Biosynthesis of D-alanyl-lipoteichoic acid: the tertiary structure of apo-D-alanyl carrier protein.

The D-alanylation of lipoteichoic acid (LTA) allows the Gram-positive organism to modulate its surface charge, regulate ligand binding, and control the electromechanical properties of the cell wall. The incorporation of D-alanine into LTA requires the D-alanine:D-alanyl carrier protein ligase (AMP-forming) (Dcl) and the carrier protein (Dcp). The high-resolution solution structure of the 81-residue (8.9 kDa) Dcp has been determined by multidimensional heteronuclear NMR. An ensemble of 30 structures was calculated using the torsion angle dynamics approach of DYANA. These calculations utilized 3288 NOEs containing 1582 unique nontrivial NOE distance constraints. Superposition of residues 4-81 on the mean structure yields average atomic rmsd values of 0.43 +/- 0.08 and 0.86 +/- 0.09 A for backbone and non-hydrogen atoms, respectively. The solution structure is composed of three alpha-helices in a bundle with additional short 3(10)- and alpha-helices in intervening loops. Comparisons of the three-dimensional structure with the acyl carrier proteins involved in fatty acid, polyketide, and nonribosomal peptide syntheses support the conclusion that Dcp is a homologue in this family. While there is conservation of the three-helix bundle fold, Dcp has a higher enthalpy of unfolding and no apparent divalent metal binding site(s), features that distinguish it from the fatty acid synthase acyl carrier protein of Escherichia coli. This three-dimensional structure also provides insights into the D-alanine ligation site recognized by Dcl, as well as the site which may bind the poly(glycerophosphate) acceptor moiety of membrane-associated LTA.     

Biosynthesis of D-alanyl-lipoteichoic acid in Lactobacillus casei: D-alanyl-lipophilic compounds as intermediates

D-Alanyl-lipoteichoic acid (D-alanyl-LTA) from Lactobacillus casei contains a poly(glycerol phosphate) moiety that is selectively acylated with D-alanine ester residues. To characterize further the mechanism of D-alanine substitution, intermediates were sought that participate in the assembly of this LTA. From the incorporation system utilizing either toluene-treated cells or a combination of membrane fragments and supernatant fraction, a series of membrane-associated D-[14C]alanyl-lipophilic compounds was found. The assay of these compounds depended on their extractability into monophasic chloroform-methanol-water (0.8:3.2:1.0, vol/vol/vol) and subsequent partitioning into chloroform. Four lines of evidence suggested that the D-alanyl-lipophilic compounds are intermediates in the synthesis of D-alanyl-LTA. First, partial degradation of the poly(glycerol phosphate) moiety of D-alanyl-LTA by phosphodiesterase II/phosphatase from Aspergillus niger generated a series of D-alanyl-lipophilic compounds similar to those extracted from the toluene-treated cells during the incorporation of D-alanine. Second, enzymatic degradation of the D-alanyl-lipophilic compounds by the above procedure gave D-alanyl-glycerol, the same degradation product obtained from D-alanyl-LTA. Third, the incorporation of D-alanine into these compounds required the same components as the incorporation of D-alanine into membrane-associated D-alanyl-LTA. Fourth, the phosphate-induced loss of D-[14C]alanine-labeled lipophilic compounds could be correlated with the stimulation of phosphatidylglycerol synthesis in the presence of excess phosphate. We interpreted these experiments to indicate that the D-alanyl-lipophilic compounds are D-alanyl-LTA with short polymer chains and are most likely intermediates in the assembly of the completed polymer, D-alanyl-LTA.     

Biosynthesis of hyaluronan: direction of chain elongation

Hyaluronan (HA), a functionally essential glycosaminoglycan in vertebrate tissues and a putative virulence factor in certain pathogenic bacteria, is an extended linear polymer composed of alternating units of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). Uncertainty regarding the mechanism of HA biosynthesis has included the directionality of chain elongation, i.e. whether addition of monosaccharide units occurs at the reducing or non-reducing terminus of nascent chains. We have investigated this problem using yeast-derived recombinant HA synthases from Xenopus laevis (xlHAS1) and from Streptococcus pyogenes (spHAS). The enzymes were incubated with UDP-[3H]GlcUA and UDP-[14C]GlcNAc, under experimental conditions designed to yield HA chains with differentially labeled reducing-terminal and non-reducing terminal domains. Digestion of the products with a mixture of beta-glucuronidase and beta-N-acetylglucosaminidase exoenzymes resulted in truncation of the HA chain strictly from the non-reducing end and release of labeled monosaccharides. The change in 3H/14C ratio of the monosaccharide fraction, during the course of exoglycosidase digestion, was interpreted to indicate whether sugar units had been added at the reducing or non-reducing end. The results demonstrate that the vertebrate xlHAS1 and the bacterial spHAS extend HA in opposite directions. Chain elongation catalyzed by xlHAS1 occurs at the non-reducing end of the HA chain, whereas elongation catalyzed by spHAS occurs at the reducing end. The spHAS is the first glycosyltransferase that has been unanimously demonstrated to function at the reducing end of a growing glycosaminoglycan chain.     

Biosynthesis of hyaluronan: direction of chain elongation

The mechanism of hyaluronan biosynthesis in vertebrates had been proposed to occur at the reducing end of growing chains. This mechanism was questioned because a recombinant synthase appeared to add new monosaccharides to the non-reducing end. I reinvestigated this problem with membranes from the eukaryotic B6 cell line. The membranes were incubated with UDP-[3H]GlcNAc and UDP-[14C]GlcA to yield differentially labelled reducing terminal and non-reducing terminal domains. Digestion of the product with a mixture of the exoglycosidases beta-glucuronidase and beta-N-acetylglucosaminidase truncated the hyaluronan chain strictly from the non-reducing end. The change in 3H/14C ratio of the remaining hyaluronan fraction, during the course of exoglycosidase digestion, confirmed the original results that the native eukaryotic synthase extended hyaluronan at the reducing end. This mechanism demands that the UDP-hyaluronan terminus is bound to the active site within the synthase and should compete with the substrates for binding. Accordingly, increasing substrate concentrations enhanced hyaluronan release from the synthase. A model is proposed that explains the direction of chain elongation at the reducing end by the native synthase and at the non-reducing end by the recombinant synthase based on a loss of binding affinity of the synthase towards the growing UDP-hyaluronan chain.     

Defects in D-alanyl-lipoteichoic acid synthesis in Streptococcus mutans results in acid sensitivity

In the cariogenic organism, Streptococcus mutans, low pH induces an acid tolerance response (ATR). To identify acid-regulated proteins comprising the ATR, transposon mutagenesis with the thermosensitive plasmid pGh9:ISS1 was used to produce clones that were able to grow at neutral pH, but not in medium at pH 5.0. Sequence analysis of one mutant (IS1A) indicated that transposition had created a 6.3-kb deletion, one end of which was in dltB of the dlt operon encoding four proteins (DltA-DltD) involved in the synthesis of D-alanyl-lipoteichoic acid. Inactivation of the dltC gene, encoding the D-alanyl carrier protein (Dcp), resulted in the generation of the acid-sensitive mutant, BH97LC. Compared to the wild-type strain, LT11, the mutant exhibited a threefold-longer doubling time and a 33% lower growth yield. In addition, it was unable to initiate growth below pH 6.5 and unadapted cells were unable to survive a 3-h exposure in medium buffered at pH 3.5, while a pH of 3.0 was required to kill the wild type in the same time period. Also, induction of the ATR in BH97LC, as measured by the number of survivors at a pH killing unadapted cells, was 3 to 4 orders of magnitude lower than that exhibited by the wild type. While the LTA of both strains contained a similar average number of glycerolphosphate residues, permeabilized cells of BH97LC did not incorporate D-[(14)C]alanine into this amphiphile. This defect was correlated with the deficiency of Dcp. Chemical analysis of the LTA purified from the mutant confirmed the absence of D-alanine-esters. Electron micrographs showed that BH97LC is characterized by unequal polar caps and is devoid of a fibrous extracellular matrix present on the surface of the wild-type cells. Proton permeability assays revealed that the mutant was more permeable to protons than the wild type. This observation suggests a mechanism for the loss of the characteristic acid tolerance response in S. mutans.     

Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme.

The D-alanine-activating enzyme (Dae; EC 6.3.2.4) encoded by the dae gene from Lactobacillus casei ATCC 7469 is a cytosolic protein essential for the formation of the D-alanyl esters of membrane-bound lipoteichoic acid. The gene has been cloned, sequenced, and expressed in Escherichia coli, an organism which does not possess Dae activity. The open reading frame is 1,518 nucleotides and codes for a protein of 55.867 kDa, a value in agreement with the 56 kDa obtained by electrophoresis. A putative promoter and ribosome-binding site immediately precede the dae gene. A second open reading frame contiguous with the dae gene has also been partially sequenced. The organization of these genetic elements suggests that more than one enzyme necessary for the biosynthesis of D-alanyl-lipoteichoic acid may be present in this operon. Analysis of the amino acid sequence deduced from the dae gene identified three regions with significant homology to proteins in the following groups of ATP-utilizing enzymes: (i) the acid-thiol ligases, (ii) the activating enzymes for the biosynthesis of enterobactin, and (iii) the synthetases for tyrocidine, gramicidin S, and penicillin. From these comparisons, a common motif (GXXGXPK) has been identified that is conserved in the 19 protein domains analyzed. This motif may represent the phosphate-binding loop of an ATP-binding site for this class of enzymes. A DNA fragment (1,568 nucleotides) containing the dae gene and its putative ribosome-binding site has been subcloned and expressed in E. coli. Approximately 0.5% of the total cell protein is active Dae, whereas 21% is in the form of inclusion bodies. The isolation of this minimal fragment without a native promoter sequence provides the basis for designing a genetic system for modulating the D-alanine ester content of lipoteichoic acid.     

Peptide chain elongation: A possible role of montmorillonite in prebiotic synthesis of protein precursors

Several studies have proven the ability of montmorillonite to catalyse amino acid condensation under assumed prebiotic conditions, simulating wetting-drying cycles. In this work, the oligomerization of short peptides gly₂, gly₃, gly₄ and ala₂ on Ca-and Cu-montmorillonite in drying-wetting cycles at 80 °C was studied. The catalytic effect of montmorillonite was found to be much higher than in the case of glycine oligomerization. From gly₂ after 3 weeks, 10% oligomers (up to gly6, with gly₃ as main products) are formed. Gly₃ and gly₄ give higher oligomers even after 1 cycle. Ala₂ produces both ala₃ and ala₄, whereas ala does not produce any oligomers under these conditions. Heteroologomerization was observed: ala-gly-gly is formed from ala and gly₂. Much higher yields are obtained using Ca-montmorillonite, because copper (II) oxidizes organic molecules. The influence of the reaction mechanism on the preferential oligomerization of oligopeptides is discussed.     

Effect of thyroid hormones on microsomal fatty acid chain elongation synthesis in rat liver.

Evidence is presented that rat liver microsomal fatty acid chain elongation synthesis and desaturation, as well as acetyl-CoA carboxylase and fatty acid synthetase, are strongly influenced by thyroid hormone level. Conversely, the fatty acid chain elongation system in mitochondria, unlike the oxidative capacity of palmitate, NADH, succinate and malate, does not seem significantly affected by the thyrotoxic state. In triiodothyronine-induced or thyroxine-induced hyperthyroidism, rat liver acetyl-CoA carboxylase, fatty acid synthetase and microsomal chain elongation and desaturation reactions are not greatly affected after the first 10 days of treatment, while after longer intervals a respective increase in these activities is shown of up to 87, 116 and 65% after 22 days. In propylthiouracil-induced hypothyroidism, all the above synthetic activities are strongly reduced immediately after three days of drug administration and diminished no further following longer periods. Although the pattern of synthesized fatty acids in the thyrotoxic state is similar to that obtained from normal subcellular rat fractions, the esterification process of fatty acids in microsomal lipids appears to be slightly inhibited in hypothyroid rats and increased following triiodothyronine or thyroxine administration. Finally, a reduction in the hepatic cyclic AMP level of about 41% is reported after 19 days of triiodothyronine-administration to rats. On the basis of the observed insensitivity of the mitochondrial fatty acid chain elongation system to the thyrotoxic state, a tentative interpretation of its role in the hepatic cell is postulated.     

Fatty acid chain elongation synthesis in eel (Anguilla anguilla) liver mitochondria

The properties of fatty acid chain elongation synthesis have been investigated in liver mitochondria of the European eel (Anguilla anguilla). The incorporation of [1-(14)C]acetyl-CoA into fatty acids shows a specific activity of 0.43+/-0.05 nmol/min x mg protein (n=6), which is more than twice higher than that previously reported in rat liver mitochondria. Label incorporation into fatty acids was, in mitochondria disrupted by freezing and thawing, much higher than in intact organelles thus suggesting a probable localization of this pathway inside mitochondria. Only a negligible acetyl-CoA incorporation into fatty acids occurs in the absence of ATP, Mg2+ or reduced pyridine nucleotides; NADH alone seems to be as effective as NADH + NADPH as a hydrogen donor for the reducing steps. CoASH, without effect up to 10 microM, showed a strong inhibition at higher concentrations. From the ratio of total radioactivity and radioactivity in carboxyl carbon it can be inferred that in eel-liver mitochondria only chain elongation of preexisting fatty acids occurs. A significant fatty acid chain elongation activity is also present when, instead of acetyl-CoA, [2-(14)C]malonyl-CoA is used as a carbon unit donor. Moreover, the synthesized fatty acids were actively incorporated into phopholipids, mainly phosphatidylcholine, phosphatidylethanolamine and sphyngomyelin.     

Template recognition and chain elongation in DNA synthesis in vitro.

DNA polymerase from Escherichia coli (Pol I) and from avian myeloblastosis virus (AMV polymerase) were compared for the manner in which they catalyze the polymerization of deoxynucleotides upon a variety of synthetic and natural templates. It was found that the rates of nucleotide incorporation with different natural RNAs were similar. Both polymerases have an associated RNA endonuclease which hydrolyses RNA templates containing double-stranded regions. This activity depends on the presence of the complementary deoxynucleoside triphosphates, and/or polymerization. Both enzymes copy natural DNA, which has been sonicated and treated with E. coli exonuclease III, at the same rate. However, avian myeloblastosis virus DNA polymerase, which has no associated DNA exonuclease activity, is unable to copy double-stranded DNA and copies DNAase-treated DNA only 10% as well as Pol I. Pol I copied all the homopolymers investigated at a greater rate than AMV polymerase with the exception of poly(C) · oligo(dG). However, the initial rate of chain elongation, as measured by gel electrophoresis, was the same for the two polymerases, approximately 300 nucleotides incorporated per minute. Template saturation experiments show a stoichiometric relationship between template and enzyme at optimal rates of nucleotide incorporation which suggests that all enzyme molecules are potential catalysts. Enzyme saturation experiments indicate that not all enzyme molecules are “effectively” bound to a template. Fewer AMV polymerase than Pol I molecules are functionally bound to a particular template. From these data, it is concluded that the two polymerases elongate DNA chains in a similar way and that the manner in which the polymerases bind to a particular template accounts for the discrepancies found in their turnover numbers.