions Phenotype

P4X Wild-typeCA 244-1 carB36 S 743 N and G 824 D arsCA 244-1 carB80 P 360 L and T 1042 I ursa

P678 carB678 T 800 F ars and urP4X carB801 P 170 L ursa

P4X carB803 A 182 V and S 948 F ursa

P4X carB1001 P 165 S ursa

a CO2 stimulation; ars, sensitive towards arginine, urs: sensitive to

the two protocols (with or without a thermodena-turation step). The kinetic parameters for CPSasespuri®ed using the two protocols were similar,indicating that heat treatment did not affect theCPSase properties. Due to its very low speci®cactivity and/or stability, the puri®cation of thecarB80 mutant enzyme could not be accom-plished.

Kinetics and allosteric properties of wild-typeand mutant CPSases

The assay used was based on coupling withATCase, given the lack of effect of the aspartateresidue on the activity of CPSase (in contrast toornithine, the substrate used in the coupling withOTCase, which stimulates CPSase activity), andthe possibility of using equimolar amounts of Mg2�

and ATP. Assays at equimolar amounts of Mg2�

and ATP allow a better observation of the in¯u-ence of the effectors on the activity (Anderson &Meister, 1966a; Anderson, 1977; Boettcher &Meister, 1982). In contrast, the widely used pyru-vate kinase/lactate dehydrogenase (PK/LDH)coupling system requires an excess of free Mg2�

(Raushel et al., 1978).Hyperbolic saturation curves were obtained with

glutamine and bicarbonate in 50 mM Hepes buffer(pH 7.5). The apparent Km values for glutamineand bicarbonate, determined at near to saturationfor Mg-ATP (40 mM Mg-ATP), are 0.12 mM and1.2 mM, respectively, in close agreement withvalues obtained by different previously publishedprocedures (Robin et al., 1989). The concentrationof Mg-ATP giving half of the maximal velocity[S]0.5 is 10.5 mM and the saturation curve forMg-ATP is sigmoidal under our experimentalconditions.

When tested at [S]0.2 of Mg-ATP (the concen-tration of Mg-ATP that gives one-®fth of the maxi-mal velocity), the [I]0.5 value (the concentration ofinhibitor giving half of the maximal inhibition)determined for the allosteric inhibitor UMP isequal to 5 mM, whereas stimulation by ornithineand IMP gives values for the activation constants([A]0.5) of 0.04 and 0.05 mM, respectively.

The allosteric inhibitor UMP acts mainly by

CPSase activities of the parental strain P4X and of carB

Minimal medium supplemented with

ÿ Arg Ura Arg � Ura

0.7 0.5 0.4 <0.10.2 n.g. 0.1 <0.10.37 0.35 n.g. 0.34

s 1.8 n.g. n.g. <0.10.67 0.3 n.g. <0.10.3 0.2 n.g. <0.10.4 0.3 n.g. <0.1

wards uracil. n.g., no growth.

Figure 1. (a) Mg-ATP saturation curves of wild-typeCPSase: (*) no effector, (&) � 1 mM ORN, (&) � 1 mM IMP, (~) � 1 mM ORN � 1 mM IMP, (*) � 0.2 mMUMP. (b) Mg-ATP saturation curves of mutant CPSaseS948F: (*) no effector, (&) � 15 mM ORN,(&) � 2.5 mM IMP, (~)� 15 mM ORN � 2.5 mM IMP,(*) � 0.2 mM UMP. (c) Mg-ATP saturation curves ofmutant CPSase T1042I: (*) no effector, (&) � 30 mMORN, (&) � 2.5 mM IMP, (~) � 30 mM ORN � 2.5 mMIMP, (*) � 1 mM UMP. Speci®c activity is de®ned asmM of carbamoylaspartate produced per hour per mgof puri®ed protein.


[S]0.5 value for Mg-ATP, whereas ornithine andIMP have essentially the reverse effect towardsMg-ATP (Figure 1(a)).

The kinetic parameters obtained for the mutantenzymes under conditions similar to those used forthe wild-type enzyme are summarized in Table 2.This Table also presents the effects of ornithine,IMP and UMP on the global reaction, when Mg-ATP is the variable substrate.

Uracil-sensitive mutants

Of the four mutants, carB80, -801, -803 and-1001, that are characterized by a pronouncedsensitivity towards uracil, carB1001 and carB801bear a single base-pair substitution convertingrespectively the proline 165 into a serine residueand the proline 170 into a leucine residue. Bothmutations are located in domain B of the carbox-yphosphate synthetic component. The alanine 182to valine substitution found in the double mutantcarB803 (A182V � S948F) is located in the sameregion; to analyze its effect separately, singly sub-stituted mutants were constructed by subcloning(see Materials and Methods). The three mutatedenzymes P165S, P170L and A182V clearly presenta reduced apparent af®nity for bicarbonate(between three- and sixfold) and a smallreduction in the af®nity for the activatorsornithine and IMP. The sensitivity of the P170Lenzyme towards UMP is increased; its [I]0.5 forUMP is about four times lower as compared tothe wild-type enzyme (Table 2). In contrast, thatof the P165S enzyme is twofold higher, whereasthat of the A182V mutant enzyme is unchanged.However, the doubly mutated carB803 CPSase(A182V � S948F) is completely insensitive to-wards pyrimidine and purine nucleosides(Table 2). This effect can clearly be attributed tothe sole S948F substitution, located in the allo-steric domain, as the same properties characterizethis single substitution construct (Table 2 andFigure 2(b)): no effect of IMP and UMP on the[S]0.5 for Mg-ATP (Figure 1(b)). In contrast, bothenzymes (S948F and A182V � S948F) are still acti-vated by ornithine although their af®nity for thisligand is ®vefold reduced (Figure 2(a)).

The fourth uracil-sensitive mutant, carB80, alsobears two amino acid substitutions, P360L andT1042I (Table 1). The sole substitution of proline360 by a leucine residue greatly reduces the af®-nity of the enzyme for Mg-ATP (about ®vefold;Table 2). UMP still inhibits the activity of theP360L enzyme; however it was not possible toquantify this effect accurately due to the verylow activities. Yet this P360L mutant enzyme wasalso affected in its allosteric properties, since areduced af®nity for the activators ornithine andIMP was observed, respectively 30 and 20-fold.However, the increase in activity in the presenceof these effectors (at saturation) is largelysuperior to that observed for the wild-typeenzyme (Figure 2(a)). The reduced af®nity for the

Table 2. Kinetic parameters of wild-type and mutant CPSases.

Km Gln Km HCO3ÿ

[A]0.5

ORN [A]0.5 IMP [I]0.5 UMP [S]0.5 of Mg-ATP (mM)

CPSases (mM) (mM) (mM) (mM) (mM) None ORN IMP ORN � IMP UMP Vmax

Wild-type 0.12 1.2 0.04 0.05 5.0 10.5 1.7 3.8 1.8 12.0 36.5P165S 0.17 6.5 0.06 0.12 12.0 6.0 2.2 4.0 2.3 12.0 21.1P170L 0.10 3.2 0.12 0.08 1.4 3.2 0.9 2.0 1.0 11.1 27.7A182V 0.10 4.4 0.08 0.07 6.0 7.1 1.5 3.2 1.7 11.5 38P360L 0.05 2.2 1.15 >1.0 n.m. 50.0 7.8 15.5 2.0 n.m. 0.8S743N 0.20 2.5 0.12 0.10 7.0 10.0 1.4 3.4 1.6 14.5 2.8T800F 0.20 1.4 0.96 0.12 0.5 8.0 2.0 7.4 1.6 28.0 33.3G824D 0.10 3.5 0.37 0.12 1.5 17.0 3.3 8.5 3.5 >50 12.4S948F 0.10 1.5 0.20 - - 8.0 1.6 7.6 1.7 7.2 18.2T1042I 0.20 1.5 - 0.35 150.0 7.2 5.0 2.3 1.0 9.5 53.3A182V 0.11 4.2 0.30 - - 5.5 1.7 5.6 1.7 5.6 14.0� S948FS743N 0.15 4.5 0.36 0.12 2.0 20.0 3.9 7.8 4.5 30.0 8.8� G824D

n.m., not measurable; -, no effect; standard deviation for [S]0.5 of Mg-ATP and for Vm, 10 %; standard deviation from triplicateassays for the other parameters, 5 %. Vmax is expressed as mM carbamoylaspartate formed � hourÿ1 � proteinÿ1 by the puri®edenzyme in the absence of effectors and in the presence of saturating concentration of both glutamine and HCO3

ÿ. Vmax was deter-mined from the saturation function of the enzyme by Mg-ATP as described in Materials and Methods.


activators presented by this mutant is thereforebalanced by the more pronounced stimulation ofenzyme activity. The second mutation present inmutant carB80, T1042I, is located in the allostericdomain. The sole substitution of threonine 1042by an isoleucine residue results in an enzymethat shows a greatly reduced activation byornithine (Table 2). Moreover, the af®nities of thismutant enzyme for both IMP and UMP arereduced, but in contrast to ornithine, theseligands still modulate the enzymatic activity(Figures 1(c), 2(a) and (b)). The observed lack ofactivation by ornithine (T1042I), the very lowVmax (P360L), and the reduced enzyme pro-duction due to partial repression of carAB tran-scription in the presence of uracil are theelements that combined, confer uracil-sensitivityto mutant carB80.

Arginine-sensitive mutants

The carB gene of the arginine-sensitive mutantcarB36 harbors two mutations that convert serine743 into an asparagine residue and glycine 824 intoan aspartate residue, respectively. The singleS743N substitution presents only minor modi®-cations of the kinetic parameters, except for aconsiderably reduced Vmax. In contrast, the singleG824D substitution mutant, located in the Cdomain of the carboxy terminal half, and thedouble mutant enzyme display a strongly reducedaf®nity for the activator ornithine (Table 2),re¯ected by a nearly tenfold increase in the [A]0.5

and a more moderate increased sensitivity forUMP (threefold reduced [I]0.5 for UMP; Figure 2(b)and Table 2).

In the presence of arginine, the synthesis ofornithine is strongly reduced due to repression ofthe biosynthetic genes and feedback inhibition ofthe N-acetyl-L-glutamate synthase, catalyzing the

®rst step of arginine biosynthesis. This reduction inornithine concentration combined with the reducedaf®nity for ornithine of the mutant enzyme, andpossibly also with the reduced Vmax, could explainthe sensitivity towards arginine of mutant carB36.

Arginine and uracil-sensitive mutants

Finally, the CPSase of mutant carB678 sensitivetowards arginine and uracil bears a single substi-tution that converts threonine 800 into a phenyl-alanine residue. This T800F replacement, likeG824D located in the C domain of the carboxy-terminal half, bears characteristics similar to thoseof this G824D substitution, i.e. reduced af®nityfor ornithine and increased sensitivity for UMP,but even more pronounced. It is most likely thisreinforcement of the effects, especially thereduction in the [I]0.5 for UMP that makes thismutant being sensitive to both arginine anduracil.

Discussion

The crystal structure of CPSase complexed withornithine, ADP, Pi, Mn2� and K� has beenreported at a resolution of 3 AÊ (Thoden et al., 1997)and more recently re®ned to a resolution of2.1 AÊ (enzyme complexed also with glutamine,H. Holden, personal communication). Therefore,the mutations reported here could be located in thestructure, and the mutational effects tentativelyanalyzed on a structural basis. The large subunit,where all the mutations are found, is folded in twosimilar halves that are de®ned by residues M1-A553 and N554-K1073. The ®rst three domains ofthe two halves superimpose very well and arerelated by an almost exact 2-fold symmetry axis,thus forming a pseudo homodimeric structure

Figure 2. (a) Effect of ornithine on (*)wild-typeCPSase, (*) P360L CPSase, (&) T1042I CPSase, (&)S948F CPSase. (b) Effect of UMP on (*) wild-typeCPSase, (&) S948F CPSase, (&) T1042I CPSase, (*)G824D CPSase. v/vo (in per cent) is the ratio of theobserved velocity with the different concentrations ofeffectors to that in their abscence.


(Figure 3). The crystal structure has been analyzedin terms of the sequential catalytic mechanism(Thoden et al., 1997). On that basis, the N-terminalhalf is described as the carboxyphosphate-formingdomain by analogy with biotin carboxylase(Climent & Rubio, 1986). This transient intermedi-ate is subsequently transferred to the C-terminalhalf, responsible for CP formation. Both halvesconsist of four well-de®ned domains denominatedfrom A to D. The two D domains diverge in termsof structure. In the carboxyphosphate part (resi-dues V404-A553), this domain comprises two glob-ular subdomains, each built from three shorta-helices connected by a longer a-helix. On thecontrary, the D domain from the CP part (residues
S937-K1073) consists of a b-sheet made up of ®ve
parallel strands and ®ve a-helices, forming aRossmann fold. This region is responsible for thebinding of the allosteric effectors, IMP, UMP andornithine.

The three mutated CPSases P165S, P170L, andA182V present a reduced af®nity for bicarbonate,which could be correlated with the observedgrowth stimulation of the original strains bearingthese mutations, by the addition of CO2 to the gasphase (Mergeay et al., 1974). Moreover, in the pre-sence of uracil, this addition is vital for growth(Table 1). Indeed, in the presence of uracil, theactivity of the strongest of the two promoters (P1)directing CPSase production is strongly repressedand, moreover, the enzyme activity is inhibited byUMP. These effects have more dramatic conse-quences in the mutants than in the wild-typebecause the mutant enzymes are either morestrongly inhibited by UMP, or less ef®ciently acti-vated by ornithine, or both, and all show a reducedaf®nity for bicarbonate. These three mutations arefound in the B domain of the carboxyphosphatehalf (residues L141-L210). P165 and P170 arelocated respectively at the beginning and at theend of the second (when counting from the N ter-minus) b-strand of this domain, a structural regionwhich actually constitutes, together with the Cdomain, the binding site of one ADP and twoMn2� (Figure 4). R169, located in the b-strand justbefore P170, interacts with the phosphoryl oxygenatoms of the a-phosphate of ADP and also forms asalt bridge with D207, a residue located at the endof the last strand of the b-sheet. Thus, it can beanticipated that the presence of these proline resi-dues in this b-strand, one of which in the cis con-formation (P165), is required to stabilize either theADP binding motif, and/or the bicarbonate bind-ing site. A182 is located at the end of the thirdb-strand of the antiparallel b-sheet. Although thisresidue is not in the direct vicinity of the ADP, itsside-chain is located in a hydrophobic clusterbetween the antiparallel b-sheet and the twoa-helices on which the B domain is built. ResiduesF164, P165, C166, I168, F188, C192, I198 and I206also participate in the cluster formation. Introdu-cing a valine residue at position 182 may interferewith side-chain packing inside the hydrophobiccluster and perturb the domain organization,resulting in a decreased af®nity for bicarbonate.

The P360L mutation is located in the C domainof the carboxyphosphate half (residues I211-E403).Replacing Pro360 by Leu results in a reducedaf®nity for Mg-ATP, although this residue is notinvolved in any direct interaction with this sub-strate, or located in the vicinity of the active site.The G824D mutation, which also results in areduced af®nity for Mg-ATP, is located at thebeginning of a b-strand of the C domain (residuesD757-N936) comprising Q829, a residue whichcoordinates one Mn2� in the active site. Introdu-cing a negatively charged side-chain in this arearequires some structural modi®cations, as this side-chain packs against the side-chain of Met669.

Figure 3. Ribbon representationof the CPSase large subunit. Shownin black are the A, B and Cdomains of the carboxyphosphateand carbamoylphosphate halves,and in light gray their D domains.The two active sites are identi®edby their respective ADP moleculeswithin the A-B-C domains.Ornithine molecules bound to theallosteric sites within the allostericD domain are also shown. Theornithine and ADP molecules aredepicted in space ®lling represen-tations. Figures 3-5 were preparedwith MOLSCRIPT (Kraulis, 1991).

Figure 4. B domain component (residues T143 toL210) of the carboxyphosphate half, where threemutations are located. Also shown are the ADP mol-ecules bound between the B and C domains, Pi, andMn2�. Labels identify P165, P170 and A182 within thesubunit. Also indicated are R169, which interacts withan a-phosphoryl oxygen of ADP, and D207, whichmakes a salt bridge with R169. In Figures 4 and 5, theN, O and C atoms are represented by white, gray andblack circles, respectively.


These modi®cations are likely to destabilize themetal coordination sphere and therefore ATP bind-ing, as it is known, from the X-ray structure, thatthe closest Mn2� is coordinated also by an a and ab-phosphoryl oxygen atom from the ADP, Q829and E841.

The T800F mutation, which is found in the Cdomain of the CP subunit, is solvent exposed. Inthis case, the allosteric behavior is related to that ofthe G824D enzyme. T800 is hydrogen-bonded toE780, and substituting T800 by a Phe residueresults in the loss of this interaction. However, adirect correlation of this mutant behavior with thethree-dimensional structure cannot be proposed sofar.

The S948F and T1042I mutations are bothlocated in the allosteric domain of the carbamoyl-phosphate half. This domain consists of a b-sheetof ®ve parallel strands and ®ve a-helices forming aRossmann fold. Two molecules of ornithine havebeen identi®ed in the crystal structure, both ofthem associated with the D domain. The ®rst bind-ing site is located at the interface between the Cand D domains, and involves residues E783, E892from the C domain and T1042 from the D domain.The second site is fully comprised in the D-domainand positioned at the carboxy-terminal end of theparallel b-sheet of this domain (Figure 5). Oneinorganic phosphate is also present in this site,approximately 3 AÊ from the side-chain aminogroup of ornithine (in the structure re®ned at

Figure 5. Close-up view of theallosteric domain and its interfacewith the C domain. This domain isorganized aroud the b-sheet of ®veparallel strands, surrounded by ®vea-helices. The two allosteric sitesare represented. The ®rst one islocated at the C-D interface, whereT1042 is interacting with the a-car-boxyl group of ornithine. Thesecond site is shown with a Glnmolecule and a Pi, as observed inthe re®ned CPSase structure at2.1 AÊ resolution (H. Holden, per-sonal communication). Ser948 is inclose proximity to one phosphateoxygen and is hydrogen-bonded to


2.1 AÊ , a glutamine residue is observed in this site).The residues interacting with this Pi are K954,T974, T977, and K993. From these observations,taken together with the fact that site-directedmutagenesis at position 977 abolishes UMP regu-lation of CPSase while retaining ornithine regu-lation (Czerwinski et al., 1995), this second site hasbeen assigned as the UMP/IMP effector site.

Mutations T1042I and S948F are located in theputative ornithine and UMP/IMP binding sites,respectively. Substituting threonine 1042 by anisoleucine residue greatly reduces ornithine acti-vation, con®rming that this site is the primaryeffector site for ornithine. T1042, located at the endof the last strand of the parallel b-sheet, is bridgingthe C and D domains through the ornithinemolecule, and E783 and E892 of the C domain(Figure 5). E783 and E892 interact with the aminogroup of ornithine. The backbone amide nitrogenatom of T1042 and its hydroxyl group are hydro-gen-bonded to the a-carboxyl group of ornithine.Our kinetic results show that T1042 is required topreserve the structural integrity of the binding site;the replacement of T1042 by an isoleucine residue
results in the loss of interactions, and also causes
steric hindrance for ornithine binding in theallosteric site.

Quite interestingly, UMP and IMP are still acti-vating and inhibiting the T1042I substitutedCPSase, although to a reduced extent. We observeda ®vefold reduction of effects of UMP and IMP,indicating that ornithine site one, although notdirectly involved in UMP and IMP binding, maybe located along the transmission pathway of theirallosteric signals.

On the other hand, the S948F mutant is totallyinsensitive towards purine and pyrimidine nucleo-tides, which is in good agreement with the X-raystructure and previous studies in identifying thebinding site for these effectors. S948 is located inthe ®rst b-strand (when counting from the N termi-nus) and its side-chain group is in close proximityto one of the inorganic phosphate oxygen atoms(3.56 AÊ ; Figure 5). Moreover, S948 is hydrogen-bonded to the hydroxyl group of Thr974, a residuealso interacting with a phosphoryl oxygen atom.Therefore, S948 is a crucial residue in de®ning thebinding site for both UMP and IMP. When examin-ing the behavior of this mutant towards ornithine,it is observed that S948F CPSase is still activated,

the hydroxyl group of T974.


although to a slightly reduced level (Table 2). Ourresults indicate that UMP and IMP, althoughhaving antagonistic effects, are acting at theoverlapping binding sites, and the allosteric signalsof such effectors are presumably transmitted viathe binding site for another allosteric effector, i.e.ornithine, at least via T1042, a residue directlyinvolved both in ornithine binding and in thede®nition of the interface between the allostericand the catalytic CP domains, suggesting thatallosteric regulation by UMP/IMP and ornithineare coupled.

A last objective of this study was to establish therelationship between the mutant phenotypes andenzymatic properties of the correspondingCPSases. The phenotype of singly substitutedmutants may be interpreted in terms of theirkinetic and allosteric characteristics and of theregulation of the carAB operon expression. Toestablish the relationships for the double substi-tuted mutants is not possible unless one of themutations would be ``silent''. Recombinantenzymes bearing the separate single amino acidsubstitution were, therefore, constructed andanalyzed.

Transcription initiation of the carAB operon(encoding the two subunits of CPSase), is drivenby two tandem promoters, P1 upstream and P2downstream, respectively, regulated by pyrimidineand arginine (for a review, see Glansdorff, 1996;Neuhard & Kelln, 1996). The addition of uracil as asource of pyrimidine nucleotides exerts a doubleeffect on CP production; it reduces transcriptioninitiation at P1 and inhibits enzyme activity. Argi-nine in the medium also directly affects the rate ofenzyme production through repression of the P2promoter and in¯uences the enzyme activity, asarginine inhibits the synthesis of ornithine, the acti-vator of CPSase. A comparison of the CPSase-speci®c activities of the wild-type strain P4X inminimal medium supplemented with arginine or

Table 3. Strains, plasmids and phages used

Strains Genotype

A. BacteriaJEF8/ld car53�B8 Hfr metB thr carB8/ldcar53 carryinJM101 F0 �(pro lac) supE thi StrA sbcB15 eC600 rÿ m�� B8 proAB lac Iq Z �M15 Fÿ hsdR hsdM

B. PlasmidspKK223-3 Apr

pMC50 pBR322-carAB

C. Phagesl199 cI857, sus xis6,b515, b519 susS7ldcar53 �B8 ld car53 carrying the carB8 deletio

D. car mutants Parental strainscarB36 CA244-1carB0 CA244-1carB678 P678carB801 P4XcarB803 P4XcarB1001 P4X

uracil illustrates these regulatory effects at the levelof enzyme production (Table 1).

The various mutants studied exhibited a reducedgrowth in minimal medium supplemented withuracil. Indeed, carB mutants having a CPSase witha high degree of af®nity for UMP (like carB801)and/or a low speci®c activity (like carB1001) areexpected not to be able to grow or to do so onlyvery slowly (like carB36). The addition of CO2 tothe gas phase is expected to stimulate the growthfor those strains having an enzyme with a lowerapparent af®nity for bicarbonate (carB80, carB801,carB803, carB1001). A reduced af®nity of theenzyme for ornithine (carB36, carB678) or an insen-sitivity towards this positive effector (carB80) willalso block growth. The double carB80-encodedmutant enzyme (P360L � T1042I) could not bestudied due to its extremely low speci®c activity; itcombines the respective handicaps of the twosingle mutant enzymes, lack of activation byornithine (T1042I) and greatly reduced af®nity forthe substrate Mg-ATP (P360L).

In view of the size of CPSase and the complexityof the reactions catalyzed, the in vivo selection ofmutants, on the basis of particular phenotypes, hasallowed the direct targeting of functionally import-ant amino acid residues, and by doing so toprogress in revealing the molecular basis for allos-tery. Here, two amino acid residues of crucialimportance for the regulation of the CPSaseactivity have been targeted. These data contributeto expand our knowledge towards a morecomplete characterization of the regulatorydomain.

Materials and Methods

Strains, plasmids, phages, constructions andgrowth conditions

All strains and plasmids used are derivatives of E. coliK-12 and of plasmid pBR322, respectively. The selection

Source of reference

g the carB8 deletion Crabeel et al. (1980)ndA hspR4 F0 haD36 Messing (1983)� thi pro carB8 recA Crabeel, this laboratory

Purchased from PharmaciaPiette et al. (1984)

R. Weisbergn Crabeel et al. (1980)

Mergeay et al. (1974)Mergeay et al. (1974)Mergeay et al. (1974)Mergeay et al. (1974)Mergeay et al. (1974)Mergeay et al. (1974)


of mutants used in this work, their genetic mapping andbehavior are described by Mergeay et al. (1974). Descrip-tions and genotypes are shown in Table 3.

Metabolic requirements were satis®ed by complement-ing minimal medium (3 g of KH2PO4, 7 g of K2HPO4,1 g of (NH4)2SO4, 0.1 g of MgSO4 7H2O, 0.5 g of Nacitrate 2H2O 0.5, 10ÿ6 M, MnSO4 , 10ÿ6 M Fe citrate10ÿ6 M made up in one litre of water) with (®nal concen-trations): 0.5 %, (w/v) glucose, 100 mg/ml each L-argi-nine, L-leucine, L-threonine, L-methionine, and L-proline,50 mg/ml L-tryptophan, 1 mg/ml thiamine, 50 mg/mluracil, 50 mg/ml ampicillin.

Cloning of carAB mutations

CarB mutations were transfered from their originalchromosomal locus to the integrated ldcar�B8 genomeby P1vir-mediated transduction at 30 �C of the doublelysogenic carB8 deletion strain C600�carB8/l199/ldcar53�B8 and selection for arginine and uracil proto-trophy. Recombinants bearing a non-biauxotrophic carBmutation on the integrated ldcar genome were identi®edby thermal-induction and spot tests of lysates on a lawnof C600�carB8 receptor cells on minimal medium platesdevoid of arginine and uracil.

Lysogens bearing a carB mutation were induced at42 �C, the cells harvested, lyzed and the phage particlespuri®ed by CsCl gradient centrifugation as described(Glansdorff et al., 1976). The approximately 10.5 kbHindIII-generated fragment was isolated by gel electro-phoresis, extracted from low melting agarose and ligatedinto the HindIII site of the expression vector pKK223-3.Transformants bearing recombinant plasmid DNA wereidenti®ed by replica plating of Apr C600�carB8 transfor-mants on minimal medium devoid of arginine anduracil.

DNA sequencing

The nucleotide sequence of carB alleles was deter-mined for both strands by the enzymatic chain termin-ation method (Sanger et al., 1977) using double-strandedpKK322-car plasmid DNA as template and a series ofregularly spaced oligonucleotides as primers.

Construction of single amino acid substitutionmutant derivatives of carB36, carB80 and carB803

In order to separate the two amino acid substitutionspresent in these mutants, pMC50 (bearing the wild-typecarAB operon), pKKcarB36, pKKcarB80 and pKKcarB803were digested with ApaI and BfrI, which cleave the vec-tor once, in the carAB operon. Fragments were puri®edand mixed pairwise such as to generate single aminoacid residue substitution mutants. Recombinant plasmidswere sequenced to verify the constructions.

Purification of wild-type and mutant enzymes

Carbamoylphosphate synthetases were puri®ed fromE. coli C600rÿmÿ�carB8 transformants carrying therecombinant plasmid pKK223-3 bearing the carB WT ora mutated carB allele. Cells were grown in minimalmedium in the presence of 50 mg/ml ampicillin in a 15 lfermentor. Logarithmic growing organisms were har-vested by centrifugation, washed with 0.9 % NaCl andstored at ÿ20 �C. Unless otherwise stated, all puri®cationprocedures were performed between 0 and 4 �C. The cell

pastes (30-50 g) were resuspended in 50 ml of 0.2 Mpotassium phosphate buffer (pH 7.5) containing 30 mMglutamine, 0.5 mM EDTA and 10ÿ5 M phenyl methanesulfonide ¯uoride (PMSF) and then disrupted by soni-cation for ten minutes. The cell extract was clari®ed bycentrifugation at 20,000 g for 15 minutes. The super-natant was either heated at 55 �C for 15 minutes andcooled in an ice bath, or precipitated in 20 % saturatedammonium sulphate, for the thermosensitive CPSases.The precipitates were discarded, after centrifugation at20,000 g for 30 minutes and the CPSase was concentratedby collecting the precipitate between 20 % and 55 % sat-uration ammonium sulphate. The resulting precipitatewas redissolved in 20 ml of 0.1 M potassium phosphatebuffer (pH 6.8) containing 10 mM glutamine, anddesalted by dialysis against the same buffer. In all fol-lowing steps, 10 mM of glutamine was included in thebuffers. The dialyzed preparation was loaded onto aDEAE-Sepharose column (2.5 cm � 40 cm) equilibratedwith 0.1 M potassium phosphate buffer (pH 6.8). Thecolumn was washed with three volumes of the same buf-fer after which the CPSase activity was eluted throughfractions by a continuous linear gradient from 0.1 M pot-assium phosphate buffer (pH 6.8) to 0.4 M potassiumphosphate buffer (pH 7.6) in a total volume of 400 ml.The CPSase activity was eluted at about 0.35 M potass-ium phosphate. The active fractions were pooled (20 ml)and concentrated tenfold in an Amicon Dia Flow using aPM 30 membrane and dialyzed against 0.1 M potassiumphosphate buffer (pH 7.6), containing 1 M ammoniumsulphate. As the last step of puri®cation, the sample wasloaded onto a phenyl-Sepharose CL6B column(1 cm � 15 cm) previously equilibrated with buffer ofthe same composition as the dialysis buffer. The proteinwas eluted by 50 ml of a linear decreasing gradient from1 M to 0 M ammonium sulphate in 0.1 M potassiumphosphate buffer (pH 7.6). CPSase is eluted near the endof the gradient. The active fractions were pooled, concen-trated (®nal volume: 2-3 ml, 2-20 mg/ml) and assessedfor purity by SDS electrophoresis of aliquots. The wild-type and mutant enzymes were puri®ed to more than90 % homogeneity, as judged from electrophoreticpatterns of SDS-polyacrylamide gels stained withCoommassie blue, and scanned to obtain densitometricpro®les. The puri®cation of the carB80 mutant enzymecould not be achieved, due to its instability.

Protein determination

The protein content was measured by the method byLowry et al. (1951).

Determination of enzyme activity

The enzyme activity was determined at 37 �C by coup-ling CP formation with OTCase or with ATCase.

The OTCase coupling system was routinely usedduring enzyme puri®cation. The rate of CP synthesiswas assayed by measuring the rate of citrulline for-mation in a coupled assay containing OTCase andornithine using a protocol derived from the method byArchibald (1944). The assay mixture contained in 2.0 ml,50 mM K-phosphate (pH 7.5), 10 mM glutamine, 10 mMNaHCO3, 5 mM ornithine, 10 units/ml OTCase (puri®edin this laboratory), 10 mM MgCl2, 10 mM ATP, 2-40 mgCPSase. The reaction was started by the addition of Mg-ATP and was stopped after ten to 30 minutes, by theaddition of 0.75 ml acid reagent (phosphoric acid and


sulfuric acid, 3/1 (v/v) and of 0.25 ml diacetyl mono-xime (1.5 % diacetylmonoxime in 10 % ethanol). Thecitrulline concentration was determined from the absor-bance at 490 nm.

In the ATCase coupling system, the rate of CP syn-thesis was assayed by measuring the rate of carbamoy-laspartate formation by the method by Prescott & Jones(1969). The assay mixture contained 50 mM Hepes(pH 7.5), 100 mM KCl, 50 mM aspartate, 10 mMNaHCO3, 10 mM glutamine, 30 units/ml ATCase (puri-®ed in this laboratory), 20 mM MgCl2 and 20 mM ATP,2-40 mg CPSase. After incubation at 37 �C for 30 minutes,the reaction was stopped by the addition of 1 ml of thePrescott and Jones reagent (5 g/liter of antipyrine in50 % (v/v) sulfuric acid). The carbamoylaspartateconcentration was determined from the absorbance at466 nm.

Determination of kinetics parameters of wild-typeand mutant CPSases

The steady state kinetic data were analyzed in termsof Lineweaver-Burk, Hanes and Eadie plots to derive themaximal velocities (Vmax) and the substrates concen-trations required for half maximal velocity (apparent Km

or [S]0.5 value).The [S]0.5 values for Mg-ATP in the presence of

effectors were obtained with concentrations of effectorsequivalent to tenfold the [A]0.5 and [I]0.5.

Acknowledgments

We acknowledge the excellent technical assistance ofN. Huysveld and D. Gigot. We thank Dr H. Holden forproviding the three-dimensional coordinates of E. coliCPSase. This work has been supported by the BelgianFund for Joint Basic Research and the Fonds pourl'Encouragement de la Recherche (FER, UniversiteÂ Librede Bruxelles).

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Edited by A. R. Fersht

(Received 24 September 1998; received in revised form 14 January 1999; accepted 14 January 1999)

serine 948 and threonine 1042 are crucial residues for allosteric regulation of escherichia coli...

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