Identification of a bifunctional enzyme MnmC involved in the biosynthesis of a hypermodified uridine in the wobble position of tRNA

The gene encoding the bifunctional enzyme MnmC that catalyzes the two last steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm5s2U) in tRNA has been previously mapped at about 50 min on the Escherichia coli K12 chromosome, but to date the identity of the corresponding enzyme has not been correlated with any of the known open reading frames (ORFs). Using the protein fold-recognition approach, we predicted that the 74-kDa product of the yfcK ORF located at 52.6 min and annotated as "putative peptidase" comprises a methyltransferase domain and a FAD-dependent oxidoreductase domain. We have cloned, expressed, and purified the YfcK protein and demonstrated that it catalyzes the formation of mnm5s2U in tRNA. Thus, we suggest to rename YfcK as MnmC.     

Folding and domain-domain interactions of the chaperone PapD measured by 19F NMR

The folding of the two-domain bacterial chaperone PapD has been studied to develop an understanding of the relationship between individual domain folding and the formation of domain-domain interactions. PapD contains six phenylalanine residues, four in the N-terminal domain and two in the C-terminal domain. To examine the folding properties of PapD, the protein was both uniformly and site-specifically labeled with p-fluoro-phenylalanine ((19)F-Phe) for (19)F NMR studies, in conjunction with those of circular dichroism and fluorescence. In equilibrium denaturation experiments monitored by (19)F NMR, the loss of (19)F-Phe native intensity for both the N- and C-terminal domains shows the same dependence on urea concentration. For the N-terminal domain the loss of native intensity is mirrored by the appearance of separate denatured resonances. For the C-terminal domain, which contains residues Phe 168 and Phe 205, intermediate as well as denatured resonances appear. These intermediate resonances persist at denaturant concentrations well beyond the loss of native resonance intensity and appear in kinetic refolding (19)F NMR experiments. In double-jump (19)F NMR experiments in which proline isomerization does not affect the refolding kinetics, the formation of domain-domain interactions is fast if the protein is denatured for only a short time. However, with increasing time of denaturation the native intensities of the N- and C-terminal domains decrease, and the denatured resonances of the N-terminal domain and the intermediate resonances of the C-terminal domain accumulate. The rate of loss of the N-terminal domain resonances is consistent with a cis to trans isomerization process, indicating that from an equilibrium denatured state the slow refolding of PapD is due to the trans to cis isomerization of one or both of the N-terminal cis proline residues. The data indicate that both the N- and C-terminal domains must fold into a native conformation prior to the formation of domain-domain interactions.     

The output of the tRNA modification pathways controlled by the Escherichia coli MnmEG and MnmC enzymes depends on the growth conditions and the tRNA species

In Escherichia coli, the MnmEG complex modifies transfer RNAs (tRNAs) decoding NNA/NNG codons. MnmEG catalyzes two different modification reactions, which add an aminomethyl (nm) or carboxymethylaminomethyl (cmnm) group to position 5 of the anticodon wobble uridine using ammonium or glycine, respectively. In and , however, cmnm⁵ appears as the final modification, whereas in the remaining tRNAs, the MnmEG products are converted into 5-methylaminomethyl (mnm⁵) through the two-domain, bi-functional enzyme MnmC. MnmC(o) transforms cmnm⁵ into nm⁵, whereas MnmC(m) converts nm⁵ into mnm⁵, thus producing an atypical network of modification pathways. We investigate the activities and tRNA specificity of MnmEG and the MnmC domains, the ability of tRNAs to follow the ammonium or glycine pathway and the effect of mnmC mutations on growth. We demonstrate that the two MnmC domains function independently of each other and that and are substrates for MnmC(m), but not MnmC(o). Synthesis of mnm⁵s²U by MnmEG-MnmC in vivo avoids build-up of intermediates in . We also show that MnmEG can modify all the tRNAs via the ammonium pathway. Strikingly, the net output of the MnmEG pathways in vivo depends on growth conditions and tRNA species. Loss of any MnmC activity has a biological cost under specific conditions.     

Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain.

Mammalian DNA (cytosine-5) methyltransferase contains a C-terminal domain that is closely related to bacterial cytosine-5 restriction methyltransferase. This methyltransferase domain is linked to a large N-terminal domain. It is shown here that the N-terminal domain contains a Zn binding site and that the N- and C-terminal domains can be separated by cleavage with trypsin or Staphylococcus aureus protease V8; the protease V8 cleavage site was determined by Edman degradation to lie 10 residues C-terminal of the run of alternating lysyl and glycyl residues which joins the two domains and six residues N-terminal of the first sequence motif conserved between the mammalian and bacterial cytosine methyltransferases. While the intact enzyme had little activity on unmethylated DNA substrates, cleavage between the domains caused a large stimulation of the initial velocity of methylation of unmethylated DNA without substantial change in the rate of methylation of hemimethylated DNA. These findings indicate that the N-terminal domain of DNA methyltransferase ensures the clonal propagation of methylation patterns through inhibition of the de novo activity of the C-terminal domain. Mammalian DNA methyltransferase is likely to have arisen via fusion of a prokaryotic-like restriction methyltransferase and an unrelated DNA binding protein. Stimulation of the de novo activity of DNA methyltransferase by proteolytic cleavage in vivo may contribute to the process of ectopic methylation observed in the DNA of aging animals, tumors and in lines of cultured cells.     

Association of the N- and C-terminal domains of phospholipase D. Contribution of the conserved HKD motifs to the interaction and the requirement of the association for Ser/Thr phosphorylation of the enzyme

Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)(4)D, denoted HKD. rPLD1 contains two HKD domains, located in the N- and C-terminal regions. The integrity of the two HKD domains is essential for enzymatic activity. Our previous studies showed that the N-terminal half of rPLD1 containing one HKD motif can associate with the C-terminal half containing the other HKD domain to reconstruct wild type PLD activity (Xie, Z., Ho, W.-T. and Exton, J. H. (1998) J. Biol. Chem. 273, 34679-34682). In the present study, we have shown by mutagenesis that conserved amino acids in the HKD domains are important for both the catalytic activity and the association between the two halves of rPLD1. Furthermore, we found that rPLD1 could be modified by Ser/Thr phosphorylation. The modification occurred at the N-terminal half of the enzyme, however, the association of the N-terminal domain with the C-terminal domain was required for the modification. The phosphorylation of the enzyme was not required for its catalytic activity or response to PKCalpha and small G proteins in vitro, although the phosphorylated form of rPLD1 was localized exclusively in the crude membrane fraction. In addition, we found that the individually expressed N- and C-terminal fragments did not interact when mixed in vitro and were unable to reconstruct PLD activity under these conditions. It is concluded that the association of the N- and C-terminal halves of rPLD1 requires their co-expression in vivo and depends on conserved residues in the HKD domains. The association is also required for Ser/Thr phosphorylation of the enzyme.     

Quaternary structure changes in a second Per-Arnt-Sim domain mediate intramolecular redox signal relay in the NifL regulatory protein

Per-Arnt-Sim (PAS) domains play a critical role in signal transduction in multidomain proteins by sensing diverse environmental signals and regulating the activity of output domains. Multiple PAS domains are often found within a single protein. The NifL regulatory protein from Azotobacter vinelandii contains tandem PAS domains, the most N-terminal of which, PAS1, contains a FAD cofactor and is responsible for redox sensing, whereas the second PAS domain, PAS2, has no apparent cofactor and its function is unknown. Amino acid substitutions in PAS2 were identified that either lock NifL in a form that constitutively inhibits NifA or that fail to respond to the redox status, suggesting that PAS2 plays a pivotal role in transducing the redox signal from PAS1 to the C-terminal output domains. The isolated PAS2 domain is a homodimer in solution and the subunits are in rapid exchange. PAS2 dimerization is maintained in the redox signal transduction mutants, but is inhibited by substitutions in PAS2 that lock NifL in the inhibitory conformer. Our results support a model for signal transduction in NifL, whereby redox-dependent conformational changes in PAS1 are relayed to the C-terminal domains via changes in the quaternary structure of the PAS2 domain.     

Deletion mutagenesis of human cystathionine beta-synthase. Impact on activity,oligomeric status,and S-adenosylmethionine regulation

Cystathionine beta-synthase is a tetrameric hemeprotein that catalyzes the pyridoxal 5'-phosphate-dependent condensation of serine and homocysteine to cystathionine. We have used deletion mutagenesis of both the N and C termini to investigate the functional organization of the catalytic and regulatory regions of this enzyme. Western blot analysis of these mutants expressed in Escherichia coli indicated that residues 497-543 are involved in tetramer formation. Deletion of the 70 N-terminal residues resulted in a heme-free protein retaining 20% of wild type activity. Additional deletion of 151 C-terminal residues from this mutant resulted in an inactive enzyme. Expression of this double-deletion mutant as a glutathione S-transferase fusion protein generated catalytically active protein (15% of wild type activity) that was unaffected by subsequent removal of the fusion partner. The function of the N-terminal region appears to be primarily steric in nature and involved in the correct folding of the enzyme. The C-terminal region of human cystathionine beta-synthase contains two hydrophobic motifs designated "CBS domains." Partial deletion of the most C-terminal of these domains decreased activity and caused enzyme aggregation and instability. Removal of both of these domains resulted in stable constitutively activated enzyme. Deletion of as few as 8 C-terminal residues increased enzyme activity and abolished any further activation by S-adenosylmethionine indicating that the autoinhibitory role of the C-terminal region is not exclusively a function of the CBS domains.     

Crystal structure and anion binding in the prokaryotic hydrogenase maturation factor HypF acylphosphatase-like domain

[NiFe]-hydrogenases require a set of complementary and regulatory proteins for correct folding and maturation processes. One of the essential regulatory proteins, HypF (82kDa) contains a N-terminal acylphosphatase (ACT)-like domain, a sequence motif shared with enzymes catalyzing O-carbamoylation, and two zinc finger motifs similar to those found in the DnaJ chaperone. The HypF acylphosphatase domain is thought to support the conversion of carbamoylphosphate into CO and CN(-), promoting coordination of these ligands to the hydrogenase metal cluster. It has been shown recently that the HypF N-terminal domain can aggregate in vitro to yield fibrils matching those formed by proteins linked to amyloid diseases. The 1.27A resolution HypF acylphosphatase domain crystal structure (residues 1-91; R-factor 13.1%) shows a domain fold of betaalphabetabetaalphabeta topology, as observed in mammalian acylphosphatases specifically catalyzing the hydrolysis of the carboxyl-phosphate bonds in acylphosphates. The HypF N-terminal domain can be assigned to the ferredoxin structural superfamily, to which RNA-binding domains of small nuclear ribonucleoproteins and some metallochaperone proteins belong. Additionally, the HypF N-terminal domain displays an intriguing structural relationship to the recently discovered ACT domains. The structures of different HypF acylphosphatase domain complexes show a phosphate binding cradle comparable to the P-loop observed in unrelated phosphatase families. On the basis of the catalytic mechanism proposed for acylphosphatases, whereby residues Arg23 and Asn41 would support substrate orientation and the nucleophilic attack of a water molecule on the phosphate group, fine structural features of the HypF N-terminal domain putative active site region may account for the lack of acylphosphatase activity observed for the expressed domain. The crystallographic analyses here reported were undertaken to shed light on the molecular bases of inactivity, folding, misfolding and aggregation of the HypF N-terminal acylphosphatase domain.     

Effect of modified nucleotides on Escherichia coli tRNAGlu structure and on its aminoacylation by glutamyl-tRNA synthetase. Predominant and distinct roles of the mnm5 and s2 modifications of U34.

Overproducing Escherichia coli tRNAGlu in its homologous host results in the presence of several distinctly modified forms of this molecule that we name modivariants. The predominant tRNAGlu modivariant in wild-type E. coli contains five modified nucleosides: Psi13, mnm5s2U34, m2A37, T54 and Psi55. Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Psi, or the presence of s2U34, or the lack of A37 methylation combined with either s2U34 or U34. Chemical probing reveals that the anticodon loop of the predominant modivariant is less reactive to the probes than that of the four others. Furthermore, the modivariant with neither mnm5s2U34 nor m2A37 has additional perturbations in the D- and T-arms and in the variable region. The lack of a 2-thio group in nucleoside 34, which is mnm5s2U in the predominant tRNAGlu modivariant, decreases by 520-fold the specificity of E. coli glutamyl-tRNA synthetase for tRNAGlu in the aminoacylation reaction, showing that this thio group is the identity element in the modified wobble nucleotide of E. coli tRNAGlu. The modified nucleosides content also influences the recognition of ATP and glutamate by this enzyme, and in this case also, the predominant modivariant is the one that allows the best specificity for these two substrates. These structural and kinetic properties of tRNAGlu modivariants indicate that the modification system of tRNAGlu optimizes the stability of tRNAGlu and its action as cofactor of the glutamyl-tRNA synthetase for the recognition of glutamate and ATP.     

Biochemical and molecular characterization of a novel type of Mutanase from Paenibacillus sp. strain RM1: identification of its mutan-binding domain, essential for degradation of Streptococcus mutans biofilms

A novel type of mutanase (termed mutanase RM1) was isolated from Paenibacillus sp. strain RM1. The purified enzyme specifically hydrolyzed alpha-1,3-glucan (mutan) and effectively degraded biofilms formed by Streptococcus mutans, a major etiologic agent in the progression of dental caries, even following brief incubation. The nucleotide sequence of the gene for this protein contains a 3,873-bp open reading frame encoding 1,291 amino acids with a calculated molecular mass of 135 kDa. The protein contains two major domains, the N-terminal domain (277 residues) and the C-terminal domain (937 residues), separated by a characteristic sequence composed of proline and threonine repeats. The characterization of the recombinant proteins for each domain which were expressed in Escherichia coli demonstrated that the N-terminal domain had strong mutan-binding activity but no mutanase activity whereas the C-terminal domain was responsible for mutanase activity but had mutan-binding activity significantly lower than that of the intact protein. Importantly, the biofilm-degrading activity observed with the intact protein was not exhibited by either domain alone or in combination with the other. Therefore, these results indicate that the structural integrity of mutanase RM1 containing the N-terminal mutan-binding domain is required for the biofilm-degrading activity.     

The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites

Protein disulfide isomerase plays a key role in catalyzing the folding of secretory proteins. It features two catalytically inactive thioredoxin domains inserted between two catalytically active thioredoxin domains and an acidic C-terminal tail. The crystal structure of yeast PDI reveals that the four thioredoxin domains are arranged in the shape of a twisted "U" with the active sites facing each other across the long sides of the "U." The inside surface of the "U" is enriched in hydrophobic residues, thereby facilitating interactions with misfolded proteins. The domain arrangement, active site location, and surface features strikingly resemble the Escherichia coli DsbC and DsbG protein disulfide isomerases. Biochemical studies demonstrate that all domains of PDI, including the C-terminal tail, are required for full catalytic activity. The structure defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds.     

Role of conserved residues of the WRKY domain in the DNA-binding of tobacco WRKY family proteins.

Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn2+. Substitution of the conserved cysteine and histidine residues of the plant-specific C2H2-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.     

Solution structure of a ubiquitin-like domain from tubulin-binding cofactor B

Proper folding and assembly of tubulin alphabeta-heterodimers involves a stepwise progression mediated by a group of protein cofactors A through E. Upon release of the tubulin monomers from the chaperonin CCT, they are acted upon by each cofactor in the folding pathway through a unique combination of protein interaction domains. Three-dimensional structures have previously been reported for cofactor A and the C-terminal CAP-Gly domain of cofactor B (CoB). Here we report the NMR structure of the N-terminal domain of Caenorhabditis elegans CoB and show that it closely resembles ubiquitin as was recently postulated on the basis of bioinformatic analysis (Grynberg, M., Jaroszewski, L., and Godzik, A. (2003) BMC Bioinformatics 4, 46). CoB binds partially folded alpha-tubulin monomers, and a putative tubulin-binding motif within the N-terminal domain is identified from sequence and structure comparisons. Based on modeling of the homologous cofactor E ubiquitin-like domain, we hypothesize that cofactors B and E may associate via their beta-grasp domains in a manner analogous to the PB1 and caspase-activated deoxyribonuclease superfamily of protein interaction domains.     

N-terminal domains of native multidomain proteins have the potential to assist de novo folding of their downstream domains in vivo by acting as solubility enhancers

The fusion of soluble partner to the N terminus of aggregation-prone polypeptide has been popularly used to overcome the formation of inclusion bodies in the E. coli cytosol. The chaperone-like functions of the upstream fusion partner in the artificial multidomain proteins could occur in de novo folding of native multidomain proteins. Here, we show that the N-terminal domains of three E. coli multidomain proteins such as lysyl-tRNA synthetase, threonyl-tRNA synthetase, and aconitase are potent solubility enhancers for various C-terminal heterologous proteins. The results suggest that the N-terminal domains could act as solubility enhancers for the folding of their authentic C-terminal domains in vivo. Tandem repeat of N-terminal domain or insertion of aspartic residues at the C terminus of the N-terminal domain also increased the solubility of fusion proteins, suggesting that the solubilizing ability correlates with the size and charge of N-terminal domains. The solubilizing ability of N-terminal domains would contribute to the autonomous folding of multidomain proteins in vivo, and based on these results, we propose a model of how N-terminal domains solubilize their downstream domains.     

pH jump studies of the folding of the multidomain ribosomal protein L9: the structural organization of the N-terminal domain does not affect the anomalously slow folding of the C-terminal domain

The folding kinetics of the multidomain ribosomal protein L9 were studied using pH jump stopped-flow fluorescence and circular dichroism (CD) in conjunction with guanidine hydrochloride (GdnHCl) jump stopped-flow CD experiments. Equilibrium CD and 1D (1)H NMR measurements demonstrated that the C-terminal domain unfolds below pH 4 while the N-terminal domain remains fully folded. Thus, the N-terminal domain remains folded during the pH jump experiments. The folding rate constant of the C-terminal domain was determined to be 3.5 s(-1) by pH jump experiments conducted in the absence of denaturant using stopped-flow CD and fluorescence. CD-detected GdnHCl jump measurements showed that the N- and C-terminal domains fold independently each by an apparent two-state mechanism. The folding rate constant for the N-terminal domain and the C-terminal domain in the absence of denaturant were calculated to be 760 and 4. 7 s(-1), respectively. The good agreement between the pH jump and the denaturant concentration jump experiments shows that the folding rate of the C-terminal domain is the same whether or not the N-terminal domain is folded. This result suggests that the slow folding of the C-terminal domain is not a consequence of unfavorable interactions with the rest of the protein chain during refolding. This is an interesting result since contact order analysis predicts that the folding rate of the C-terminal domain should be noticeably faster. The folding rate of the isolated N-terminal domain was also measured by stopped-flow CD and was found to be the same as the rate for the domain in the intact protein.     

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45–50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.     

Hydrophilic C-terminal domain of the Escherichia coli mannitol permease: phosphorylation, functional independence, and evidence for intersubunit phosphotransfer

The mannitol-specific enzyme II (mannitol permease) of the Escherichia coli phosphotransferase system (PTS) catalyzes the concomitant transport and phosphorylation of D-mannitol. Previous studies have shown that the mannitol permease (637 amino acid residues) consists of 2 structural domains of roughly equal size: an N-terminal, hydrophobic, membrane-bound domain and a C-terminal, hydrophilic, cytoplasmic domain. The C-terminal domain can be released from the membrane by mild proteolysis of everted membrane vesicles [Stephan, M.M., & Jacobson, G.R. (1986) Biochemistry 25, 8230-8234]. In this report, we show that phosphorylation of the intact permease by [32P]HPr (a general phosphocarrier protein of the PTS) followed by tryptic separation of the two domains resulted in labeling of only the C-terminal domain. Phosphorylation of the C-terminal domain occurred even in the complete absence of the N-terminal domain, showing that the former contains most, if not all, of the critical residues comprising the interaction site for phospho-HPr. The phosphorylated C-terminal domain, however, could not transfer its phospho group to mannitol, suggesting that the N-terminal domain is necessary for mannitol binding and/or phosphotransfer from the enzyme to the sugar. The elution profile of the C-terminal domain after molecular sieve chromatography showed that the isolated domain is monomeric, unlike the native permease which is likely a dimer in the membrane. Experiments employing a deletion mutation of the mtlA gene, which encodes a protein lacking the first phosphorylation site in the C-terminal domain (His-554) but retaining the second phosphorylation site (Cys-384), demonstrated that a phospho group could be transferred from phospho-HPr to Cys-384 of the deletion protein, and then to mannitol, only in the presence of the full-length permease.(ABSTRACT TRUNCATED AT 250 WORDS)