Macrocyclic intermediates in the biosynthesis of porphyrins.

The hepta-, hexa- and penta-carboxylic porphyrins found in the faeces of rats poisoned with hexachlorobenzene have been separated by high-pressure liquid chromatography and characterized largely by spectroscopie methods. Their structures were confirmed by total synthesis, as part of a programme in which eleven of the fourteen hepta-, hexa- and penta-carboxylic porphyrins derived from uroporphyrin III have now been synthesized as their methyl esters. The four isomeric heptacarboxylic and three of the pentacarboxylic porphyrinogens have been incubated with haemolysates of chicken erythrocytes, and they are all converted into protoporphyrin IX but at different rates. On the basis of this and other evidence we conclude that the decarboxylation of uroporphyrinogen III to coproporphyrinogen III is a stepwise process taking place by a preferred pathway (both in normal and abnormal metabolism); the acetic acid groups are decarboxylated in a sequential clockwise fashion starting with that on the D ring and followed by those on the A, B and C rings. In the poisoned rats the uroporphyrinogen decarboxylase enzyme (or group of enzymes) is probably partially inhibited and the pentacarboxylic porphyrinogen with an acetic acid group on ring C accumulates. The latter is then transformed by a side pathway into dehydroisocoproporphyrinogen and thence into dehydroisocoproporphyrin and its congeners.     

Enzyme-bound intermediates in the biosynthesis of bacitracin.

1. Bacitracin synthetase, a three-component enzyme complex which catalyzes synthesis of the dodecapeptide bacitracin A, has been prepared from Bacillus licheniformis strains ATCC 10716, AL and SB 319. During synthesis of bacitracin, the amino acids (smaller amounts) and peptides are covalently bound to the enzyme complex. The nature of the bindings suggest that the amino acids and peptides are thioester linked. 2. The peptides, identified by thin-layer chromatography after performic acid liberation were Ile-Cys, Ile-Cys-Leu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu-Ile, Ile-Cys-Leu-Glu-Ile-Lys-Orn, Ile-Cys-Leu-Glu-Ile-Ile-Orn-Ile, Ile-Cys-L-EU-Glu-Ile-Lys-Orn-Ile-Phe, Ile-Cys-Leu-Glu-Ile-L-YS-Orn-Ile-Phe-His-Phe-His and Ile-Cys-Leu-Glu-Ile-Lys-Orn-Ile-Phe-His-Asp. 3. The labelled peptides covalently bound to bacitracin synthetase were intermediates in bacitracin synthesis. 4. Chain growth is initiated on one enzyme component (A) by the addition of isoleucine and cysteine. The sequential addition of the other amino acids proceeds in the C-terminal direction until the pentapeptide is formed. Further addition of amino acids and production of bacitracin are obtained by adding the other enzyme components (B and C) to the incubation mixture.     

Studies on the nature of microsome-bound substances involved in the biosynthesis of acidic glycoproteins of guinea-pig serum

1. In further studies on the biosynthesis of components of fraction I, an acidic glycoprotein-containing fraction from guinea-pig serum, an investigation was made of the substances bound to liver microsomes that had earlier been implicated to participate in the synthesis of components of fraction I present in serum. These substances were normally liberated by ultrasonic vibrations. Antisera to subfractions of fraction I were used for characterization. 2. At pH8.6, most of the microsome-bound substances showed electrophoretic mobilities lower than components of fraction I but similar to components of sialic acid-free fraction I. 3. The sialic acid/protein ratios of immune precipitates formed by microsome extracts were similar to those of precipitates formed by sialic acid-free fraction I. 4. On chromatography on Sephadex G-150, most of the microsomal substances were eluted at an essentially similar volume to the main components of fraction I. 5. It was concluded that most of the microsome-bound substances lack sialic acid residues, and, as appreciable degradation of completed molecules is unlikely, these substances appear to be precursors of serum glycoprotein molecules with incomplete prosthetic groups. 6. Evidence was obtained on the submicrosomal localization of incomplete and complete serum glycoprotein molecules.     

Lipid intermediates in the biosynthesis of bacterial peptidoglycan.

This review is an attempt to bring together and critically evaluate the now-abundant but dispersed data concerning the lipid intermediates of the biosynthesis of bacterial peptidoglycan. Lipid I, lipid II, and their modified forms play a key role not only as the specific link between the intracellular synthesis of the peptidoglycan monomer unit and the extracytoplasmic polymerization reactions but also in the attachment of proteins to the bacterial cell wall and in the mechanisms of action of antibiotics with which they form specific complexes. The survey deals first with their detection, purification, structure, and preparation by chemical and enzymatic methods. The recent important advances in the study of transferases MraY and MurG, responsible for the formation of lipids I and II, are reported. Various modifications undergone by lipids I and II are described, especially those occurring in gram-positive organisms. The following section concerns the cellular location of the lipid intermediates and the translocation of lipid II across the cytoplasmic membrane. The great efforts made since 2000 in the study of the glycosyltransferases catalyzing the glycan chain formation with lipid II or analogues are analyzed in detail. Finally, examples of antibiotics forming complexes with the lipid intermediates are presented.     

The biosynthesis of peptidoglycan lipid-linked intermediates

The biosynthesis of bacterial cell wall peptidoglycan is a complex process involving many different steps taking place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner and outer sides of the cytoplasmic membrane (assembly and polymerization of the disaccharide-peptide monomer unit, respectively). This review summarizes the current knowledge on the membrane steps leading to the formation of the lipid II intermediate, i.e. the substrate of the polymerization reactions. It makes the point on past and recent data that have significantly contributed to the understanding of the biosynthesis of undecaprenyl phosphate, the carrier lipid required for the anchoring of the peptidoglycan hydrophilic units in the membrane, and to the characterization of the MraY and MurG enzymes which catalyze the successive transfers of the N-acetylmuramoyl-peptide and N-acetylglucosamine moieties onto the carrier lipid, respectively. Enzyme inhibitors and antibacterial compounds interfering with these essential metabolic steps and interesting targets are presented.     

Investigation of tomato (Solanum lycopersicum) aminotransferases involved in biosynthesis of branched-chain-amino-acids

This study presents evidence for the role of BCAT3 and BCAT4 proteins in the synthesis of branched-chain-amino-acids in tomato Solanum lycopersicum. BCAT3 and BCAT4 genes were located on tomato chromosomal map by RFLP method (restriction fragment length polymorphism). Using confocal microscopy it was shown that BCAT3-GFP and BCAT4-GFP fusion proteins were localised in chloroplasts. It was observed that these aminotransferase isoforms exhibited distinct kinetic properties and a differential expression pattern of mRNA levels in various tomato tissues.     

Mechanisms involved in the biosynthesis of polysaccharides

The mechanisms for the biosynthesis of three polysaccharides are presented: (i) starch synthesized by starch synthase and adenosine diphospho glucose; (ii) dextran synthesized by Leuconostoc mesenteroides B-512FMC dextransucrase and sucrose; and (iii) Acetobacter xylinum cellulose synthesized by cellulose synthase, uridine diphospho glucose, and bactoprenol phosphate. All three enzymes were pulsed with substrates, containing ¹⁴C-glucose and chased with the same nonlabeled substrates. When the polysaccharides were isolated, reduced, and hydrolyzed, the pulsed reactions gave ¹⁴C-glucitol, which was significantly decreased in the chase reaction. These experiments definitively show that all three polysaccharides are biosynthesized by the addition of glucose to the reducing-ends of the growing polysaccharides and not by the addition to the nonreducing-ends of primers. Additional evidence indicates that glucose and the polysaccharides are covalently attached to the active-sites of the enzymes. A two catalytic-site insertion mechanism at one active-site is proposed for the biosyntheses. Two of the polysaccharides are α-linked glucans, starch and dextran, and cellulose is a β-linked glucan, known for several years to require a bactoprenol lipid phosphate intermediate. It is shown how this intermediate is involved in determining that β-linkages are synthesized. Other β-linked polysaccharides: bacterial cell wall peptidomurein, Salmonella O-antigen polysaccharide, and Xanthanomonas camprestris xanthan, are heteropolysaccharides, with the later two also being hetero-linked polysaccharides, with the β-linkage at the reducing-end of the repeating unit. All three require bactoprenol lipid phosphate intermediates and are biosynthesized by the addition of the repeating units to the reducing-end of a growing polysaccharide chain, with the formation of a β-linkage.