Source of amino acids for tRNA acylation in growing chicks

Summary Specific radioactivity in three amino acid compartments was examined in broiler chicks following a flooding dose of leucine or phenylalanine. In general, specific radioactivity of leucine and phenylalanine in deproteinated plasma (SA_e) and tissue (SA_i) compartments, exceeded that in acylated-tRNA (SA_t). In most tissues, SA_e and SA_i rapidly reached a similar peak level by 5 min followed by a slow decline for the next 30 minutes. Many tissues (eg. GI tract, liver, skin, and thigh) failed to maintain equilibrium between SA_e and SA_i over time. More metabolically active tissues, such as GI and liver had the greatest differences between these compartments. The difference between SA_e and SA_i for both leucine and phenylalanine were due to SA_i decreasing faster than SA_e, indicating dilution with unlabelled amino acids from proteolysis. Plasma and tissue specific radioactivity overestimated tRNA specific radioactivity by as much as 5 and 2.8 fold using leucine or 2.7 and 1.4 fold using phenylalanine, respectively. These data suggest that intracellular compartmentation of protein metabolism and the coupling of protein degradation and synthesis occur, in vivo.     

The balance between pre- and post-transfer editing in tRNA synthetases

The fidelity of tRNA aminoacylation is dependent in part on amino acid editing mechanisms. A hydrolytic activity that clears mischarged tRNAs typically resides in an active site on the tRNA synthetase that is distinct from its synthetic aminoacylation active site. A second pre-transfer editing pathway that hydrolyzes the tRNA synthetase aminoacyl adenylate intermediate can also be activated. Pre- and post-transfer editing activities can co-exist within a single tRNA synthetase resulting in a redundancy of fidelity mechanisms. However, in most cases one pathway appears to dominate, but when compromised, the secondary pathway can be activated to suppress tRNA synthetase infidelities.     

Aminoacylation of tRNA 2′- or 3′-hydroxyl by phosphoseryl- and pyrrolysyl-tRNA synthetases

Class I and II aminoacyl-tRNA synthetases (AARSs) attach amino acids to the 2′- and 3′-OH of the tRNA terminal adenosine, respectively. One exception is phenylalanyl-tRNA synthetase (PheRS), which belongs to Class II but attaches phenylalanine to the 2′-OH. Here we show that two Class II AARSs, O-phosphoseryl- (SepRS) and pyrrolysyl-tRNA (PylRS) synthetases, aminoacylate the 2′- and 3′-OH, respectively. Structure-based-phylogenetic analysis reveals that SepRS is more closely related to PheRS than PylRS, suggesting that the idiosyncratic feature of 2′-OH acylation evolved after the split between PheRS and PylRS. Our work completes the understanding of tRNA aminoacylation positions for the 22 natural AARSs.     

Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.     

Question 6: Coevolution Theory of the Genetic Code: A Proven Theory

The coevolution theory proposes that primordial proteins consisted only of those amino acids readily obtainable from the prebiotic environment, representing about half the twenty encoded amino acids of today, and the missing amino acids entered the system as the code expanded along with pathways of amino acid biosynthesis. The isolation of genetic code mutants, and the antiquity of pretran synthesis revealed by the comparative genomics of tRNAs and aminoacyl-tRNA synthetases, have combined to provide a rigorous proof of the four fundamental tenets of the theory, thus solving the riddle of the structure of the universal genetic code. Presented at: International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Using the inner membrane of Escherichia coli as a scaffold to anchor enzymes for metabolic flux enhancement

Clustering enzymes in the same metabolic pathway is a natural strategy to enhance productivity. Synthetic protein, RNA and DNA scaffolds have been designed to artificially cluster multiple enzymes in the cell, which require complex construction processes and possess limited slots for target enzymes. We utilized the Escherichia coli inner cell membrane as a native scaffold to cluster four fatty acid synthases (FAS) and achieved to improve the efficiency of fatty acid synthesis in vivo. The construction strategy is as simple as fusing target enzymes to the N-terminus or C-terminus of the membrane anchor protein (Lgt), and the number of anchored enzymes is not restricted. This novel device not only presents a similar efficiency in clustering multiple enzymes to that of other artificial scaffolds but also promotes the product secretion, driving the entire metabolic flux forward and further increasing the gross yield compared with that in a cytoplasmic scaffold system.     

Disaccharide nucleosides as an important group of natural compounds

The main structural features of disaccharide nucleosides, an important group of natural compounds, are reviewed. The preparation and properties of modified oligonucleotides constructed on their basis and the incorporation of reactive groups are summarized. Several examples are given for the use of the compounds to investigate the enzymes of nucleic acid metabolism.     

Characterization of a triazole scaffold compound as an inhibitor of Mycobacterium tuberculosis imidazoleglycerol-phosphate dehydratase

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), employs ten enzymes including imidazoleglycerol-phosphate dehydratase (IGPD) for de novo biosynthesis of histidine. The absence of histidine-biosynthesis in humans combined with its essentiality for Mtb makes the enzymes of this pathway major anti-TB drug targets. We explored the inhibitory potential of a small molecule β-(1,2,4-Triazole-3-yl)-DL-alanine (DLA) against Mtb IGPD. DLA exhibits an in vitro inhibitory efficacy in the lower micromolar range. Higher-resolution crystal structures of native and substrate-bound Mtb IGPD provided additional structural features of this important drug target. Crystal structure of IGPD-DLA complex at a resolution of 1.75 Å, confirmed that DLA locks down the function of the enzyme by binding in the active site pocket of the IGPD mimicking the substrate-binding mode to a high degree. In our biochemical study, DLA showed an efficient inhibition of Mtb IGPD. Furthermore, DLA also showed bactericidal activity against Mtb and Mycobacterium smegmatis and inhibited their growth in respective culture medium. Importantly, owing to the favorable ADME and physicochemical properties, it serves as an important lead molecule for further derivatizations.     

Aspartate-phobia of thermophiles as a reaction to deleterious chemical transformations

Prokaryotes growing at high temperatures have a high proportion of charged residues in their proteins to stabilize their 3D structure. By mining 175 disparate bacterial and archaeal proteomes we found that, against the general trend for charged residues, the frequency of aspartic acid residues decreases strongly as natural growth temperature increases. In search of the explanation, we hypothesized that the reason for such unusual correlation is the deleterious consequences of spontaneous chemical transformations of aspartate at high temperatures. Our subsequent statistical analysis supported this hypothesis. This finding reveals that organisms have likely adapted to high temperatures by minimizing the harmful consequences of spontaneous chemical transformations.     

Growth and shape transformations of giant phospholipid vesicles upon interaction with an aqueous oleic acid suspension

The interaction of two types of vesicle systems was investigated: micrometer-sized, giant unilamellar vesicles (GUVs) formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and submicrometer-sized, large unilamellar vesicles (LUVs) formed from oleic acid and oleate, both in a buffered aqueous solution (pH 8.8). Individual POPC GUVs were transferred with a micropipette into a suspension of oleic acid/oleate LUVs, and the shape changes of the GUVs were monitored using optical microscopy. The behavior of POPC GUVs upon transfer into a 0.8 mM suspension of oleic acid, in which oleic acid/oleate forms vesicular bilayer structures, was qualitatively different from the behavior upon transfer into a 0.3 mM suspension of oleic acid/oleate, in which oleic acid/oleate is predominantly present in the form of monomers and possibly non-vesicular aggregates. In both cases, changes in vesicle morphology were observed within tens of seconds after the transfer. After an initial increase of the vesicle cross-section, the vesicle started to evaginate, spawning dozens of satellite vesicles connected to the mother vesicle with narrow necks or tethers. In 60% of the cases of transfer into a 0.8 mM oleic acid suspension, the evagination process reversed and proceeded to the point where the membrane formed invaginations. In some of these cases, several consecutive transitions between invaginated and evaginated shapes were observed. In the remaining 40% of the cases of transfer into the 0.8 mM oleic acid suspension and in all cases of vesicle transfer into the 0.3 mM oleic acid suspension, no invaginations nor subsequent evaginations were observed. An interpretation of the observed vesicle shape transformation on the basis of the bilayer-couple model is proposed, which takes into account uptake of oleic acid/oleate molecules by the POPC vesicles, oleic acid flip-flop processes and transient pore formation.     

In vitro validation of acetyltransferase activity of GlmU as an antibacterial target in Haemophilus influenzae

GlmU is a bifunctional enzyme that is essential for bacterial growth, converting D-glucosamine 1-phosphate into UDP-GlcNAc via acetylation and subsequent uridyl transfer. A biochemical screen of AstraZeneca's compound library using GlmU of Escherichia coli identified novel sulfonamide inhibitors of the acetyltransferase reaction. Steady-state kinetics, ligand-observe NMR, isothermal titration calorimetry, and x-ray crystallography showed that the inhibitors were competitive with acetyl-CoA substrate. Iterative chemistry efforts improved biochemical potency against gram-negative isozymes 300-fold and afforded antimicrobial activity against a strain of Haemophilus influenzae lacking its major efflux pump. Inhibition of precursor incorporation into bacterial macromolecules was consistent with the antimicrobial activity being caused by disruption of peptidoglycan and fatty acid biosyntheses. Isolation and characterization of two different resistant mutant strains identified the GlmU acetyltransferase domain as the molecular target. These data, along with x-ray co-crystal structures, confirmed the binding mode of the inhibitors and explained their relative lack of potency against gram-positive GlmU isozymes. This is the first example of antimicrobial compounds mediating their growth inhibitory effects specifically via GlmU.     

Epoxyqueuosine Reductase Structure Suggests a Mechanism for Cobalamin-dependent tRNA Modification

Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5′-GUN-3′ tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes.     

The structural gene for aspartokinase II in Bacillus subtilis is closely linked to the sdh operon

The aecA and aecB loci map at 250 and 290 degrees, respectively, on the Bacillus subtilis chromosomal genetic map. The aecB locus has been proposed as the structural gene for aspartokinase II. From DNA sequence analyses and comparisons to the sequence of the aspartokinase II gene, it can be concluded that the structural gene for aspartokinase II is located close to sdh at 250 degrees and cannot be aecB. A detailed map over 7 kbp in the 250 degree region is presented.     

Tryptophan-based fluorophores for studying protein conformational changes

With the continuing interest in deciphering the interplay between protein function and conformational changes, small fluorescence probes will be especially useful for tracking changes in the crowded protein interior space. Presently, we describe the potential utility of six unnatural amino acid fluorescence donors structurally related to tryptophan and show how they can be efficiently incorporated into a protein as fluorescence probes. We also examine the various photophysical properties of the new Trp analogues, which are significantly redshifted in their fluorescence spectra relative to tryptophan. In general, the Trp analogues were well tolerated when inserted into Escherichia coli DHFR, and did not perturb enzyme activity, although substitution for Trp22 did result in a diminution in DHFR activity. Further, it was demonstrated that D and E at position 37 formed efficient FRET pairs with acridon-2-ylalanine (Acd) at position 17. The same was also true for a DHFR construct containing E at position 79 and Acd at position 17. Together, these findings demonstrate that these tryptophan analogues can be introduced into DHFR with minimal disruption of function, and that they can be employed for the selective study of targeted conformational changes in proteins, even in the presence of unmodified tryptophans.     

Convenient solid-phase synthesis of oligopeptides using pentacoordinated phosphoranes with amino acid residue as building blocks

The reactive intermediates of pentacoordinated phosphoranes with amino acids (P(5)-AA) as building blocks, which were obtained by the reaction of O-phenylene phosphorochloridate with N,O-bis(trimethylsilyl)amino acids, were linked to a solid-phase support containing a hydroxymethyl polystyrene functional group. The first amino acid residue was coupled to the solid-phase support after washing the resin with organic solvent. Repeating the procedure led to oligopeptides linked on the resin. A series of free oligopeptides including tetra-Gly, di-Val, tri-Val, di-Leu, di-Phe, and Phe-Leu were obtained after cleavage from solid-phase support. The structure of these oligopeptides were determined by IR, (1)H NMR, FAB-MS, and HPLC.     

Fluorescence in blue light (FLU) is involved in inactivation and localization of glutamyl‐tRNA reductase during light exposure

Fluorescent in blue light (FLU) is a negative regulator involved in dark repression of 5‐aminolevulinic acid (ALA) synthesis and interacts with glutamyl‐tRNA reductase (GluTR), the rate‐limiting enzyme of tetrapyrrole biosynthesis. In this study, we investigated FLU‘s regulatory function in light‐exposed FLU‐overexpressing (FLUOE) Arabidopsis lines and under fluctuating light intensities in wild‐type (WT) and flu seedlings. FLUOE lines suppress ALA synthesis in the light, resulting in reduced chlorophyll content, but more strongly in low and high light than in medium growth light. This situation indicates that FLU's impact on chlorophyll biosynthesis depends on light intensity. FLU overexpressors contain strongly increased amounts of mainly membrane‐associated GluTR. These findings correlate with FLU‐dependent localization of GluTR to plastidic membranes and concomitant inhibition, such that only the soluble GluTR fraction is active. The overaccumulation of membrane‐associated GluTR indicates that FLU binding enhances GluTR stability. Interestingly, under fluctuating light, the leaves of flu mutants contain less chlorophyll compared with WT and become necrotic. We propose that FLU is basically required for fine‐tuned ALA synthesis. FLU not only mediates dark repression of ALA synthesis, but functions also to control balanced ALA synthesis under variable light intensities to ensure the adequate supply of chlorophyll.     

Nutritional requirements for the production of dithiolopyrrolone antibiotics by Saccharothrix algeriensis NRRL B-24137

The amino acid and humic acid requirements of Saccharothrix algeriensis NRRL B-24137 for growth and production of the dithiolopyrrolone antibiotics were studied in a semi-synthetic medium (SSM). Nature and concentration of amino acids and humic acid strongly influenced the growth and dithiolopyrrolone specific production.

The highest value of thiolutin (acetyl-pyrrothine) specific production was obtained in the presence of 1 g/l humic acid (336 mg/g DCW), and in the presence of 5 mM l-cystine (309 mg/g DCW) as compared to 19 mg/g DCW obtained with the control. Furthermore, thiolutin production was increased about six-fold, four-fold and three-fold in the presence of l-proline, l-glutamic acid and dl-histidine, respectively. In contrast, the production of thiolutin was reduced by addition of other amino acids such as l-glutamine, dl-ethionine, l-methionine and l-arginine. The highest value of isobutyryl-pyrrothine production was obtained in the presence of 2,6-diaminopimelic acid and l-lysine (7.8 and 1.0 mg/g DCW, respectively). However, the highest value of butanoyl-pyrrothine production was obtained in the presence of humic acid (6.6 mg/g DCW), followed by l-cysteine and l-proline (3.6 and 3.2 mg/g DCW, respectively). In addition, the maximum specific production of senecioyl-pyrrothine (29 mg/g DCW) and tigloyl-pyrrothine (21 mg/g DCW) was obtained in the presence of humic acid. We found that, except for isobutyryl-pyrrothine, production of all dithiolopyrrolones was favoured by addition of l-proline. The maximum specific production was obtained with l-proline at concentrations of 2.50 mM for thiolutin (133 mg/g DCW), 1.25 mM for senecioyl-pyrrothine, tigloyl-pyrrothine and butanoyl-pyrrothine production (29, 23 and 3.9 mg/g DCW, respectively). Production of all dithiolopyrrolones strongly decreased as the l-methionine or dl-ethionine concentration was increased in the culture medium.     

Do ribosomal RNAs act merely as scaffold for ribosomal proteins?

Investigations that are being carried out in various laboratories including ours clearly provide the answer which is in the negative. Only the direct evidences obtained in this laboratory will be presented and discussed. It has been unequivocally shown that the interaction between 16S and 23S RNAs plays the primary role in the association of ribosomal subunits. Further, 23S RNA is responsible for the Binding of 5S RNA to 16S.23S RNA complex with the help of three ribosomal proteins, L5, L18, L15/L25. The 16S.23S RNA complex is also capable of carrying out the following ribosomal functions, although to small but significant extents, with the help of a very limited number of ribosomal proteins and the factors involved in protein synthesis: (a) poly U-Binding, (B) poly U-dependent Binding of phenylalanyl tRNA, (c) EF-G-dependent GTPase activity, (d) initiation complex formation, (e) peptidyl transferase activity (puromycin reaction) and (f) polyphenylalanine synthesis. These results clearly indicate the direct involvement of rRNAs in the various steps of protein synthesis. Very recently it has Been demonstrated that the conformational change of 23S RNA is responsible for the translocation of peptidyl tRNA from the aminoacyl (A) site to the peptidyl (P) site. A model has Been proposed for translocation on the Basis of direct experimental evidences. The new concept that ribosomal RNAs are the functional components in ribosomes and proteins act as control switches may eventually turn out to Be noncontroversial.     

Effect of Loading Doses of l-Valine on Relative Contributions of Valine Derived from Protein Degradation and Plasma to the Precursor Pool for Protein Synthesis in Rat Brain

"Flooding" amino acid pools with high doses of labeled amino acids of low specific activity has been proposed to minimize the effects of recycling of amino acids derived from protein degradation on the specific activity of the amino acid precursor pool for protein synthesis. We have examined the influence of recycling on the precursor pool for protein synthesis under conditions in which plasma valine concentrations were normal (0.19 mM) and "flooded" (10-28 mM) by comparing the steady-state specific activity of the tRNA-bound valine with that of the plasma valine. Under normal and "flooding" conditions, the relative contributions of valine from protein degradation to the precursor pool were 63 and 26%, respectively; "flooding" with a plasma level of 28 mM raised the brain acid-soluble pool level to 3.1 mM but was no more effective in decreasing the relative contribution of valine from protein degradation to the precursor pool than "flooding" with a plasma level of 17 mM valine, which raised the brain acid-soluble level only to 2.3 mM. The results of these studies show that "flooding" amino acid pools does indeed reduce the effect of recycling on the precursor amino acid pool for protein synthesis, but it does not totally eliminate it.     

Interactions of membrane lipoproteins with the murein sacculus of Escherichia coli as shown by chemical crosslinking studies of intact cells

Proteins that were closely associated with murein in intact cells of Escherichia coli were studied by treating [3H]leucine and [3H]palmitate-labeled cells with the chemical crosslinking reagent dithiobis(succinimidylpropionate). Murein was purified and crosslinked peptides were released from the murein by treatment with beta-mercaptoethanol. Nine murein-associated [3H]leucine-labeled peptides were identified. Five of the nine peptides were lipoproteins, based on labeling with [3H]palmitate, protease sensitivity and gel electrophoretic correspondence to membrane lipoproteins present in uncrosslinked cell envelope preparations. The results suggest that these membrane lipoproteins may play a significant role in the structural integration of the murein and membrane layers of the cell envelope.