Role of cysteine residues in pseudouridine synthases of different families.

The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.     

Ribosomal RNA pseudouridines and pseudouridine synthases

Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui pseudouridines on the three-dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA-protein complex. No unique specific role for ribosomal pseudouridines has been identified. We propose that pseudouridine's function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.     

Functional importance of motif I of pseudouridine synthases: mutagenesis of aligned lysine and proline residues

On the basis of sequence alignments, the pseudouridine synthases were grouped into four families that share no statistically significant global sequence similarity, though some common sequence motifs were discovered [Koonin, E. V. (1996) Nucleic Acids. Res. 24, 2411-2415; Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762]. We have investigated the functional significance of these alignments by substituting the nearly invariant lysine and proline residues in Motif I of RluA and TruB, pseudouridine synthases belonging to different families. Contrary to our expectations, the altered enzymes display only very mild kinetic impairment. Substitution of the aligned lysine and proline residues does, however, reduce structural stability, consistent with a temperature sensitive phenotype that results from substitution of the cognate proline residue in Cbf5p, a yeast homologue of TruB [Zerbarjadian, Y., King, T., Fournier, M. J., Clarke, L., and Carbon, J. (1999) Mol. Cell. Biol. 19, 7461-7472]. Together, our data support a functional role for Motif I, as predicted by sequence alignments, though the effect of substituting the highly conserved residues was milder than we anticipated. By extrapolation, our findings also support the assignment of pseudouridine synthase function to certain physiologically important eukaryotic proteins that contain Motif I, including the human protein dyskerin, alteration of which leads to the disease dyskeratosis congenita.     

Degradation of aspartic acid and asparagine residues in human growth hormone-releasing factor.

Products of the degradation of human growth hormone-releasing factor (GRF) in aqueous solutions (15-200 microM) have been isolated and fully characterized. The cleavage product, GRF(4-44)-NH2, and the isomerization product, [beta-Asp3]GRF(1-44)-NH2, from the degradation of GRF(1-44)-NH2 in acidic solution and the corresponding products, GRF(4-29)-NH2 and [beta-Asp3]GRF(1-29)-NH2, from the degradation of GRF(1-29)-NH2 have been isolated and characterized. The products, [beta-Asp8]GRF(1-44)-NH2 and [Asp8]GRF(1-44)-NH2, from the deamidation of GRF(1-44)-NH2 at pH 8.0 and the corresponding products, [beta-Asp8]GRF(1-29)-NH2 and [Asp8]GRF(1-29)-NH2, from the deamidation of GRF(1-29)-NH2 have been isolated and characterized. All the degradation products of GRF(1-44)-NH2 and GRF(1-29)-NH2 were evaluated for biological activity and found to have much lower in vitro potencies than the parent peptides. Degradation occurs at Asp3 and Asn8 and the kinetics of these various transformations versus pH and temperature have been studied. GRF is most stable at pH 4-5. At pH below the pKa of the Asp3 side-chain (pH less than 4), cleavage at Asp3-Ala4 is the major route of degradation. For pH greater than 4, isomerization of Asp3 to beta-Asp3 (iso-Asp3) predominates. The rates of cleavage and isomerization are simple first order and vary with pH, independent of buffer concentration, such that the protonated (COOH) form of Asp3 undergoes cleavage while the ionized (COO-) form isomerizes. The more rapid deamidation of Asn8 to generate beta-Asp8 and Asp8 in about a 4:1 ratio, presumably via a cyclic imide intermediate, occurs at pH greater than or equal to 5 and is general base-catalyzed. Evidence was also obtained for direct hydrolysis of protonated Asn8 in GRF(1-29)-NH2 at pH less than or equal to 2 to give exclusively [Asp8]GRF(1-29)-NH2. The deamidation of Asn8 in GRF(1-29)-NH2 at pH 8.0, 22-55 degrees C, is relatively insensitive to temperature for T less than 37 degrees C, possibly due to conformational constraints. Asp25 and Asn35 are sterically, conformationally, or otherwise hindered with respect to these changes as no degradation at these sites was observed under the conditions employed.     

Modification of aspartic acid residues to induce trypsin cleavage

1,2-Diaminoethane and diaminomethane were coupled to aspartic acid residues in small peptides by means of a water-soluble carbodiimide. The resulting modified side chains sufficiently resembled lysine for trypsin to cleave the peptides. Similar modification of glutamic acid residues in peptides gave little or no susceptibility to trypsin.     

Two consecutive aspartic acid residues conferring herbicide resistance in tobacco acetohydroxy acid synthase

Acetohydroxy acid synthase (AHAS) catalyzes the first common step in the biosynthesis pathway of the branch chain amino acids in plants and microorganisms. A great deal of interest has been focused on AHAS since it was identified as the target of several classes of potent herbicides. In an effort to produce a mutant usable in the development of an herbicide-resistant transgenic plant, two consecutive aspartic acid residues, which are very likely positioned next to the enzyme-bound herbicide sulfonylurea as the homologous residues in AHAS from yeast, were selected for this study. Four single-point mutants and two double mutants were constructed, and designated D374A, D374E, D375A, D375E, D374A/D375A, and D374E/D375E. All mutants were active, but the D374A mutant exhibited substrate inhibition at high concentrations. The D374E mutant also evidenced a profound reduction with regard to catalytic efficiency. The mutation of D375A increased the K(m) value for pyruvate nearly 10-fold. In contrast, the D375E mutant reduced this value by more than 3-fold. The double mutants exhibited synergistic reduction in catalytic efficiencies. All mutants constructed in this study proved to be strongly resistant to the herbicide sulfonylurea Londax. The double mutants and the mutants with the D375 residue were also strongly cross-resistant to the herbicide triazolopyrimidine TP. However, only the D374A mutant proved to be strongly resistant to imidazolinone Cadre. The data presented here indicate that the two residues, D374 and D375, are located at a common binding site for the herbicides sulfonylurea and triazolopyrimidine. D375E may be a valuable mutant for the development of herbicide-resistant transgenic plants.     

Functional roles of aspartic acid residues at the cytoplasmic surface of bacteriorhodopsin.

The functions of the four aspartic acid residues in interhelical loops at the cytoplasmic surface of bacteriorhodopsin, Asp-36, Asp-38, Asp-102, and Asp-104, were investigated by studying single and multiple aspartic acid to asparagine mutants. The same mutants were examined also with the additional D96N residue replacement. The kinetics of the M and N intermediates of the photochemical cycles of these recombinant proteins were affected only in a minor, although self-consistent, way. When residue 38 is an aspartate and anionic, it makes the internal proton exchange between the retinal Schiff base and Asp-96 about 3 times more rapid, and events associated with the reisomerization of retinal to all-trans about 3 times slower. Asp-36 has the opposite effect on these processes, but to a smaller extent. Asp-102 and Asp-104 have even less or none of these effects. Of the four aspartates, only Asp-36 could play a direct role in proton uptake at the cytoplasmic surface. In the 13 bacterioopsin sequences now available, only this surface aspartate is conserved.     

Critical residues in D-amino acid oxidase. A pulse-radiolysis and inactivation study.

The enzyme D-amino acid oxidase and its apoenzyme have been irradiated at pH 5.5--10 under conditions designed to assess the inactivating effect of OH radicals and the selective free radicals Br2- and (SCN)2-. Near neutral pH, removal of the coenzyme FAD from the enzyme results in greater inactivation by selective free-radical attack. From pulse-radiolysis spectra, this increase is associated with attack on tyrosine and tryptophan residues in the protein. A large increase in inactivation of both the haloenzyme and apoenzyme by selective free-radical attack is seen with increasing alkalinity. This is consistent with attack on tyrosine being of major importance.     

Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli.

There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, respectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseudouridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(Ile1) and tRNA(Asp). Deletion of each of these synthase genes individually had no effect on exponential growth in rich media at 25 degrees C, 37 degrees C, or 42 degrees C. A strain lacking RluB and RluF also showed no growth defect under these conditions. Mutation of a conserved aspartate in a common sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, also caused a loss of in vivo activity in all four of the synthases studied in this work.     

Modification of human U4 RNA requires U6 RNA and multiple pseudouridine synthases.

Small nuclear RNAs (snRNA), cofactors in the splicing of pre-mRNA, are highly modified. In this report the modification of human U4 RNA was studied using cell extracts and in vitro synthesized, and therefore unmodified, U4 RNA. The formation of pseudouridine (Psi) at positions 4, 72 and 79 in U4 RNA was dependent on an RNA-containing cofactor, since the activities in the extracts were micrococcal nuclease (MN) sensitive. Extracts were fractionated on glycerol gradients and there was a broad peak of reconstitution activity centered at 14 S. Reconstitution was not due to additional enzymatic activity, since the peak fraction was MN sensitive. Oligodeoxynucleotide-mediated RNase H digestion of U6 RNA in the extracts inhibited formation of Psi in U4 RNA. From glycerol gradient analysis we determined that exogenously added U4 RNA that is associated with U6 RNA (sedimentation velocity 16 S) was significantly higher in Psi content than U4 RNA not associated with U6 RNA (8 S). Competitive inhibitors of Psi synthases, 5-fluorouridine-containing (5-FU) wild-type and mutant U4 RNAs, were used to investigate formation of Psi in U4 RNA. Deletions and point mutations in these 5-FU-containing U4 RNAs affected their ability to inhibit Psi synthase in vitro. With the aid of these potent inhibitors it was determined that at least two separate activities modify the uridines at these positions.     

Factors contributing to decreased protein stability when aspartic acid residues are in beta-sheet regions

Asp residues are significantly under represented in beta-sheet regions of proteins, especially in the middle of beta-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a beta-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V(L)) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a beta-strand, and that of Q89D, located in the middle of a beta-strand, reduced the stability of the parent immunoglobulin V(L) domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V(L)-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V(L)-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V(L)-Len domain. The structures of mutants V(L)-Len Q38D and V(L)-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these Q-->D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a beta-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a beta-strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo.     

Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli: catalytic role of conserved aspartic acid residues

Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His6) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.     

Function of aspartic acid residues in optimum pH control of l-arabinose isomerase from Lactobacillus fermentum

l-Arabinose isomerase (l-AI) catalyzes the isomerization of l-arabinose to l-ribulose and d-galactose to d-tagatose. Most reported l-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial d-tagatose production. Lactobacillus fermentum l-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for d-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, d-galactose was isomerized into d-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other l-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of l-AIs that can be modified for desired optimum pH and better pH stability, which are useful in d-tagatose bioproduction.     

Selective modification of cytidine, uridine, guanosine and pseudouridine residues in Escherichia coli leucine transfer ribonucleic acid

The specificity of methoxyamine for the cytidine residues in an Escherichia coli leuoine transfer RNA (tRNA₁^leu is described in detail. Of the six non-hydrogen-bonded cytidine residues in the clover-leaf model of this tRNA, four are very reactive (C-35, 53, 85 and 86) and two are unreactive (C-67 and 79).The specificity of l-cyclohexyl-3-[2-morpholino-(4)-ethyl]carbodiimide methotosylate for the uridine, guanosine and pseudouridine residues in the leucine tRNA was also investigated. The carbodiimide completely modified four uridine residues (U-33, 34, 50 and 51) and partially modified G-37 and Ψ-39. For technical reasons, the sites of partial modification in loop I of the tRNA were difficult to establish. There was no modification of base residues in loop IV nor of U-59 at the base of stem e of the tRNA.The modification patterns described for the leucine tRNA are compared with those observed for the E. coli initiator tRNA₁^met and su⁺_III tyrosine tRNA. Several general conclusions regarding tRNA conformation are made. In particular, the evidence supporting a diversity of anticodon loop structures amongst tRNAs is discussed.