The Presence and Distribution of Reduced Folates in Escherichia coli Dihydrofolate Reductase Mutants*

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Escherichia
coli DNA photolyase was overproduced and purified from each of two mutant E. coli strains lacking dihydrofolate reductase.
The extent of overproduction in the mutants was comparable to that seen in the wild type strain.
Examination of the isolated photolyase from these strains revealed that the folate cofactor, 5,10-methenyltetrahydrofolate, was present in these proteins at a level of 60-80% compared to that purified from the wild type strain.Further examination of the dihydrofolate reductase-deficient strains revealed the presence of other tetrahydrofolate derivatives.These findings demonstrate that dihydrofolate reductase is not essential for the production of tetrahydrofolates in E. coli.
Folate cofactors are of critical importance in intermediary metabolism.These essential cofactors are synthesized de nouo in bacteria and plants but are derived from dietary sources in mammals, including man.In Escherichia coli, folates are known to be derived from GTP as shown in Scheme 1.The reduction of 7,8-H*folate' to H4folate is an obligatory step in the synthesis of the single carbon carrier forms of the molecule (Scheme 2).This conversion is catalyzed by dihydrofolate reductase, and the reaction is considered to be the sole source of cellular H4folates in E. coli.
Dihydrofolate reductase has long been the focus of mechanistic studies, particularly the targeting of drugs such as methotrexate and trimethoprim which are thought to cause cell death by inhibiting the formation of Hlfolate.To facilitate structure-function studies on the enzyme by the technique of site-directed mutagenesis, construction of a dihydrofolate reductase-deficient (fol-) strain is desirable.Because of the cellular importance of this enzyme for generating a multitude of essential compounds for the folate-dependent reactions in the cell, the survival of such a mutant would appear to be difficult.Nevertheless, the construction of two dihydrofolate reductase mutants by deletion of the fol gene was reported recently by two separate laboratories (2, 4).This fol deletion was stable only in the presence of an accompanying mutation in thymidylate synthetase.
In order to understand the details of folate metabolism in these mutants, we have analyzed the folate pool in each mutant, LH18 and PA414.Our findings are that both strains contain Hlfolates.Thus, it appears that another enzyme or pathway exists in E. coli for the production of Hlfolates in addition to the reaction or pathway involving dihydrofolate reductase.Twenty-five-ml cultures were inoculated with single colonies and grown overnight, and 5-ml aliquots of the culture were added to 2-liter flasks containing 500 ml of the appropriate media.The flasks were shaken at 37 "C until the A was approximately 0.6, induced with 1 mM isopropyl-P-D-thiogalactoside, and shaken 12-16 h before harvesting.

Chemicals
For the construction of UNC414, PA414 was mated with CSR603 (5) and transformed with pMS1310.(9,1'7).Although the normal copy number of this enzyme is 15-20 molecules per cell, when a plasmid containing the photolyase gene, phr, is placed in various E. coli hosts and induced, the enzyme can constitute 5-10% of the total cellular protein (5).Photolyase purified from such an overproducing strain contains approximately 0.3 mol of the folate cofactor per mol of protein with some variation from preparation to preparation (12).Since the enzyme readily binds exogenously added 5,10-CH+-H4folate, the less than stoichiometric amount of folate in the isolated enzyme appears to be due to an insufficiency of intracellular folate to meet the demand of enzyme overproduction.
By overproducing the photolyase in a folstrain, we hoped to obtain enzyme completely free of folate.Although the folate cofactor can be removed by chemical treatment (12), we wished to obtain enzyme free of folate without the additional chemical treatment.
When LH18 was transformed with pMSl310 to give SHAlO and photolyase was induced and purified from this strain, the isolated enzyme displayed the spectrum shown in Fig. 1.The presence of the peak at 384 nm in this enzyme and its relative amplitude compared to the radical flavin peak at 580 nm indicated that the protein contained CH+-H,folate.
The presence of this reduced folate was verified by HPLC; Fig. 2 shows the HPLC chromatogram of the acid-released cofactors from photolyase purified from SHAlO.The spectrum shown in Fig. 2 was obtained by an on-line scan of the material in the single peak indicated by an arrow on the HPLC chromatogram and is identical with standard CH+-H*folate.From analysis of these results, it is evident that photolyase from this particular preparation was approximately 20% saturated with the re- Strain UNC414 was constructed from the fol-strain PA414 in order to determine whether photolyase overproduction in this strain also yielded an enzyme preparation partly saturated with the reduced folate cofactor.Our analysis revealed that the photolyase purified from UNC414 was 25% saturated with CH'-H,folate (data not shown).Previous experiments have indicated that the overproduced photolyase is only partially saturated with folate in uiuo in wild type MS09; supplementation of the MS09 crude extract with CH+-H,folate resulted in isolation of photolyase containing virtually a full complement of the folate cofactor after purification ( 12).Therefore, it appears unlikely that the extent of overproduction in the fol-mutants would be affected by intracellular folate levels.In fact, photolyase purified from the folmutants is saturated with the folate cofactor to 60-80% of the level seen for photolyase purified from the wild type strain, and both of the fol-mutants overproduce photolyase to approximately the same extent as the wild type strain.As shown in Table II, the extent of overproduction of photolyase in SHAlO was 3-4%.Even though the extent of overproduction for SHAlO is slightly less than that obtained with MS09, this lower yield probably reflects the poorer growth of this particular folstrain.The more vigorous PA414-derived strain, UNC414, overproduces photolyase as well as the wild type strain, MS09 (5.8% for UNC414 compared to 5.5% for MS09).It is evident that the amount of the CH+-H,folate present in both folmutants is considerable.
There is no evidence that photolyase can produce CH'-H,folate from a H,folate precursor; our findings are that it binds only 5,10-CH+-H4folate/10-CHO-Hrfolate (12).The results of photolyase overproduction in both of these folstrains have led to the conclusion that another source for fully reduced folates besides the dihydrofolate reductase must exist. in the text.Before HPLC, the extracts in 5 mM sodium ascorbate, 5 mM 2-mercaptoethanol were passed through Spin-X columns by centrifugation in an Eppendorf microcentrifuge.Chromatography was in 5 mM tetrabutylammonium phosphate, 10 mM ammonium dihydrogen phosphate, 22% methanol, pH 6.1, at a flow rate of 0.9 ml/min.The numbered peaks are labeled as follows: I, lo-CHO-H,folate; ZZ, H,folate; III, 5-CHO-H,folate; ZV, Hpfolate; V, folate; and VZ, CHs-H,folate.cell paste.The chromatography conditions were slightly different for the chromatograms in Fig. 4, and all folates exhibit shorter retention times relative to the chromatograms in Fig. 3 as a result.Under these conditions, the H,folate peak, indicated by the arrows in Fig. 4, is obscured by UV-absorbing material.The chromatography was repeated under conditions that better resolved the H,folate peaks in the mutants, and a distinct Hdfolate peak was observed for each of the fol-mutants with a magnitude of about 50% less than that of the Hlfolate peak shown in Fig. 3 for the parent strains (data not shown).By these methods, we have verified the presence of H4folate, CHB-H,folate, folic acid, and lo-CHO-H,folate in the fol-strains.
The relative amounts and the distribution of these folates can be seen by a comparison of the different HPLC chromatograms in Fig. 3 and Fig. 4, keeping in mind that the chromatograms for the fol-strains were obtained using 1.6-fold more sample.lo-CHO-H,folate is the major folate species in both of the for strains and the parent strains.However, there appears to be slightly more of this species in the parent strains, since the amounts are approximately equal in the chromatograms but more material was injected for the folstrain analysis.The serum y-glutamyl hydrolase controls indicated that there was not any folate material in the serum preparations (data not shown).Extracts of the strains containing internal folate standards were analyzed for folate content, and the recovery of the internal standards was com- parable between the strains (data not shown).These recoveries were also comparable to yields of the folate standard controls.
The yields of each standard when carried through the procedure with and without added E. eoli crude extract are somewhat lower than those reported by Duch et al. (14) and vary between 20 and 50% for all species but I&folate and 5-CHO-H,folate which are on the order of 10%.The low yields of Hzfolate and 5-CHO-H4folate were expected, although the yields of the other folates have been reported to be higher than ours (14).We attribute our lower yields to the fact that the ionic strengths of our extracts were certainly higher due to the relative impurity and proportionally greater volume of y-glutamyl hydrolase that was used.It was disappointing that we could not obtain better yields of Hafolate, since it would have been useful to see how much Hpfolate was present in the fol-strains relative to the fol' strains.

Dihydrofolate
Reductase Assays of PA414, LH18, AB11.57, and N&&522--Both PA414 and LH18 were constructed by deleting the fol gene so that spontaneous reversions could not occur (2, 4).The fol gene was in fact replaced with the gene coding for kanamycin resistance so that the fol-phenotype was easily verifiable.The phenotypes of both mutants have been confirmed by enzymatic assays and Southern blot experiments (2,4).We have also confirmed that crude extracts of PA414 and LH18 contain no dihydrofolate reductase activity.Assays of crude extracts of PA414 and LH18 and the two parent strains AB1157 and NM522 as well as another fol+ strain, MJ7, are shown in Table III.The folstrains show a very small and inconsistent rate of change in absorbance at 340 nm.This rate is similar to the background rate of NADPH oxidation in foZ+ strains.If this rate is significant, then the observation that it is not eliminated completely in assays of PA414 incubated with methotrexate might indicate that it represents an "alternative" reductase activity with dihydrofolate that might be present in all strains but elevated in PA414.In separate assays, extracts of SHAlO and UNC414 were also shown to contain no dihydrofolate reductase activity (data not shown).
Growth Requirements for PA414 and LHlS-The reports of the growth requirements for PA414 and LH18 (2, 4) seemed inconsistent.PA414 reportedly required only added thymine for growth, but LH18 required all of the folate-dependent end products (thymine, adenine, glycine, methionine, and pantothenic acid) for growth.Both of the strains were constructed with mutations in the gene coding for thymidylate synthetase since the fol mutations were otherwise lethal.Both mutants would be expected to require thymine for growth because of the thymidylate synthetase mutation.However, the report that LH18 requires all folate end products for growth is inconsistent with the presence of the reduced folates in the cell that we observed.We have found that both LH18 and PA414 demonstrate a requirement for thymine but not for the additional folate end products.Both grew on minimal plates containing kanamycin and thymine or kanamycin, thymine, adenine, glycine, methionine, and pantothenate.Neither grew on minimal plates containing only kanamycin.This is consistent with the fact that each strain contains Rfolates.

DISCUSSION
Our results show unambiguously that two E. coli strains that are devoid of dihydrofolate reductase activity contain significant quantities of reduced folates.The presence of these reduced folates was first inferred in LH18; photolyase, an enzyme containing a tightly bound reduced folate cofactor, was overproduced and purified from SHAlO, a derivative of LH18, and shown to contain the reduced folate cofactor.This reduced folate cofactor was also isolated from photolyase purified from UNC414, a derivative of PA414.This finding, that a H*folate was present in sufficient quantity in these folstrains to partly saturate the overproduced photolyase, was FIG. 3. HPLC profile at 280 nm of folate cofactors from 2.5 g of NM522 cells (top) and from 2.5 g of AB115'7 cells (bottom) treated as described folate compound.The numerals indicate the folates identified in each of the extracts.In these extracts, lo-CHO-H,folate, H4folate, CHs-H,folate, and traces of folic acid were identified.Representative chromatograms of extracts from LH18 and PA414 as well as a chromatogram of several folate standard compounds are shown in Fig. 4.Each of the chromatograms for the folextracts represents approximately 4 g of E. coli FIG. 4. HPLC profile at 280 nm of standard folates (top) and folates from 4 g of LHl8 cells (middle) or 4 g of PA414 cells (bottom).Extracts in 5 mM sodium ascorbate, 5 mM Z-mercaptoethanol were centrifuged through Spin-X columns and immediately injected.Chromatography was in 98% solvent A and 2% solvent B at a flow rate of 1.8 ml/min.Solvent A consisted of 5 mM tetrabutylammonium phosphate, 10 mM ammonium dihydrogen phosphate, 24% methanol, pH 6.1, and solvent B consisted of 100% methanol.The labeled peaks are as follows: Z, lo-CHO-H1folate; ZI, Hlfolate; IZZ, 5CHO-Hlfolate; ZV, H*folate; V, folate; and VZ, CHa-Hlfolate.The arrows indicate the position of the H,folate peaks which were obscured by UV-absorbing material in these chromatograms.

TABLE I E
. coli strains and plasmids methanol with a final pH of 6.1-6.2, and O-2% of 100% methanol.The flow rate was 0.9-2.0ml/min.RESULTSOverproduction of E. coli DNA Photolyase in an LH18-

TABLE III Activities
of E. coli extracts Assays are composite.Values were normalized to 1 mg of protein from crude extract added per assay.The concentration of methotrexate (MTX) was 1 WM.