The avidin-biotin complex in bioanalytical applications

Bacteria and fungi use large multifunctional enzymes, the so-called nonribosomal peptide synthetases (NRPSs), to produce peptides of broad structural and biological activity. Biochemical studies have contributed substantially to the understanding of the key principles of these modular enzymes that can draw on a much larger number of catalytic tools for the incorporation of unusual features compared with the ribosomal system. Several crystal structures of NRPS-domains have yielded deep insight into the catalytic mechanisms involved and have led to a better prediction of the products assembled and to the construction of hybrid enzymes. In addition to the structure-function relationship of the core- and tailoring-domains of NRPSs, which is the main focus of this review, different biosynthetic strategies and essential enzymes for posttranslational modification and editing are discussed.

Biosynthesis of nonribosomal peptides1.

Genetic Contributions to Understanding Polyketide Synthases.

Regulation of fatty acid biosynthesis in Escherichia coli.

Although phenolic compounds are present in both the plant and animal worlds, most of them are of plant origin.1 This heterogeneous group of natural compounds is still, in many textbooks, described as “secondary metabolites”, which suggests their less important role in cellular physiology and biochemistry. It includes both simple phenols and polyphenols as well as their derivatives. In general, the term “phenol” can be defined chemically as a substance that possesses an aromatic ring bearing a hydroxyl substituent and functional derivatives. The natural plant phenols arise biogenetically from two main pathways: the shikimate pathway, which directly provides phenylpropanoids such as the hydroxycinnamic acids and coumarins, and the polyketide (acetate) pathway, which can produce simple phenols and also lead to quinones. The flavonoids, by far the largest group of phenolics, are derived by combination of these two pathways. Among plant single-ring phenolics the group of various compounds that has recently shown growth is the derivatives described as phenolic lipids or long-chain phenols.2 They are amphiphilic in nature due to the non-isoprenoid side chains attached to the hydroxybenzene ring and are believed to be also derived from the polyketide (acetate) pathway, as, for example, 6-pentadecylsalicylic acid. Non-isoprenoid phenolic lipids are relatively uncommon and can be considered for simplicity as fatty acids, with the carboxyl group replaced by the hydroxybenzene ring. Therefore, they are derivatives of monoand dihydroxyphenols, namely, catechol, resorcinol, and hydroquinone. The biogenetic pathway of these compounds, described in textbooks and recent reviews,2-5 is based on incomplete experimental data supplemented with chemical considerations. The biosynthetic aspects of resorcinolic lipids Volume 99, Number 1 January 1999

http://www.halsanet.com/pdf/340516159.pdf

Resorcinolic Lipids, the Natural Non-isoprenoid Phenolic Amphiphiles and Their Biological Activity.

Acetyl Coenzyme A Carboxylase System of Escherichia coli PURIFICATION AND PROPERTIES OF THE BIOTIN CARBOXYLASE, CARBOXYLTRANSFERASE, AND CARBOXYL CARRIER PROTEIN COMPONENTS

An extract was prepared from Penicillium patulum grown in submerged culture, which converted acetyl-CoA and malonyl-CoA into 6-methylsalicylic acid in the presence of NADPH. The enzyme was very labile in the crude extract, but appeared stable after acetone precipitation. A 100-fold purification was achieved by ammonium sulfate fractionation, sedimentation in the ultracentrifuge, and sucrose-density gradient centrifugation. The enzyme was a single particle with a molecular weight of 1.1–1.5 × 106 as determined by gradient centrifugation with catalase and fatty acid synthetase as standards. In the first steps of purification, fatty acid synthetase from Penicillium patulum migrated with the methylsalicylic acid synthetase complex. A separation of the two enzyme systems was accomplished by sucrose-density gradient centrifugation. The purified enzyme represents a new multienzyme complex which shares several common properties with fatty acid synthetase. Methylsalicylic acid synthetase is inhibited by iodoacetamide and N-ethylmaleinimide. The incubation of enzyme with acetyl-CoA and malonyl-CoA in the absence of NADPH led to triacetic acid lacton. This indicates that in 6-methylsalicylic acid synthesis reduction occurs at the triacetic acid level before the final condensation with malonyl-CoA. Triacetic acid lacton was produced by the enzyme at 10% of the rate of 6-methylsalicylic acid synthesis in the complete system. This rate of lacton synthesis, however, is an order of magnitude greater than that catalysed by fatty acid synthetase. A possible explanation for this difference in behavior is given. Covalent linkage of substrates and intermediates to SH-groups of the protein during the synthesis of 6-methylsalicyslic acid is suggested. A reaction scheme leading to 6-methylsalicylic acid is proposed and the partial reactions of this synthesis are discussed with respect to similar reactions of fatty acid biosynthesis.

Peter Dimroth

Papers

Acetyl Coenzyme A Carboxylase System of Escherichia coli PURIFICATION AND PROPERTIES OF THE BIOTIN CARBOXYLASE, CARBOXYLTRANSFERASE, AND CARBOXYL CARRIER PROTEIN COMPONENTS

Biosynthese von 6‐Methylsalicylsäure