ARCHIVES

Vol.

OF BIOCHEMISTRY

284, No. 1, January,

AND

BIOPHYSICS

pp. 9-16,

1991

Mutagenesis of the fo/C Gene Encoding Folylpolyglutamate Synthetase-Dihydrofolate Synthetase in Escherichia co/i’ Lenka

J. Kimlova,

Received

Caron

of Microbiology,

Department

February

Pyne,

University

1, 1990, and in revised

Karim

Keshavjee,

of Toronto,

form

August

John Huy,

Press,

Inc.

1 This work was supported search Council of Canada. * To whom correspondence

by Grant

MA-9822

should

be addressed.

0003-9861/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

from

Ontario

and Andrew

L. Bognar2

Canada M5S IA8

28, 1990

The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagen&d by progressive deletions from both the 5’ and the 3’ ends and by TAB linker insertion. A number of 5’-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site is at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the lz,, of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the foZC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme. o 1991 Academic

Glen Beebakhee,

150 College Street, Toronto,

the Medical

Re-

The Escherichia coli folC gene product, folylpolyglutamate synthetase-dihydrofolate synthetase (EC 6.3.2.17), is an enzyme catalyzing two distinct reactions in the folate biosynthetic pathway. The dihydrofolate synthetase activity adds L-glutamate to dihydropteroate to form dihydrofolate. After reduction of dihydrofolate to tetrahydrofolate by dihydrofolate reductase, the folylpolyglutamate synthetase activity of the enzyme adds a second and third glutamate in y-linkage to form polyglutamates. Although tetrahydrofolate is an effective substrate for folylpolyglutamate synthetase, the preferred one-carbon form of the folate substrate of the enzyme is lo-formyl-tetrahydrofolate (1, 2). The polyglutamate products of the enzyme are the predominant forms of folate in cells and the in uiuo cofactors of folate-dependent enzymes. Kinetic studies of the enzyme suggested that the two synthetase activities are catalyzed at separate sites and the enzyme is bifunctional (2). A differential effect on the two enzyme activities cannot be demonstrated by selective inhibition or chemical modification (K. Keshavjee and A. Bognar, unpublished observations). Although the folC gene is cloned (3) and its sequence has been determined (4), the absence of a three-dimensional structure for the enzyme makes mutagenesis studies directed at the active site of the enzyme difficult. We are using in vitro mutagenesis to determine which regions of the folC gene product are essential for the function of its two enzyme activities. In this report, we describe a series of progressive deletion mutants from both the 3’ and the 5’ ends of the gene, resulting in protein products truncated at the carboxy or amino terminals. We also describe a series of two amino acid insertions (produced by TAB linkers) in various parts of the gene product. The effect of the mutations on the ability of the plasmid-encoded gene to complement the folC mutant, SF4, and a folC deletion mutant are reported. The specific activities for both enzyme activities of the mutant enzymes are investigated. 9

10 EXPERIMENTAL

KIMLOVA

PROCEDURES

Materials. [a3’P]dATP (7000 Ci/mmol), [&?,]dATP (1000 Ci/ mmol), [y3’P]ATP (3000 Ci/mmol), and L-[Ur4C]glutamic acid (10 mCi/ mmol) were obtained from Amersham Corp. [35S]Methionine Translabel was obtained from ICN Pharmaceuticals. Fluorography reagents (Er?Hance) were from New England Nuclear. Tetrahydrofolate, folinic acid, ATP, glutamic acid, 5-bromo-4-chloro-3-indolyl-/3-D-galactopyranoside and isopropyl-P-D-thiogalacto-pyranoside were purchased from Sigma Chemical Co. Polyethylene glycol (Carbowax 20,000) was from Fisher Scientific. Modified T7 DNA polymerase and all deoxy and dideoxy nucleotides were purchased as a Sequenase kit from United States Biochemical Corp. The Geneclean kit was purchased from BiolOl. Restriction endonucleases, ribonucleases, and DNA modifying enzymes were purchased from Boehringer-Mannheim, Pharmacia, or Bethesda Research Laboratories. TAB linkers were purchased from Pharmacia. Oligonucleotide primers and linkers were synthesized and purified as described previously (5). lo-Formyl-tetrahydrofolate and dihydropteroate were prepared and purified as described previously (6). E. coli mutants SF2 (folC Bacterial strains, media, and plasmids. &A) and SF4 ( folC s&A recA TnlO::srlC) have been previously described (3,7). SF4 was used as the recipient strain for complement&ion analysis of plasmids containing mutations in the jolC gene. The strain SF2AfolC, whose construction will be described elsewhere (C. Pyne and A. Bognar, in preparation), contains a chromosomal deletion of the folC gene complemented by a copy of the folC gene cloned into an unstable plasmid, pPM103 (8, 9), and was also used as a recipient for complementation tests. JF1754 (hsdR lac gal metB 1euB hisB) (10) was used as a highly competent host for all mutagenized plasmids. DR1984 (recA uurB) (11) was used for maxicell experiments. E. coli strains were grown in YT or LB (12) medium, supplemented with 50 ag/ml ampicillin, where indicated. SF4 grew on Vogel-Bonner minimal medium (13) supplemented with methionine (50 ag/ml) and glycine (100 ag/ml). Unsupplemented minimal medium on solid agar medium was used in complementation analysis. DR1984 was grown on YT or Hershey medium (14). All plasmids used in these studies were constructed from pAC5 (4) containing the folC gene in a pUC9 (15) vector. In some cases the gene was cloned into pUC19 (16). Plasmid Plusmid isolation, DNA modification, and transformation. preparations were by the alkaline lysis procedure of Birnboim and Doly (17). Restriction endonuclease digestions, nuclease digestions, and ligations were carried out according to the manufacturer’s instructions. Transformations were done by the Hanahan procedure (18). A 14-base Bglll linker, Construction of 5’ and 3’ deletion mutants. 5’-CCTGAGATCTCAGG-3’, which has been previously described (5), was inserted into the Stul site at the 5’ end of the folC gene. The gene was then excised as a Bglll-BamHI fragment and inserted into pUC19 previously digested with BumHI. The orientation of the insert in the plasmids was determined by restriction analysis. Plasmids with the gene in either orientation were digested with Pstl. The plasmids in the orientation to produce 5’ deletions were then digested with Xbal, while those used for 3’ deletions were digested with BumHI. Progressive unidirectional deletions were made in the DNA inserts using exonuclease III/S1 nuclease, as described by McNeil et al. (19). The 14-base Bglll linker was added to the deleted plasmids prior to ligation to recyclize the plasmids. This linker provided a site which could be used to subclone the deleted folC gene and, in the case of 3’ deletions, provided a stop codon in one of the reading frames. Two of the five 3’ deletion mutants described had this stop codon immediately after the point of deletion. The others were digested with BglII and Hind111 and the 5’ overhanging ends were removed using Sl nuclease, followed by DNA polymerase I Klenow fragment. The extent of each deletion was determined for each construct by dideoxy nucleotide DNA sequencing. TAB linker mutagenesis. Plasmid pAC5 was partially digested with Hhal, Hpull or Accl and the products of the digestions were electrophoresed on an agarose gel. The bands corresponding to the linearized

ET

AL.

plasmid were excised and purified from the gel using the Geneclean kit (BiolOl) as described by the manufacturer. The plasmids were recyclized in the presence of the appropriate linker to produce an Apul site at the point of insertion. These were 5’-GGCCCG-3’ for Hhal and 5’.CGGGCC3’ for Hpull and Accl. The ligated plasmids were used to transform JF1754 to ampicillin resistance. Plasmid DNA from transformants was digested with Apul. Those plasmids which were linearized were religated at high dilution and used to transform JF1754 to ampicillin resistance. This served to remove multiple copies of the linkers that may be present at the site of insertion. The position of linker insertion was determined by restriction analysis using Hindlll, BamHI, and Apul triple digests to determine the approximate position in the gene of the insertion and DNA sequencing with the plasmid DNA template using a series of sequence-specific primers which have been previously described (4). In the case of the Hpull digests, a strong selective digestion was observed. The majority of linkers were inserted into a site in the multiple cloning region and a minority at the site within the folC gene described in the Results section. No other sites of insertion were observed. Complementution analysis. All mutated plasmids were used to transform E. coli strain SF4 to ampicillin resistance. Transformants were grown in YT medium and used to inoculate minimal medium plates (13), lacking methionine, which were selective against the parental SF4 strain. Only those transformants would grow which expressed sufficient folylpolyglutamate synthetase activity to complement the methionine auxotrophy of the parental strain. We have recently constructed a strain of E. coli, SF2AfolC, in which the chromosomal foC gene has been deleted (C. Pyne and A. Bognar, in preparation). Folylpolyglutamate synthetase activity must be provided in this strain by a plasmid-encoded copy of the folC gene. We have transduced this mutation into strain SF2 containing the folC gene on a plasmid, pPM103, which is temperature-sensitive for replication and tends to segregate at 37°C in the absence of selection. It is maintained in all strains containing the chromosomal folC deletion, regardless of temperature, suggesting that this gene is essential in E. coli. Mutants of the folC gene in pUC plasmids were transformed into this strain. If the product of the mutant gene contains sufficient folylpolyglutamate synthetase-dihydrofolate synthetase activity to maintain cell viability, the pPM103 plasmid, containing the complementing wild type gene, will segregate at high frequency, leaving the strain with only the mutant gene. If the activity of the mutant gene product is too low to maintain the cell, this results in a selection for the gene on the pPMl03 plasmid and it does not segregate. This complementation test is more rigorous than complementation in SF4 because it eliminates the background enzyme activity. This deletion strain will be very useful for studying the kinetics of the products of mutant genes constructed in uitro. The mutants described in this report were used to transform the SF2A folC strain to ampicillin resistance. Single colony transformants were grown in the absence of tetracycline selection to test for segregation of the pPM103 plasmid. The presence of Amp’ Tet” single colonies indicated that the mutants had sufficient enzyme activity to support the growth of E. coli. Plasmid DNA was prepared for double-stranded DNA sequencing. sequencing as described (20). The dideoxy nucleotide method of Sanger et al. (21) was used with [a-35S]deoxyadenosinethiotriphosphate or [o32P]dATP label. Reactions were primed using the universal or reverse sequencing primers or sequence-specific oligonucleotide primers (4). Regions of compression were verified by synthesis reactions using dITP in place of dGTP. Enzyme extraction and assay. Cells of strain SF4 transformed with wild type or mutagenized plasmids were grown up in l-liter cultures of YT medium supplemented with 100 ag/ml ampicillin. Cells were harvested by centrifugation and lysed by sonication in a Branson sonifier. The cleared lysate (50 ml) was made 60% in ammonium sulfate and the precipitate collected by centrifugation. This fraction was resuspended in 5 to 10 ml of 50 mM Tris-HCl, pH 7.5. All values reported were corrected for the background activity of SF4 cells prepared in the same way. Although the chromatographic properties of the mutant enzymes

MUTAGENESIS

OF

THE

FOLYLPOLYGLUTAMATE TABLE

Kinetic

Constants

Varied

substrate

Dihydrofolate synthetase H,Pte ATP Glutamate Folylpolyglutamate synthetase 10.Formyl-H,Pteglu ATP Glutamate

Terminal

Deleted

Mutant ND11

type

Mutant

V max pmol/h mg

1.1

4.1

0.9 * 0.2” 10 Ifr 1.5O 2800 k 500” 17 54 300

f + f

5” 14” 50”

’ The error values shown are the error in the K,,, as determined by the kinetics experiment. The absolute error in the biological determination is at least 20%.

were similar to those of the wild type, including binding to phosphocellulose, the key step in the purification procedure (4), many of the mutant proteins, particularly the amino terminal-deletion mutants, were found to be considerably less stable than the wild type and further steps failed to increase the specific activity of the preparations. A purification of 22.fold was achieved by phosphocellulose chromatography but this purification resulted in a variable amount of inactivation with different mutants and introduced uncertainty in the contribution of the host strain enzyme to activity. As a result, all enzyme specific activities reported were for ammonium sulfate fractions, as these gave the most reliable relative activities. Folylpolyglutamate synthetase activity was measured by the incorporation of [‘4C]glutamate (1 mM) into folate products as described previously (3, 6). Both tetrahydrofolate (100 pM) and lo-formyltetrahydrofolate (10 and 200 PM) were used as folate substrates to determine folylpolyglutamate synthetase activity of mutants relative to wild type. Dihydrofolate synthetase activity was measured using dihydropteroate (25 pM) as substrate as previously described (3). The reactions were incubated for 1 h, during which the assay is linear with time. In the experiments to determine kinetic constants, the amount of enzyme used was adjusted so that no more than 10% of the pteroate substrate was converted to product and the activities therefore represent initial rates. The kinetic constants reported in Table I for the wild type and amino terminal-deleted mutants were determined using the Enzyme program (R. A. Lutz and D. Robard, Laboratory of Theoretical and Physical Biology, NIH and Laboratory of Clinical Chemistry, Kantonospital, Winterthur, Switzerland). Analysis ofplasmid-encodedgeneproducts in maxicells. E. coli strain DR1984 was transformed with various plasmids and UV irradiated and plasmid-encoded proteins were labeled with [35S]methionine as described by Sancar et al. (14). SDS-polyacrylamide3 gel electrophoresis was performed using 13% polyacrylamide gels according to the method of Laemmli (22). The labeled products were visualized by fluorography. Densitometry of maxicell fluorographs. The relative intensity of the radioactive bands corresponding to the plasmid-encoded gene products from maxicell experiments was quantitated using densitometry. To determine the relative intensities of the protein bands of mutant and wild type FPGS, the densitometry values were normalized to the fl-lactamase bands. Since all of the mutagenized genes were in the same vector and the constructs varied by only the mutagenized regions, the copy numbers

3 Abbreviations used: SDS, glutamate synthetase; DHFS,

11

GENE

I

of an Amino Wild

SYNTHETASE

sodium dodecyl sulfate; FPGS, dihydrofolate synthetase.

folylpoly-

1200 110 0.4

0.22

3.1 * 0.2” 180 + 50” 870 f 90”

70 1.2 0.3

240 40 14

0.75

63 210 160

12 3.6 4.7

program

and represent

the scatter

k 19” AT 80’ + 40’ in the points

in the kinetic

and transcription signals (all genes were transcribed from the lac promoter in the vector) should be similar for all the plasmids. The relative intensities of the bands should therefore be directly proportional to the relative expression of the plasmid-dependent proteins in the transformed strains. Since we are looking at relative intensity, the use of different strains in the maxicell experiments and the expression studies should not affect these comparisons, as they would affect the expression of mutant and wild type genes in a similar manner. Protein assays. Protein concentration was measured using the dyebinding assay of Bradford (23) with bovine serum albumin as the standard. The predicted secondary structures for the wild type Data analysis. enzyme and the linker insertion mutants were determined by the Microgenie program (Beckman instruments). RESULTS

Amino terminal deletions. A series of Exonuclease III/ Sl nuclease-generated deletions originating at a linker inserted into the StuI site 77 base pairs upstream of the initiation codon for the folC gene were prepared. DNA sequence analysis revealed that a number of these deletions extended into the coding sequence of the amino terminal of the folC gene. The positions of the deletions of a number of these plasmids are shown in Fig. 1. Unexpectedly, it was found that mutants with up to the first 85 bases of the coding sequence of the folC gene deleted (NDl-11) were able to complement the folC mutation in strain SF4 and the folC-deletion strain, even though the mutants had lost the ATG codon for the initiator methionine as a result of the deletions. Genes containing deletions of 149 bp (ND12) or greater, however, no longer complemented either strain and therefore did not produce a functional product. The expression of these mutants (with the exception of NFl and NF2, see below) was not due to an in-frame fusion with the ATG codon of the la& gene in the multiple cloning region of the vector. Some of the mutants which produced functional gene products (e.g., ND2, ND7) had in-frame stop codons just a few base pairs upstream of the point of deletion of the coding

12

KIMLOVA NFl(-) .

ND1 ND2 NIX (+) (+) (+I . . .

MIIKRTP AE ATT ATC AAA 0.X ACT TAAOWLATCA -TACC No4 ND5 NF2 (+I m(+) (+) (+I . . . . PLASWLSYLENLHSKT ccTcx;GcTTa;TGGcITTcTTATcx;GAAFACcIGcACRGTppAAcT NLnO(C) ND9 Null (f) (f) .. I IDLGLERVSLVAARLG A~GATcpCGGCCrr~Q;cGn;AGCcn;GIcccGGa;CGTCITt%C

03

Q

A

CAA

Gee

A Ga:

S TOG

ND7 ND8 (+I (+I . .

NLn2(-)

NLm(-)

.

1

ND14 C-1 .

VLKPAPFVFTVAGTQG GTCCTGAAACCAG‘GCCATITcn;TITAfXGITGO;GGTACGAATGGC ND15(-) . KGTTCRTLGSILMAAG AAAGGCA~A~n;c~AfficI~Ta;ATTCTGpLR;Go:GCAGGG

FIG. 1. Deletions from the 5’end of the folC gene. The DNA sequence of the 5’ upstream region and the codons for the first 75 amino acids of folylpolyglutamate synthetase are shown. Deletion endpoints are indicated by the triangles. Codons which may be used as downstream translation initiation sites are highlighted. Putative Shine-Dalgarno sequences for these sites are underlined. NF, endpoint of 5’ deletion known to be fused to vector sequences in-frame with the initiation site of P-galactosidase; ND, endpoint of 5’ deletions having no in-frame translation start site in the vector.

sequence, thus precluding a chance fusion to an in-frame ATG codon in the vector. Maxicell analysis of the products expressed from these deleted plasmids is shown in Fig. 2. A band corresponding to full-length folylpolyglutamate synthetase, M, 47,000 (see lanes 1, 7), is not expressed from these mutants. A new, much fainter, band appears in the lanes corresponding to the N-terminal deletion mutants, indicating a protein that migrates at a greater mobility of approximately M, 43,000 (lanes 2-4). There are only two products encoded by these plasmids, FPGS and /3-lactamase. The new band could only be a truncated form of FPGS, since plactamase is much smaller. All the deletion mutants which express a functional product by complementation analysis, produce this M, 43,000 band in maxicell experiments. In contrast, those plasmids with larger deletions which do not complement the SF4 mutation do not produce this protein product (e.g., lane 5). This direct correspondence between the presence of the faint band and a functional FPGS expressed from N-terminal-deleted plasmids indicates that this band represents the mutant FPGS gene product. Since a band of identical mobility is produced by plasmids which have 15 to 85 bp deleted from the coding sequence, representing 5 to 28 amino acid codons, and a difference of 5 amino acids would be detectable as a difference in the mobility of the product, it suggests that all of these deletion mutants produce the same gene product. We have determined the k, values for lo-formyl tetrahydrofolate, of two distinct deletion mutants (ND3 and ND4), and found these to be identical, within experimental error, to that of the deletion mutant (NDll) whose

ET

AL.

kinetic constants are shown in Table I, providing evidence that these deletions produce a mutant protein with the same properties. Since the deletion is at the amino terminal, an explanation consistent with all the data is that a secondary site of translation initiation is utilized in the mutants, producing a functional product truncated at the amino terminal and that the same product is produced by all the mutants. When the extent of deletion is correlated to complementation of the folC mutation in SF4 (Fig. l), it is clear that the functional mutant proteins do not initiate at the second methionine ATG codon in the gene, since this codon is present in a number of deleted plasmids which do not complement the SF4 mutant. However, it is known that translation can sometimes be initiated in bacteria from codons other than ATG and the minor codons most frequently used are GTG codons coding for valine (24). The efficiency of initiation at such codons is lower than for ATG codons. There are two GTG codons in the region affected by these deletions, one at +103 bp and one at fl51 bp relative to the normal initiation site (Fig. 1). Since deletion ND12 contains the GTG codon at +151 bp but does not produce a functional product, the secondary initiation site for the functional truncated proteins is likely the GTG codon at $103 bp. We have not, however, been able to sequence the amino terminal of the mutant protein to verify our hypothesis because of its low expression and instability upon purification. There is a sequence upstream of this site resembling a Shine-Dalgarno sequence for a ribosome binding site. Initiation at

1234567

Mr,103 -

FPGS

93

-68

-

-

43

- 21

WT

ND1

ND4

ND8

ND15

NF2

WT

FIG. 2. Expression of plasmid-dependent proteins in maxicells. DR1984 cells were transformed with the indicated plasmids and extracts were analyzed by electrophoresis on 13% polyacrylamide gels in the presence of sodium dodecyl sulfate and fluorography to detect [35S]methionine-labeled proteins. The positions of plasmid-encoded gene products are shown at left. fl-lactamase bands derived from the vectors are present in all lanes. The arrow denotes the gene product expressed from the 5’.deleted plasmids. The positions of coelectrophoresed standards of known molecular weight are indicated at right. Lanes: 1 and 7, pAC5, expressing wild-type folylpolyglutamate synthetase; 2 to 5, derivatives of pAC5 containing 5’ deletions shown in Fig. 1; 6, derivative of pAC5 containing a 5’ deletion whose product is fused in-frame with the N-terminal of P-galactosidase.

MUTAGENESIS

OF

THE

FOLYLPOLYGLUTAMATE

this site would produce a protein shorter by 34 amino acids with a predicted difference in molecular weight of 3740. This is in good agreement with the difference in M, between the wild type and mutant enzymes, which are 47,000 and 43,000, respectively. A cell extract of the SF4 mutant transformed with deleted plasmid ND11 was partially purified and concentrated by precipitation with 60% ammonium sulfate. Kinetic analysis of this fraction is shown in Table I. The values shown were corrected for the contribution of the SF4 product. The K,,, for lo-formyl-tetrahydrofolate and for dihydropteroate were 3-fold higher than that of the wild type enzyme. The K, for glutamate was 2- to 3-fold lower than that of the wild type for both activities but the K,,, for ATP was 5 to 20-fold higher. The relative amount of the mutant protein expressed, was determined by densitometry of the fluorographs in maxicells and by the folylpolyglutamate synthetase specific activity of the extracts of cells transformed with the mutant genes. The amount of expression varied somewhat among the mutants with deletions of different extent but the variation in specific activity was at most threefold (see Table II). This may reflect differences in the amount of mRNA produced, its stability, or the strength of ribosome binding, due to the differences in the DNA sequences. The specific activity of the ND11 mutant was 13 to 19% that of the wild type enzyme while that of the ND4 mutant was 4 to 6% of wild type. The relative amount of protein expressed in maxicells was determined, compared to that of the wild type gene product (pAC5 in Fig. 2). The expression of the mutants was about lo%, and the highest value obtained for a mutant was 16%, that of the wild type expression. As the genes are present in the same vector and transcribed from the same promoter, this suggests that the translation of the mutant product is much less efficient. A comparison of the specific activity of the extracts of strains containing the mutant and wild type enzymes and the relative expression of the mutant and wild type gene products suggests that the catalytic activity (iz,,,) of the N-terminal deletion mutants is comparable to that of the wild type gene. Two mutants were obtained in which the 10 amino acids of ,B-galactosidase and the multiple cloning region up to the PstI site were fused in frame to the deleted 5’ end of the folC gene. Mutant NF2 has the fusion after a deletion of 17 codons and NFl after a deletion of 3 codons (Fig. 3). Analysis of the NF2 gene product in maxicells showed that it was highly expressed, similar in intensity to the wild type protein and migrated slightly faster than the wild type protein (Fig. 2, lane 6). Complementation analysis showed that the gene product of the fusion with the more deleted folC gene, NF2, could complement the mutation in SF4, whereas the product of fusion to the less deleted gene (NFl) did not complement. The latter fusion protein, although produced in amounts similar to NF2

SYNTHETASE

GENE

(not shown), has virtually no folylpolyglutamate tase or dihydrofolate synthetase activity.

13 synthe-

Carboxy terminal deletions. Exonuclease III/S1 nuclease digests were also done on the fob2 gene in the opposite orientation in the same vector, producing deletions from the carboxy terminal of folylpolyglutamate synthetase. A linker containing a stop codon was inserted at the 3’ end of the deleted gene. Sequence analysis was used to determine the extent of deletion in each mutant. The mutants investigated are shown in Fig. 3. The sequence of the vector-derived amino acids added after each deletion until a stop codon is reached are also shown. Plasmids CDl, with 5 amino acids deleted and 1 added, CD2, with 13 amino acids deleted and 10 added, and CD3, with 14 amino acids deleted and 14 added, all complemented the mutation in SF4, while those with 25 or more amino acids deleted (CD4, CD5, others not shown) did not complement. None of these deletion mutants could complement the folC-deletion strain. The specific activities of the 60% ammonium sulfate fractions of extracts from transformants containing these mutant plasmids are shown in Table II (CD1 to CD5). Folylpolyglutamate synthetase activity with tetrahydrofolate (100 pM) and lo-formyl-tetrahydrofolate (10 pM and 200 pM) as well as dihydrofolate synthetase activity (25 pM dihydropteroate) were measured. The carboxy terminal-deleted gene products which did not complement the mutation in SF4 (CD4, CD5) had no measurable enzyme activity above the SF4 background under any assay conditions. Even those gene products which could complement the mutant had greatly decreased activities for both enzyme activities of the protein. CDl, which had only five codons deleted and only one codon added, had less than 0.1% of wild type folylpolyglutamate synthetase activity (at low lo-formyl-tetrahydrofolate concentrations, which minimize background activity from the SF4 mutant) and less than 0.05% dihydrofolate synthetase activity. TAB linker mutagenesis. Six-base pair linkers were inserted into seven HhaI sites, one HpaII site and the sole AccIA site of the folC gene. The sites of two-amino acid insertion are scattered throughout the gene (Fig. 3), although none are found in the amino terminal region, which was deleted in the experiments described above without much effect on catalysis. The amino acids inserted are glycine and proline in all but one mutant, Ll, which has arginine and alanine inserted. All mutants were sequenced to identify their sites of insertion. The gene products of each mutant plasmid were expressed in maxicells (not shown). All the mutants shown expressed products with electrophoretic mobilities indistinguishable from those of the wild type. The amount of the product expressed, determined by densitometry, normalized to the amount of the P-lactamase present, was within 10% of the amount of wild type protein present. The specific ac-

14

KIMLOVA

ET

TABLE Specific

Activities

FPGS

Mutant SF4 pAC5/SF4 (wt) ND4/SF2AfolC NDll/SF4 (+) Ll/SF4 (+) * L2/SF4 (+) L4/SF4 (+) L5/SF4 (+) L6/SF4 (+) LB/SF4 (+) L9/SF4 (-) LlO/SF4 (+) CDl/SF4 (+) CD2/SF4 (+) CD3/SF4 (+) CD4/SF4 (-) CD5/SF4 (-)

under

II of

folc Mutants

activity”

DHFS

activity

10.formyl-HIPteGlu (10 PM)

10.formyl-H,PteGlu

(200PM)

(100PM)

(25PM)

0.09 392 30 25 0.46 0.25 0.37 0.08 0.36 0.15 0.02 0 0.35 0.24 0.37 0 0

1.67 3578 590 224 0.1 0 1.4 0.8 0.4 0 0 0 0.6 0 0 0 0

0.04 2511 335 106 0.35 0.18 0.72 0.06 0.09 0.04 0.04 0.0 0.21 0.08 0.28 0 0

0.4 1032 195 66 0.2 0.3 1.6 3.1 0.2 0 0 2.2 0.3 0 0.3 0 0

(+)

’ Determined as described subtracted from all values. * (+) Indicates the ability medium.

AL.

Experimental

of the plasmid

Procedures.

to complement

Units

are nmol

the mutation

tivities reported in Table II are therefore directly proportional to the amount of enzyme protein present in the extracts and the differences in the activities directly reflect differences in the catalytic activities of the enzymes. None of the protein bands corresponding to the mutants showed downward streaking, which would indicate rapid degradation of the mutant gene product. The effect of the insertion mutations on enzyme function was investigated by complementation analysis in strain SF4, which is also shown in Fig. 3. Seven mutants could complement the mutation in strain SF4 and three could not. The inactive mutants were not localized to a particular region of the gene but were interspersed among the mutations which allowed complementation. It was found that one or seven linkers, each coding for a GlyPro pair, could be inserted at the HpaII site (L4) and still produce a product which complemented the mutant phenotype. Of the seven mutants which could complement the defect in SF4, all but L4 were unable to complement at 42°C indicating that their products are temperaturesensitive. Interestingly, these mutants were also cold sensitive, since their growth rates on minimal medium at 30°C were greatly reduced. None of the linker-insertion mutants were able to complement the foZC-deletion strain. These results suggest that the mutations have significant effects on the structure of the proteins, leading to gene products less stable to temperature. The predicted secondary structure of the wild type FPGS-DHFS protein (calculated using the Microgenie

h-i mg-‘.

in SF4, allowing

H,PteGlu

The

specific

the strain

activity to grow

H,Pte

of the SF4 mutant in the absence

background

of methionine

is in the

program) is also shown in Fig. 3. The perturbations of this secondary structure predicted to be caused by each linker addition mutation are also shown. These effects on structure are quite significant, particularly for the nine insertions of glycine, proline pairs, which introduce or extend stretches of random coil in all cases.This disrupts both the putative P-sheet structures (Ll, L2, L3, L6, L9) and predicted regions of a-helix (L2, L5, L8, LlO). The insertions L4 and L7 are in regions of the protein where random coil is predicted, yet these mutations also have a large effect on the activity of the proteins (Table II). Two of the three mutants which do not complement the folC mutation in strain SF4 (L3 and L7) occur in regions of the protein that are highly conserved when compared with the L. caseiFPGS (25), both being within stretches where there are four identical amino acid residues in sequence. All the other insertions are in regions of the protein that are less conserved, compared to the L. casei protein. The third noncomplementing mutant, L9, is in a region where both proteins contain a short stretch of P-sheet followed by a turn, with an identical proline residue. The mutation destroys the P-sheet, which may be essential for structure or catalysis. Enzyme activities of extracts of strain SF4 transformed with the mutagenized plasmids are shown in Table II. All mutants show large decreases in both enzyme activities as a result of the amino acid insertions. The mutants which did not complement the mutation in SF4 (eg. L9) had little or no detectable activity above the background

MUTAGENESIS

OF

ml(+) WC-) LJ.o(+) c?=.GFPLEGpRGA tt--t----* E$gYz . . F--K C pp-t---t-aammaawum--~~~~~Q~Qa~~~~a~

FE22 .

cw(-) -c

THE

FOLYLPOLYGLUTAMATE

cDz(i-) an(+) PaIJmwmvsms-c -lT-C .. . 422 cmtttt-

FIG. 3. Deletions and amino acid insertions in mutant folylpolyglutamate synthetases. The amino acid sequence of folylpolyglutamate synthetase is shown. The secondary structures predicted from the primary structure according to the Microgenie program are shown below the amino acid sequence. Deletions from the amino and carboxy terminals are indicated. The point of deletion is to the right of the n- for amino terminal deletions and to the left of the -c for carboxy terminal deletions. The sequences of vector-derived amino acids added to various deletions are shown. The point of fusion is to the right of the last amino acid for amino terminal deletions and to the left of the first amino acid for carboxy terminal deletions. The position of linker additions is indicated by the triangles. The inserted amino acid pair is shown in the context of the surrounding residues immediately above the point of insertion. The predicted secondary structure in the region of the insert is shown immediately below this sequence. The surrounding residues shown include all those whose predicted secondary structure is altered by the insertion as well as the first residues on either side whose predicted structure is unaffected. The (+) or (-) after each mutation indicates whether these mutants, expressed from high copy number pUC vectors, utilizing the lac promoter can complement the folC mutation in E. coli strain SF4. A mutant at the position of L5, containing an insertion of seven copies of glycine, proline pairs, was also able to complement SF4. The predicted secondary structure of this mutant had a more extended section of random coil than the structure shown. The secondary structures are: 01, alpha helix; 0, beta sheet; t, turn; -, random coil.

for either enzyme activity. Mutants which did complement (Ll, L2, L4, L5, L6, L8, LlO) all had less than 0.2% of wild type activity for folylpolyglutamate synthetase and less than 0.4% of wild type activity for dihydrofolate synthetase. These values are 20- to 60-fold lower than the specific activities of the amino terminal-deletion mutants. In all cases, both enzyme activities were severely affected. DISCUSSION Our studies indicate that a number of amino terminal amino acids of folylpolyglutamate synthetase-dihydro-

SYNTHETASE

GENE

15

folate synthetase are quite dispensable and can be deleted without a drastic effect on either enzyme activity. The genetic evidence of our deletion studies shows that the secondary site of translation initiation is not the second methionine in the reading frame. Since the GTG codon of valine is the most likely alternate initiation codon, this suggests that our mutant protein begins at valine 35 and lacks the first 34 amino acids of the wild type protein. The predicted size of a protein initiated at this point agrees well with the electrophoretic mobility of the unique protein expressed in maxicells of transformants containing deletion mutants. This region of the enzyme has little or no homology to the amino terminal regions of folylpolyglutamate synthetase from Lactobacillus casei, although sequences just downstream have strong homology (25). This suggests that this part of the enzyme is not directly involved in catalysis. The deletion does, however, decrease the stability of the enzyme to purification. Replacement of deleted codons in this region with random sequences is also tolerated if this does not increase the size of the resultant protein. The fusion of 10 amino acids to the protein missing the first 3 amino acids results in a completely inactive enzyme, either due to the disruption of the normal folding of the enzyme or a steric effect on the accessibility of substrates. The enzyme truncated at the amino terminal has a catalytic rate similar to the wild type for both enzyme activities. The affinity for pteroate and folate is about threefold lower, while the affinity for glutamate is two- to threefold higher. The greatest effect is the substantial decrease in affinity for ATP with both enzyme activities. Interestingly, the amino terminal of the truncated protein is now near the putative ATP binding site of folylpolyglutamate synthetase (4). The removal of the amino terminal amino acids may destabilize the ATP binding site, leading to the higher K,,, values for ATP binding. All the other mutants we constructed, including the carboxy terminal deletions and the linker additions, resulted in greater than 95% inactivation of both enzyme activities. The amino acids inserted in 9 out of 10 mutants, glycine and proline, have particularly strong effects on protein secondary structure. Glycine tends to destabilize both a-helix and P-sheet structures (26), while proline tends to create turns in the protein, terminate a-helices, and disrupt other secondary structures. This suggests that altering the structure of the folylpolyglutamate synthetase enzyme in any part of the enzyme except the amino terminal region is deleterious to its activity. None of the mutations affected only the folylpolyglutamate synthetase or only the dihydrofolate synthetase activity without affecting the other. There was never a differential loss of activity of one of the two enzymes as a result of any mutation. Taken together with the similar extent of inactivation of both activities upon treatment with several reagents for chemical modification (K. Keshavjee and A. Bognar, unpublished observations) these results suggest

16

KIMLOVA

that the enzyme has a single active site required for both of its activities. The entire enzyme may be considered one functional domain. However this conclusion must be viewed with caution because the mutations described appear to have major effects on the structure and folding of the protein, apart from the active site. In our recent studies with site-directed mutagenesis of the codon that is the locus of the SF4 mutation, we found that when conservative amino acid substitutions were made, which resulted in proteins which retained 5 to 90% of wild type activity, the mutations consistently affected both activities of the enzyme similarly, while less conservative substitutions abolished both activities (C. Pyne, K. Keshavjee, and A. Bognar, in preparation). These results provide additional evidence for a single active site for the enzyme. Our results show that complementation analysis in strain SF4 is a very sensitive method for detecting residual folylpolyglutamate synthetase activity in mutants. The mutants which failed to complement the SF4 auxotrophy all had less than 0.01% of wild type activity. Mutants having as little as 0.04% of wild type folylpolyglutamate synthetase activity or 0.02% folylpolyglutamate synthetase activity in the presence of 0.3% dihydrofolate synthetase activity, could complement the mutation. This sensitivity is due in part to the leaky nature of the SF4 mutant, which produces sufficient enzyme to provide the cell with folates for all cellular functions except methionine synthesis. The mutant must provide enough additional activity to supplement the host activity in making polyglutamates for methionine synthesis. The high copy number of the plasmid also amplifies expression of the mutant protein, allowing products with lower activities to produce sufficient polyglutamates to complement the mutation. We have predicted that the dihydrofolate synthetase activity is probably the essential activity of this enzyme, since it is required for folate biosynthesis and E. coli does not transport exogenous folate (24). Our recent results confirm that folC is an essential gene in E. coli. We have deleted this gene in a polAt” strain and replaced it with a kanamycin resistance marker (C. Pyne and A. Bognar, in preparation). This chromosomal deletion is only possible if the folC gene is complemented on a plasmid. We used a complementing plasmid, derived from pSC101, which is unstable and can be replaced with wild type and mutant folC genes on plasmids such as those described in this work. The wild type gene and the amino terminal deletion mutants can complement this deletion mutant but none of the linker insertion or carboxy terminal-deletion mutants can do so. This indicates that these mutants, even when expressed from multicopy vectors cannot complement a nonleaky folylpolyglutamate synthetase mutant. We are currently investigating the growth and intracellular folate pools of the foZC-deleted strain con-

ET

AL.

taining the amino terminal-deletion mutant and other in uitro-constructed mutants capable of complementing this strain. These studies will help us define the metabolic role of the folC gene product in E. coli. ACKNOWLEDGMENTS The authors thank Dr. V. L. Chan for the use of the Microgenie program and Dr. S. McCraken for reading the manuscript.

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