Parameters affecting the restoration of activity to inactive mutants of thymidylate synthase via subunit exchange: further evidence that thymidylate synthase is a half-of-the-sites activity enzyme.

In a previous study we demonstrated that Escherichia coli thymidylate synthase activity could be restored completely by incubating basically inactive mutants of this enzyme at room temperature with R(126)E, another inactive mutant [Maley, F., Pedersen-Lane, J., and Changchien, L.-M. (1995) Biochemistry 34, 1469-1474]. Since only one of the enzyme's two subunits possessed a functional active site and the restoration of activity could be titrated to be equivalent to that of the wild-type enzyme's specific activity, it was proposed that thymidylate synthase was a half-of-the-sites activity enzyme. We now provide additional support for this thesis by presenting an in-depth analysis of some conditions affecting the restoration of enzyme activity. For this purpose, we employed two mutants with marginal thymidylate synthase activity, Y(94)A and R(126)E. The parameters that were examined included pH, concentration of protein, temperature, and urea concentration, all of which influenced the rate of activity restoration. It was found, surprisingly, that by maintaining the amount of each protein constant, while increasing the volume of solution, the rate and total activity restored was greatly enhanced. Increasing the pH from 6.0 to 9.0 markedly increased the rate at which the optimal activity was restored, as did increasing the temperature from 4 to 40 degrees C. A similar effect was obtained when the incubation of the mutants was conducted at 4 degrees C in the presence of 1.5 M urea, a temperature at which activity is restored extremely slowly. Raising the pH to 9.0 resulted in an almost instantaneous restoration of activity at 4 degrees C. The manner in which thymidylate synthase activity is restored from the mutants in the presence of varying concentrations of ethanol, ethylene glycol, and glycerol suggests that changes in subunit interaction and enzyme conformation are in part responsible for the observed differences. Most significantly, at solution levels of 10%, ethanol was found to activate, while ethylene glycol inhibited slightly and glycerol was somewhat more inhibitory. At a concentration of 20%, ethanol inhibited rather strikingly, ethylene glycol was slightly more inhibitory than at 10%, and glycerol was strongly inhibitory. Since the net result of these findings is the suggestion that the restoration of thymidylate synthase activity is due to a separation of the mutant dimers into their respective subunits, followed by their recombination to an active heterodimer, evidence for this phenomenon was sought by separating the recombined dimers using nondenaturating polyacrylamide gel electrophoresis. Sequence analysis of the isolated homo- and heterodimers clearly demonstrated that the active enzyme is a product of subunit exchange, one that is very efficient relative to the wild-type enzyme, which did not exchange subunits unless denatured.

High-level expression of Escherichia coli and Bacillus subtilis thymidylate synthases.

Procedures are described for the preparation of highly purified thymidylate synthases from Escherichia coli and Bacillus subtilis. The yields in each case are quite high with about 350 mg of pure protein obtained from 1 liter of cells. Basically all that is required to obtain pure enzyme is an induction step from a high-expression vector, followed by a DE-52 column elution. Both enzymes appeared to be fairly stable in that incubation at 43 degrees C for 10 min resulted in the loss of 50% of the E. coli thymidylate synthase activity, while 50 degrees C for 10 min was required to obtain the same effect with the B. subtilis enzyme. In the presence of the substrate, dUMP, each protein was stabilized further by 6 to 7 degrees C, which was increased to 9 to 10 degrees C on addition of dihydrofolate. It was shown also that the E. coli thymidylate synthase could be maintained at 4 degrees C for at least 4 months with little or no loss in activity provided that mercaptoethanol was not present. The presence of the latter led to a progressive loss in activity until little activity could be detected after 18 weeks, which was due, in part, to the formation of a disulfide bond with the active site cysteine. Addition of dithiothreitol restored the enzyme activity to its original state.

Glycosylation of the overlapping sequons in yeast external invertase: effect of amino acid variation on site selectivity in vivo and in vitro.

Yeast invertase contains 14 sequons, all of which are glycosylated to varying degrees except for sequon 5 which is marginally glycosylated, if at all. This sequon overlaps with sequon 4 in a sequence consisting of Asn92-Asn93-Thr94-Ser95(Reddy et al., 1988, J. Biol. Chem., 263, 6978-6985). To determine whether glycosylation at Asn93is sterically hindered by the oligosaccharide on Asn92, the latter amino acid was converted to a glutamine residue by site-directed mutagenesis of the SUC2 gene in a plasmid vector which was expressed in Saccharomyces cerevisiae. A glycopeptide encompassing sequons 3 through 6 was purified from a tryptic digest of the mutagenized invertase and sequenced by Edman degradation, which revealed that Asn93 of sequon 5 contained very little, if any, carbohydrate, despite the elimination of sequon 4. When Ser and Thr were inverted to yield Asn-Asn-Ser-Thr carbohydrate was associated primarily with the second sequon, in agreement with numerous studies indicating that Asn-X-Thr is preferred to Asn-X-Ser as an oligosaccharide acceptor. However, when the invertase overlapping sequons were converted to Asn-Asn-Ser-Ser, both sequons were clearly glycosylated, with the latter sequon predominating. These findings rule out steric hindrance as a factor involved in preventing the glycosylation of sequon 5 in invertase. Comparable results were obtained using an in vitro system with sequon-containing tri- and tetrapeptides acceptors, in addition to larger oligosaccharide acceptors.

Crystal structure of thymidylate synthase A from Bacillus subtilis.

Thymidylate synthase (TS) converts dUMP to dTMP by reductive methylation, where 5,10-methylenetetrahydrofolate is the source of both the methylene group and reducing equivalents. The mechanism of this reaction has been extensively studied, mainly using the enzyme from Escherichia coli. Bacillus subtilis contains two genes for TSs, ThyA and ThyB. The ThyB enzyme is very similar to other bacterial TSs, but the ThyA enzyme is quite different, both in sequence and activity. In ThyA TS, the active site histidine is replaced by valine. In addition, the B. subtilis enzyme has a 2.4-fold greater k(cat) than the E. coli enzyme. The structure of B. subtilis thymidylate synthase in a ternary complex with 5-fluoro-dUMP and 5,10-methylenetetrahydrofolate has been determined to 2.5 A resolution. Overall, the structure of B. subtilis TS (ThyA) is similar to that of the E. coli enzyme. However, there are significant differences in the structures of two loops, the dimer interface and the details of the active site. The effects of the replacement of histidine by valine and a serine to glutamine substitution in the active site area, and the addition of a loop over the carboxy terminus may account for the differences in k(cat) found between the two enzymes.

Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA.

Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.

Molecular cloning and sequence analysis of Flavobacterium meningosepticum glycosylasparaginase: a single gene encodes the alpha and beta subunits.

A full-length insert for the Flavobacterium meningosepticum N4-(N-acetyl-beta-glucosaminyl)-L-asparagine amidase gene was located on a 2500-bp HindIII fragment and cloned into the plasmid vector pBluescript. DNA sequencing revealed an open reading frame of 1020 nucleotides encoding a putative 45-amino-acid leader sequence and a deduced precursor polypeptide of 295 amino acids. In F. meningosepticum this precursor polypeptide undergoes proteolytic processing by an as yet unknown mechanism to generate an alpha-subunit and a beta-subunit, which constitute the active form of the heterodimeric mature glycosylasparaginase. The Flavobacterium glycosylasparaginase gene was expressed in Escherichia coli and found to be enzymatically active. The recombinant enzyme was purified from crude lysates and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist of the typical alpha- and beta-subunits. The recombinant beta-subunit cross-reacted to antibody specific for the rat liver beta-subunit, and Edman analysis demonstrated that its amino-terminus corresponded exactly to that of the mature native glycosylasparagine beta-subunit. A comparison of the Flavobacterium glycosylasparaginase with a mammalian glycosylasparaginase revealed 30% structural identity and 60% overall similarity between the prokaryotic and eukaryotic forms of the enzyme. Even more striking was the conservation of the amino acid sequence in both proteins where the post-translational cleavage to generate the active enzyme occurs. Our data demonstrate that deglycosylation of asparagine-linked glycans via hydrolysis of the AspNHGlcNAc linkage is an important reaction which has been preserved during evolution.

Identification of a site necessary for allosteric regulation in T4-phage deoxycytidylate deaminase.

An allosteric inhibitor of dCMP deaminase, dTTP, forms a photolabile covalent bond with T4-phage dCMP deaminase in the presence of UV light at 254 nm. The importance of the methyl group in this process is supported by the findings that dUTP, also an allosteric inhibitor, does not photofix to the enzyme and that tritium is released from [methyl-3H dTTP during the course of the photofixation. That the bond formed is photolabile is demonstrated by the fact that tritium is released by about 10-fold over the amount of nucleotide that is photofixed. The amino acid that covalently binds dTTP in T4-dCMP deaminase was identified as Phe112. On conversion of Phe112 to an alanine by site-directed mutagenesis, there was a dramatic change in the enzyme's response to its allosteric effectors when measured early in the reaction, in that the mutant enzyme was as active as the wild-type even in the absence of dCTP and was only weakly inhibited by dTTP. However, after 10-15% of the substrate had been deaminated, the reaction rate fell off rather markedly, indicating either that an inhibitor was being accumulated on the enzyme or that the enzyme was being irreversibly inactivated with time. That the latter was not the case was shown by the addition of dCTP to the reaction, which restored the rate to that expected when it was present initially. Furthermore, we showed that, consistent with the observed loss of allosteric regulation by dCTP and dTTP, the affinity of the mutant enzyme for dTTP and dCTP as determined by binding studies was greatly reduced relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple endoglycosidase F activities expressed by Flavobacterium meningosepticum endoglycosidases F2 and F3. Molecular cloning, primary sequence, and enzyme expression.

The genes for Flavobacterium meningosepticum Endo (endoglycosidase) F2 and Endo F3 were cloned, and their nucleotide sequences were determined. The deduced amino acid sequences were verified independently to a large extent by direct peptide microsequencing of 66 and 84% of native Endo F2 and Endo F3, respectively. Structurally, the Endo F2 and Endo F3 genes code for a typically long leader sequence of 45 and 39 amino acids, respectively, and, in both cases, a mature protein of 290 amino acids. Comparative structural analysis demonstrated minimum overall homology (15-30%) between Endo F1, Endo F2, and Endo F3, but revealed distinct clusters of identical residues distributed throughout the entire sequence, which represent motifs for binding and hydrolysis of beta 1,4-di-N-acetylchitobiosyl linkages in complex carbohydrates. The mobility of native Endo F2 and Endo F3 on SDS-polyacrylamide gel electrophoresis, unlike Endo F1, did not correlate with the molecular weights determined from the coding region of the corresponding genes. Mass spectrometry confirmed that Endo F2 and Endo F3 were heterogeneous and contained approximately 4000 and 1200 daltons of mass not accounted for in the gene structure. We presume that Endo F2 and Endo F3 are variably post-translationally modified during secretion by possible linkage to the hydroxyl of serine.

Multiple endoglycosidase (Endo) F activities expressed by Flavobacterium meningosepticum. Endo F1: molecular cloning, primary sequence, and structural relationship to Endo H.

A full-length insert for the endo-beta-N-acetylglucosaminidase (Endo) F1 gene was located on a 2,200-base pair EcoRI fragment of genomic DNA and cloned into the plasmid vector Bluescript. Transformed Escherichia coli cells expressed Endo F1 activity very well, but the enzyme apparently was not processed and secreted into the medium as it normally is in Flavobacterium meningosepticum. DNA sequencing revealed an open reading frame of 1,017 nucleotides encoding a putative 50-amino acid signal sequence, and a mature protein (31,667 Da) of 289 amino acids. The deduced amino acid sequence was verified by direct Edman microsequencing of 88% of the purified protein as tryptic and V8 protease peptides. Alignment of Endo F1 (289 amino acids) with the established amino acid sequence of Streptomyces plicatus Endo H (271 amino acids) revealed a 32% structural identity over the entire sequence and a high degree of conservative replacements. Potential catalytic domains identified in other proteins that hydrolyze the beta 1,4 glycosidic linkage between N-acetylglucosamine residues are also conserved for amino acid identity and relative spacing in Endo F1.

Molecular cloning and amino acid sequence of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from flavobacterium meningosepticum.

A 3,000-base pair EcoRI fragment containing the Flavobacterium meningosepticum gene for peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was cloned into the Bluescript plasmid vector and expressed in Escherichia coli. The gene consists of an open reading frame of 1,062 base pairs coding for a 354-amino acid protein; the first 40 amino acids are presumed to be the natural secretory signal sequence, with the remaining 314 amino acids (34,779 Da) representing the catalytically active protein. The deduced amino acid sequence was verified independently by direct microsequencing of over 94% of the pure protein (Flavobacterium peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase) as tryptic and cyanogen bromide peptides. Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was not secreted by E. coli; molecular weight analysis of the partially purified recombinant enzyme suggested incomplete processing of the putative leader sequence.

Evidence that the intron open reading frame of the phage T4 td gene encodes a specific endonuclease.

The phage T4 thymidylate synthase (td) gene contains an intron open reading frame that encodes a 245-amino acid-long basic protein (Chu, F. K., Maley, G. F., West, D. K., Belfort, M., and Maley, F. (1986) Cell 45, 157-166). The open reading frame (Irf) has been cloned as a fusion protein behind a phage T7 promoter and overexpressed in Escherichia coli. The amplified Irf protein is associated with insoluble inclusion bodies and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis about 7 kDa smaller than expected. Data obtained from DNA sequencing, amino acid sequencing of the fusion protein, and carboxypeptidase Y digestion suggest that although the cloned gene is not altered and the protein is made from the expected start codon, it appears to terminate about 90 amino acids before the encoded stop codon. Proteolytic cleavage during or soon after synthesis appears to be responsible for the truncated Irf. The expressed protein is solubilized in guanidine HCl and renatured by dialysis against high salt. This partially purified preparation has been found to contain a DNA endonuclease activity specific for the td delta I gene, which contains a precise deletion of the intron.

RNA-protein interactions in 30S ribosomal subunits: folding and function of 16S rRNA.

Chemical probing methods have been used to "footprint" 16S ribosomal RNA (rRNA) at each step during the in vitro assembly of twenty 30S subunit ribosomal proteins. These experiments yield information about the location of each protein relative to the structure of 16S rRNA and provide the basis for derivation of a detailed model for the three-dimensional folding of 16S rRNA. Several lines of evidence suggest that protein-dependent conformational changes in 16S rRNA play an important part in the cooperativity of ribosome assembly and in fine-tuning of the conformation and dynamics of 16S rRNA in the 30S subunit.

Identification of Escherichia coli ribosomal proteins by an alternative two-dimensional electrophoresis system.

We have developed a two-dimensional gel electrophoretic system for the identification of Escherichia coli ribosomal proteins that involves the use of acid-urea in the first dimension and sodium dodecyl sulfate in the second dimension. This system has high sensitivity, resolution, and speed, and it is more convenient than others previously described. We have identified individual E. coli ribosomal proteins by this system.

Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA.

We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690-720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5'-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.

Probing the assembly of the 3' major domain of 16 S rRNA. Interactions involving ribosomal proteins S2, S3, S10, S13 and S14.

We have used rapid probing methods to follow the changes in reactivity of residues in 16 S rRNA to chemical and enzymatic probes as ribosomal proteins S2, S3, S10, S13 and S14 are assembled into 30 S subunits. Effects observed are confined to the 3' major domain of the RNA and comprise three general classes. (1) Monospecific effects, which are attributable to a single protein. Proteins S13 and S14 each affect the reactivities of different residues which are adjacent to regions previously found protected by S19. S10 effects are located in two separate regions of the domain, the 1120/1150 stem and the 1280 loop; both of these regions are near nucleotides previously found protected by S9. Both S2 and S3 protect different nucleotides between positions 1070 and 1112. In addition, S2 protects residues in the 1160/1170 stem-loop. (2) Co-operative effects, which include residues dependent on the simultaneous presence of both proteins S2 and S3 for their reactivities to appear similar to those observed in native 30 S subunits. (3) Polyspecific effects, where proteins S3 and S2 independently afford the same protection and enhancement pattern in three distal regions of the domain: the 960 stem-loop, the 1050/1200 stem and in the upper part of the domain (nucleotides 1070 to 1190). Proteins S14 and S10 also weakly affect the reactivities of several residues in these regions. We believe that several of the protected residues of the first class are likely sites for protein-RNA contact while the third class is indicative of conformational rearrangement in the RNA during assembly. These results, in combination with the results from our previous study of proteins S7, S9 and S19, are discussed in terms of the assembly, topography and involvement in ribosomal function of the 3' major domain.

Interaction of proteins S16, S17 and S20 with 16 S ribosomal RNA.

We have used rapid chemical probing methods to examine the effect of assembly of ribosomal proteins S16, S17 and S20 on the reactivity of individual residues of 16 S rRNA. Protein S17 strongly protects a compact region of the RNA between positions 245 and 281, a site previously assigned to binding of S20. Protein S20 also protects many of these same positions, albeit more weakly than S17. Strong S20-dependent protections are seen elsewhere in the 5' domain, most notably at positions 108, and in the 160-200 and 330 loop regions. Enenpectedly, S20 also causes protection of several bases in the 1430-1450 region, in the 3' minor domain. In the presence of the primary binding proteins S4, S8 and S20, we observe a variety of effects that result from assembly of the secondary binding protein S16. Most strongly protected are nucleotides around positions 50, 120, 300 to 330 and 360 in the 5' domain, and positions 606 to 630 in the central domain. In addition, numerous nucleotides in the 5' and central domains exhibit enhanced reactivity in response to S16. Interestingly, the strength of the S20-dependent effects in the 1430-1450 region is attenuated in the presence of S4 + S8 + S20, and restored in the presence of S4 + S8 + S20 + S16. Finally, the previously observed rearrangement of the 300 region stem-loop that occurs during assembly is shown to be an S16-dependent event. We discuss these findings with respect to assignment of RNA binding sites for these proteins, and in regard to the co-operativity of ribosome assembly.

Interaction of ribosomal proteins, S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA.

We have constructed complexes of ribosomal proteins S8, S15, S8 + S15 and S8 + S15 + S6 + S18 with 16 S ribosomal RNA, and probed the RNA moiety with a set of structure-specific chemical and enzymatic probes. Our results show the following effects of assembly of proteins on the reactivity of specific nucleotides in 16 S rRNA. (1) In agreement with earlier work, S8 protects nucleotides in and around the 588-606/632-651 stem from attack by chemical probes; this is supported by protection in and around these same regions from nucleases. In addition, we observe protection of positions 573-575, 583, 812, 858-861 and 865. Several S8-dependent enhancements of reactivity are found, indicating that assembly of this protein is accompanied by conformational changes in 16 S rRNA. These results imply that protein S8 influences a much larger region of the central domain than was previously suspected. (2) Protein S15 protects nucleotides in the 655-672/734-751 stem, in agreement with previous findings. We also find S15-dependent protection of nucleotides in the 724-730 region. Assembly of S15 causes several enhancements of reactivity, the most striking of which are found at G664, A665, G674, and A718. (3) The effects of proteins S6 and S18 are dependent on the simultaneous presence of both proteins, and on the presence of protein S15. S6 + S18-dependent protections are located in the 673-730 and 777-803 regions. We observed some variability in our results with these proteins, depending on the ratio of protein to RNA used, and in different trials using enzymatic probes, possibly due to the limited solubility of protein S18. Consistently reproducible was protection of nucleotides in the 664-676 and 715-729 regions. Among the latter are three of the nucleotides (G664, G674 and A718) that are strongly enhanced by assembly of protein S15. This result suggests that an S15-induced conformational change involving these nucleotides may play a role in the co-operative assembly of proteins S6 and S18.

Probing the assembly of the 3' major domain of 16 S ribosomal RNA. Quaternary interactions involving ribosomal proteins S7, S9 and S19.

We have studied the effect of assembly of ribosomal proteins S7, S9 and S19 on the accessibility and conformation of nucleotides in 16 S ribosomal RNA. Complexes formed between 16 S rRNA and S7, S7 + S9, S7 + S19 or S7 + S9 + S19 were subjected to a combination of chemical and enzymatic probes, whose sites of attack in 16 S rRNA were identified by primer extension. The results of this study show that: (1) Protein S7 affects the reactivity of an extensive region in the lower half of the 3' major domain. Inclusion of proteins S9 or S19 with S7 has generally little additional effect on S7-specific protection of the RNA. Clusters of nucleotides that are protected by protein S7 are localized in the 935-945 region, the 950/1230 stem, the 1250/1285 internal loop, and the 1350/1370 stem. (2) Addition of protein S9 in the presence of S7 causes several additional effects principally in two structurally distal regions. We observe strong S9-dependent protection of positions 1278 to 1283, and of several positions in the 1125/1145 internal loop. These findings suggest that interaction of protein S9 with 16 S rRNA results in a structure in which the 1125/1145 and 1280 regions are proximal to each other. (3) Most of the strong S19-dependent effects are clustered in the 950-1050 and 1210-1230 regions, which are joined by base-pairing in the 16 S rRNA secondary structure. The highly conserved 960-975 stemp-loop, which has been implicated in tRNA binding, appears to be destabilized in the presence of S19. (4) Protein S7 causes enhanced reactivity at several sites that become protected upon addition of S9 or S19. This suggests that S7-induced conformational changes in 16 S rRNA play a role in the co-operativity of assembly of the 3' major domain.