Critical aspartic acid residues in pseudouridine synthases.

The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.     

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.     

Leishmania donovani methionine adenosyltransferase. Role of cysteine residues in the recombinant enzyme.

Methionine adenosyltransferase (MAT, EC 2.5.1.6)-mediated synthesis of S-adenosylmethionine (AdoMet) is a two-step process consisting of the formation of AdoMet and the subsequent cleavage of the tripolyphosphate (PPPi) molecule, a reaction induced, in turn, by AdoMet. The fact that the two activities, AdoMet synthesis and tripolyphosphate hydrolysis, can be measured separately is particularly useful when the site-directed mutagenesis approach is used to determine the functional role of the amino acid residues involved in each. The present report describes the cloning and subsequent functional refolding, using a bacterial expression system, of the MAT gene (GenBank accession number AF179714) from Leishmania donovani, the etiological agent of visceral leishmaniasis. The absolute need to include a sulfhydryl-protection reagent in the refolding buffer for this protein, in conjunction with the rapid inactivation of the functionally refolded protein by N-ethylmaleimide, suggests the presence of crucial cysteine residues in the primary structure of the MAT protein. The seven cysteines in L. donovani MAT were mutated to their isosterical amino acid, serine. The C22S, C44S, C92S and C305S mutants showed a drastic loss of AdoMet synthesis activity compared to the wild type, and the C33S and C47S mutants retained a mere 12% of wild-type MAT activity. C106S mutant activity and kinetics remained unchanged with respect to the wild-type. Cysteine substitutions also modified PPPi cleavage and AdoMet induction. The C22S, C44S and C305S mutants lacked in tripolyphosphatase activity altogether, whereas C33S, C47S and C92S retained low but detectable activity. The behavior of the C92S mutant was notable: its inability to synthesize AdoMet combined with its retention of tripolyphosphatase activity appear to be indicative of the specific involvement of the respective residue in the first step of the MAT reaction.     

Difference between uracilylalanine synthases and cysteine synthases in Pisum sativum

Purification of cysteine synthase from seedlings of pea (Pisum sativum) reveals the presence of three forms of this enzyme, separated by chromatography on DEAE-Sephadex A-50, and also differences between the cysteine- and uracilylalanine-synthases. Isoenzymes A and B of pea cysteine synthase were purified about 1200-fold and had specific activities of 933 U/mg protein and 892 U/mg protein, respectively. Both isoenzymes were found to have the same M_r (52 000) and to dissociate into two identical subunits (M_r 26 000). The K_m value of isoenzyme A is 2.1 mM for O-acetyl-l-serine (OAS) and 36 μM for sulphide, while that of isoenzyme B is 2.3 mM for OAS and 38 μM for sulphide. None of the three isoenzymes from pea seedlings catalyses the formation of the uracilylalanines l-willardiine and l-isowillardiine from OAS and uracil, although isoenzyme A catalyses the formation of β-cyano-l-alanine, and isoenzyme C catalyses the formation of l-quisqualic acid and l-mimosine. Other significant differences occur in the substrate specificity of the three isoenzymes. Several properties, including the amino acid composition of the purified cysteine synthase isoenzymes, are also described.     

Role of cysteine residues in tryptophanase for monovalent cation-induced activation

We cloned and sequenced the tryptophanase structural gene of Escherichia coli B/1t7-A strain. The results indicate that tryptophanase proteins of E. coli B/1t7-A and K-12 are identical. When cysteine residues in tryptophanase were chemically modified with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), the stabilizing effect of the active cations such as K+ and NH4+ was abolished. In consideration of our previous results that Cys-298 was selectively modified by SH reagents [Honda T. et al. (1986) J. Chromatogr. 371, 353-360], Cys-298 seems to have a close relation to the expression of the effect of monovalent cations. Fluorescence decay measurement of the holoenzyme revealed that the fluorescence lifetime derived from the coenzyme, pyridoxal 5'-phosphate (PLP), was dependent on coexisting monovalent cations, whereas that of the tryptophyl residue was not, in either the apo- or the holoenzyme preparation. The results of the synchrotron small-angle X-ray scattering measurements showed that radii of gyration which reflect the size and shape of the enzyme were constant at around 38 A irrespective of the presence or absence of the K+ ion. These results suggest that the monovalent cations interact specifically with the PLP-binding site, and that the conformational change of enzyme protein caused by the monovalent-cation binding is limited to a small range. The above results are compatible with the possibility that Cys-298 is involved in the formation of "monovalent cation binding site" in the holoenzyme.     

Role of cysteine residues in the lac permease of Escherichia coli

Oligonucleotide-directed, site-specific mutagenesis has been utilized to replace cysteine residues 117, 333, or 353 and 355 with serine in the lac permease of Escherichia coli. Replacement of Cys-117 or Cys-333 has no significant effect on permease activity, while permease with serine residues in place of Cys-353 and Cys-355 has about 50% of wild-type permease activity. The results provide a clear demonstration that cysteine residues at positions 117, 333, 353, and 355 are not obligatory for lactose/H+ symport. When considered in conjunction with previous findings, the results indicate that, of the eight cysteine residues in the lac permease, only Cys-154 is important for lactose transport. As discussed, the conclusion has important implications for the hypothesis that sulfhydryl-disulfide interconversion plays an important role in the symport mechanism.     

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.     

Role of cysteine residues in ribonuclease H from Escherichia coli. Site-directed mutagenesis and chemical modification.

The role of the three cysteine residues at positions 13, 63 and 133 in Escherichia coli RNAase H, an enzyme that is sensitive to N-ethylmaleimide [Berkower, Leis & Hurwitz (1973) J. Biol. Chem. 248, 5914-5921], was examined by using both site-directed mutagenesis and chemical modification. Novel aspects that were found are as follows. First, none of the cysteine residues is required for activity. Secondly, chemical modification of either Cys-13 or Cys-133 with thiol-blocking reagents inactivates the enzyme, but that of Cys-63 does not. Thus the sensitivity of E. coli RNAase H to N-ethylmaleimide arises not from blocking of the thiol group but from steric hindrance by the modifying group incorporated at either Cys-13 or Cys-133.     

Four families of folate-independent methionine synthases

Although most organisms synthesize methionine from homocysteine and methyl folates, some have “core” methionine synthases that lack folate-binding domains and use other methyl donors. In vitro, the characterized core synthases use methylcobalamin as a methyl donor, but in vivo, they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.     

Neutral sphingomyelinase 2 is palmitoylated on multiple cysteine residues. Role of palmitoylation in subcellular localization

The neutral sphingomyelinases (nSMases) are considered major candidates for mediating the stress-induced production of ceramide. nSMase2, which has two hydrophobic segments near the NH(2)-terminal region, has been reported to be located at the plasma membrane and play important roles in ceramide-mediated signaling. In this study, we found that nSMase2 is palmitoylated on multiple cysteine residues via thioester bonds. Site-directed mutagenesis of cysteine residues to alanine indicated that two cysteine clusters of the enzyme are multiply palmitoylated; one cluster is located between the two hydrophobic segments, and the second one is located in the middle of the catalytic region of the protein. When overexpressed in the confluent phase of MCF-7 cells, wild-type nSMase2 was strictly localized in the plasma membranes, and the cysteine mutants of each palmitoylated cysteine cluster were seen not only at the plasma membrane but also in some punctate structures. Furthermore, mutation of all potential palmitoylation sites resulted in a dramatic reduction in the plasma membrane distribution and an increase in the punctate structures. The palmitoylation-deficient mutant was directed to lysosomes and rapidly degraded. Palmitoylation had no effect on enzyme activity but affected membrane-association properties of the protein. Finally, the catalytic region of nSMase2 where palmitoylation occurs was found to be localized at the inner leaflet of the plasma membrane. In summary, the results from this study reveal for the first time the palmitoylation of nSMase2 via thioester bonds and its importance in the subcellular localization and stability of this protein.     

Role of cysteine residues in thermal inactivation of fungal Cel6A cellobiohydrolases

Numerous protein engineering studies have focused on increasing the thermostability of fungal cellulases to improve production of fuels and chemicals from lignocellulosic feedstocks. However, the engineered enzymes still undergo thermal inactivation at temperatures well below the inactivation temperatures of hyperthermophilic cellulases. In this report, we investigated the role of free cysteines in the thermal inactivation of wild-type and engineered fungal family 6 cellobiohydrolases (Cel6A). The mechanism of thermal inactivation of Cel6A is consistent with disulfide bond degradation and thiol–disulfide exchange. Circular dichroism spectroscopy revealed that a thermostable variant lacking free cysteines refolds to a native-like structure and retains activity after heat treatment over the pH range 5–9. Whereas conserved disulfide bonds are essential for retaining activity after heat treatment, free cysteines contribute to irreversible thermal inactivation in engineered thermostable Cel6A as well as Cel6A from Hypocrea jecorina and Humicola insolens.     

Strong hydrophobic nature of cysteine residues in proteins.

The differences between disulfide-bonding cystine (Cys_SS) and free cysteine (Cys_SH) residues were examined by analyzing the statistical distribution of both types of residue in proteins of known structure. Surprisingly, Cys_SH residues display stronger hydrophobicity than Cys_SS residues. A detailed survey of atoms which come into contact with the sulfhydryl group (sulfur atom) of Cys_SH revealed those atoms are essentially the same in number and variety as those of the methyl group of isoleucine, but are quite different to those of the hydroxyl group of serine. Moreover, the relationships among amino acids were also determined using the 3D-profile table of known protein structures. Cys_SH was located in the hydrophobic cluster, along with residues such as Met, Trp and Tyr, and was clearly separated from Ser and Thr in the polar cluster. These results imply that free cysteines behave as strongly hydrophobic, and not hydrophilic, residues in proteins.     

Role of cysteine residues in 4-oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1

4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wild- and mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the K(m)-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl(2); and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.     

The streptococcal hyaluronan synthases are inhibited by sulfhydryl-modifying reagents, but conserved cysteine residues are not essential for enzyme function.

Hyaluronan (HA) synthase (HAS) is a membrane-bound enzyme that utilizes UDP-glucuronic acid (GlcUA) and UDP-GlcNAc to synthesize HA. The HAS from Streptococcus pyogenes (spHAS, 419 amino acids) contains six Cys residues, whereas the enzyme from Streptococcus equisimilis (seHAS, 417 amino acids) contains four Cys residues. These Cys residues of seHAS are highly conserved in all Class I HAS family members. Here we investigated the structural and functional roles of these conserved cysteines in seHAS by using site-directed mutagenesis and sensitivity to sulfhydryl modifying reagents. Both seHAS and spHAS were inhibited by sulfhydryl reagents such as N-ethylmaleimide (NEM) and iodoacetamide in a dose-dependent and time-dependent manner. These inhibition curves were biphasic, indicating the presence of sensitive and insensitive components. After treatment of seHAS with NEM, the V(max) value was decreased approximately 50%, and the K(m) values changed only slightly. All the Cys-to-Ala mutants of seHAS were partially active. The least active single (C226A), double (C226A,C262A), or triple (C226A,C262A,C367A) Cys mutants retained 24, 3.2, and 1.4% activity, respectively, compared with wild-type enzyme. Surprisingly, the V(max) value of the seHAS(cys-null) mutant was approximately 17% of wild-type, although the K(m) values for both substrates were increased 3-6-fold. Cys residues, therefore, are not involved in a critical interaction necessary for either substrate binding or catalysis. However, the distribution of HA products was shifted to a smaller size in approximately 25% of the seHAS Cys mutants, particularly the triple mutants. Mass spectroscopic analysis of wild-type and Cys-null seHAS as well as the labeling of all double Cys-to-Ala mutants with [(14)C]NEM demonstrated that seHAS contains no disulfide bonds. We conclude that the four Cys residues in seHAS are not directly involved in catalysis, but that one or more of these Cys residues are located in or near substrate binding or glycosyltransferase active sites, so that their modification hinders the functions of HAS.     

Role of cysteine residues in structural stability and function of a transmembrane helix bundle

To study the structural and functional roles of the cysteine residues at positions 36, 41, and 46 in the transmembrane domain of phospholamban (PLB), we have used Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis to prepare alpha-amino-n-butyric acid (Abu)-PLB, the analogue in which all three cysteine residues are replaced by Abu. Whereas previous studies have shown that replacement of the three Cys residues by Ala (producing Ala-PLB) greatly destabilizes the pentameric structure, we hypothesized that replacement of Cys with Abu, which is isosteric to Cys, might preserve the pentameric stability. Therefore, we compared the oligomeric structure (from SDS-polyacrylamide gel electrophoresis) and function (inhibition of the Ca-ATPase in reconstituted membranes) of Abu-PLB with those of synthetic wild-type PLB and Ala-PLB. Molecular modeling provides structural and energetic insight into the different oligomeric stabilities of these molecules. We conclude that 1) the Cys residues of PLB are not necessary for pentamer formation or inhibitory function; 2) the steric properties of cysteine residues in the PLB transmembrane domain contribute substantially to pentameric stability, whereas the polar or chemical properties of the sulfhydryl group play only a minor role; 3) the functional potency of these PLB variants does not correlate with oligomeric stability; and 4) acetylation of the N-terminal methionine has neither a functional nor a structural effect in full-length PLB.     

Adjacent cysteine residues as a redox switch.

Oxidation of adjacent cysteine residues into a cystine forms a strained eight-membered ring. This motif was tested as the basis for an enzyme with an artificial redox switch. Adjacent cysteine residues were introduced into two different structural contexts in ribonuclease A (RNase A) by site-directed mutagenesis to produce the A5C/A6C and S15C/S16C variants. Ala5 and Ala6 are located in an alpha-helix, whereas Ser15 and Ser16 are located in a surface loop. Only A5C/A6C RNase A had the desired property. The catalytic activity of this variant decreases by 70% upon oxidation. The new disulfide bond also decreases the conformational stability of the A5C/A6C variant. Reduction with dithiothreitol restores full enzymatic activity. Thus, the insertion of adjacent cysteine residues in a proper context can be used to modulate enzymatic activity.