红霉素生物合成的生物化学和基因学

从基因重组技术问世以来,对红霉素(大环内酯类抗生素)生物合成的生物化学和基因学的研究到目前已经累积了大量的研究结果。就这2个领域中对红霉素生物合成的研究历史、研究现状和研究前景(组合生物合成)作以简要的介绍。     

The Biosynthesis of Thiol- and Thioether-containing Cofactors and Secondary Metabolites Catalyzed by Radical S-Adenosylmethionine Enzymes

Sulfur atoms are present as thiol and thioether functional groups in amino acids, coenzymes, cofactors, and various products of secondary metabolic pathways. The biosynthetic pathways for several sulfur-containing biomolecules require the substitution of sulfur for hydrogen at unreactive aliphatic or electron-rich aromatic carbon atoms. Examples discussed in this review include biotin, lipoic acid, methylthioether modifications found in some nucleic acids and proteins, and thioether cross-links found in peptide natural products. Radical S-adenosyl-l-methionine (SAM) enzymes use an iron-sulfur cluster to catalyze the reduction of SAM to methionine and a highly reactive 5′-deoxyadenosyl radical; this radical can abstract hydrogen atoms at unreactive positions, facilitating the introduction of a variety of functional groups. Radical SAM enzymes that catalyze sulfur insertion reactions contain a second iron-sulfur cluster that facilitates the chemistry, either by donating the cluster''s endogenous sulfide or by binding and activating exogenous sulfide or sulfur-containing substrates. The use of radical chemistry involving iron-sulfur clusters is an efficient anaerobic route to the generation of carbon-sulfur bonds in cofactors, secondary metabolites, and other natural products.     

Transformed Roots of Lupinus mutabilis: Induction, Culture and Isoflavone Biosynthesis

Transformed roots of Lupinus mutabilis cv. Potosi induced by Agrobacterium rhizogenes strain R1601 were cultured on Murashige and Skoog-based medium lacking kanamycin sulphate, or with this antibiotic at 40 mg l⁻¹. The neomycin phosphotransferase gene in the genome of transformed roots was confirmed by non-radioactive Southern hybridisation. Neomycin phosphotransferase protein was detected by ELISA. Transformed roots synthesised isoflavones, but not quinolizidine alkaloids; the latter are typical secondary metabolites of lupin normally produced in aerial parts of the plant. Genistein and 2′-hydroxygenistein, were the main secondary metabolites in cultured, transformed roots, whereas the glycoside genistin was more abundant in roots of non-transformed plants. Wighteone concentrations in transgenic roots were higher than those of non-transformed roots. Transformed roots produced twice the concentration of isoflavones compared with roots from non-transformed plants, indicating that Ri plasmid T-DNA genes modified isoflavone concentration and pattern of biosynthesis.     

The Redox Biochemistry of Protein Sulfenylation and Sulfinylation

Controlled generation of reactive oxygen species orchestrates numerous physiological signaling events (Finkel, T. (2011) Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7–15). A major cellular target of reactive oxygen species is the thiol side chain (RSH) of Cys, which may assume a wide range of oxidation states (i.e. −2 to +4). Within this context, Cys sulfenic (Cys-SOH) and sulfinic (Cys-SO₂H) acids have emerged as important mechanisms for regulation of protein function. Although this area has been under investigation for over a decade, the scope and biological role of sulfenic/sulfinic acid modifications have been recently expanded with the introduction of new tools for monitoring cysteine oxidation in vitro and directly in cells. This minireview discusses selected recent examples of protein sulfenylation and sulfinylation from the literature, highlighting the role of these post-translational modifications in cell signaling.     

Parathyroid Hormone-Related Protein: Biochemistry and Molecular Biology

Abstract

This article critically reviews the current state of knowledge regarding the recently identified and cloned novel hormone parathyroid hormone-related protein (PTHrP). PTHrP is produced by tumors associated with the syndrome of humoral hypercalcemia of malignancy giving rise to the parathyroid hormone (FTH)-like symptoms characteristic of the syndrome.

Areas that will be reviewed include identification, purification and cloning, localization, actions, and significance of PTHrP in cancers and normal physiology. The structure and regulation of the PTHrP gene that may be ancestrally related to the PTH gene will also be discussed.

Studies in vivo and in vitro with synthetic and recombinant PTHrP sequences and antibodies developed against them have established that the PTH-like actions of PTHrP are mediated via the N-terminal sequences, which show some limited sequence homology with PTH.

Evidence for PTH and non-PTH-like actions of PTHrP in normal physiology, which implicate a role for FTHrP in fetal and neonatal development, is also presented.     

DMSO, an Organic Cleanup Solvent for TCA/Acetone-Precipitated Proteins, Improves 2-DE Protein Analysis of Rice Roots

Protein extraction is a critical step in a two-dimensional electrophoresis (2-DE)-based proteomic analysis. Dimethyl sulfoxide (DMSO), a small organic molecule, is widely used as a solvent in biological sciences. In this study, we modified the cleanup step of the commonly used trichloroacetic acid (TCA)/acetone method of protein extraction by using 20?% DMSO/acetone as a solvent to wash TCA?Cacetone-precipitated pellets. The improved protocol (TCA/acetone?CDMSO) was compared with the TCA/acetone and phenol extraction method based on the protein yield, the number of spots, and resolution on 2-DE maps. TCA/acetone?CDMSO washing increased protein yield, and improved the resolution in 2-DE with less background, streaking, and smearing. This method also produced the highest number of protein spots. To our knowledge, the present study is the first to use DMSO as a cleanup solvent for TCA/acetone-precipitated proteins to enhance their quality prior to 2-DE.     

Roots: Molecular basis of biological regulation: Origins from feedback inhibition and allostery

One observes regulation at every biological level. Organisms, cells, and biochemical processes operate efficiently, normally wasting neither material nor energy, and adjusting their functions to external influences. Nature evidently has evolved mechanisms specifically dedicated to regulation at many levels. What is the molecular basis of this control? In the 1950s these molecular control mechanisms began to be explored seriously. The discoveries of feedback inhibition of enzyme activity were important because they gave an initial example of how regulation is achieved at the molecular level. We showed that certain enzymes are composed of two parts, one for catalysis and another distinct part present only to regulate this catalysis. Catalytic activity can be either stimulated or inhibited when a small molecule in the environment combines with the enzyme's regulatory site. In particular, a final product of a metabolic pathway generates a feedback loop which automatically limits its own excessive production, by combining with the regulatory site of an early enzyme in that pathway. Later studies showed that catalytic and regulatory structures of some enzymes could be separated physically. The discovery of regulatory sites and their interaction with molecules unrelated to their substrates was the basis for the generalized allosteric concept. This concept extends the regulatory site idea to a wide variety of processes such as control of gene expression, hormone action, and cellular growth.     

New Developments in Selenium Biochemistry: Selenocysteine Biosynthesis in Eukaryotes and Archaea

We used comparative genomics and experimental analyses to show that (1) eukaryotes and archaea, which possess the selenocysteine (Sec) protein insertion machinery contain an enzyme, O-phosphoseryl-transfer RNA (tRNA)^[Ser]Sec kinase (designated PSTK), which phosphorylates seryl-tRNA^[Ser]Sec to form O-phosphoseryl-tRNA^[Ser]Sec and (2) the Sec synthase (SecS) in mammals is a pyridoxal phosphate-containing protein previously described as the soluble liver antigen (SLA). SecS uses the product of PSTK, O-phosphoseryl-tRNA^[Ser]Sec, and selenophosphate as substrates to generate selenocysteyl-tRNA^[Ser]Sec. Sec could be synthesized on tRNA^[Ser]Sec from selenide, adenosine triphosphate (ATP), and serine using tRNA^[Ser]Sec, seryl-tRNA synthetase, PSTK, selenophosphate synthetase, and SecS. The enzyme that synthesizes monoselenophosphate is a previously identified selenoprotein, selenophosphate synthetase 2 (SPS2), whereas the previously identified mammalian selenophosphate synthetase 1 did not serve this function. Monoselenophosphate also served directly in the reaction replacing ATP, selenide, and SPS2, demonstrating that this compound was the active selenium donor. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that contain selenoproteins. X.-M. Xu and B. A. Carlson contributed equally to the studies described herein.     

Biosynthesis of cyclic carotenoids: Biochemistry and molecular genetics of the reaction sequence

Cyclic carotenids are found in the photosynthetic apparatus of higher plants. The biosynthesis of these pigments has been under investigation for two decades. Especially with in vitro systems from chromoplasts and unicellular cynaobacteria. new information has been obtained on the enzymatic reactions in carotenogenesis. Very recently, progress in the identification and cloning of several genes from the carotenogenic pathway has been made with bacteria, cyanobacteria and fungi. The new developments in biochemistry and molecular biology of cyclic carotenoids are reviewed, and the consequences of the results obtained by molecular genetics on the general understanding of the biosynthetic pathway of carotenoids are discussed, especially the desaturation and isomerization reactions.     

Iron-Sulfur (Fe/S) Protein Biogenesis: Phylogenomic and Genetic Studies of A-Type Carriers

Iron sulfur (Fe/S) proteins are ubiquitous and participate in multiple biological processes, from photosynthesis to DNA repair. Iron and sulfur are highly reactive chemical species, and the mechanisms allowing the multiprotein systems ISC and SUF to assist Fe/S cluster formation in vivo have attracted considerable attention. Here, A-Type components of these systems (ATCs for A-Type Carriers) are studied by phylogenomic and genetic analyses. ATCs that have emerged in the last common ancestor of bacteria were conserved in most bacteria and were acquired by eukaryotes and few archaea via horizontal gene transfers. Many bacteria contain multiple ATCs, as a result of gene duplication and/or horizontal gene transfer events. Based on evolutionary considerations, we could define three subfamilies: ATC-I, -II and -III. Escherichia coli, which has one ATC-I (ErpA) and two ATC-IIs (IscA and SufA), was used as a model to investigate functional redundancy between ATCs in vivo. Genetic analyses revealed that, under aerobiosis, E. coli IscA and SufA are functionally redundant carriers, as both are potentially able to receive an Fe/S cluster from IscU or the SufBCD complex and transfer it to ErpA. In contrast, under anaerobiosis, redundancy occurs between ErpA and IscA, which are both potentially able to receive Fe/S clusters from IscU and transfer them to an apotarget. Our combined phylogenomic and genetic study indicates that ATCs play a crucial role in conveying ready-made Fe/S clusters from components of the biogenesis systems to apotargets. We propose a model wherein the conserved biochemical function of ATCs provides multiple paths for supplying Fe/S clusters to apotargets. This model predicts the occurrence of a dynamic network, the structure and composition of which vary with the growth conditions. As an illustration, we depict three ways for a given protein to be matured, which appears to be dependent on the demand for Fe/S biogenesis.     

Biosynthesis of Violacein: Origins of Hydrogen,Nitrogen and Oxygen Atoms in the 2-Pyrrolidone Nucleus

The biosynthetic origins of the hydrogen, nitrogen and oxygen atoms in the pyrrolidone ring of violacein'were established by an anaylses of the ¹H, ¹³C NMR and MS spectra of its isotope-enriched metabolites. Feeding experiments of [2-²H] and [3-²H₂]tryptophans have revealed that the hydrogen in the pyrrolidone ring was derived from the methylene protons at the 3-position of tryptophan. The stereochemical fate of the prochiral hydrogens was determined to be in the retention of the pro-S hydrogen by these feeding experiments using [3R-²H] and [3S”-²H]tryptophans. The incorporation experiment of [α-¹⁵N]tryptophan demonstrated that the nitrogen atom in the ring originated from the α-amino group of tryptophan. The incorporation experiment of ¹⁸O₂ gas verified that all the oxygen atoms of violacein were derived from the molecular oxygen.     

Roots: Molecular basis of gene expression: Origins from the Pajama experiment

The Pajama (Pardee, Jacob, Monod) experiment provided a breakthrough in our understanding of the molecular mechanisms by which gene expression is regulated. Today, twenty-five years later it provides a paradigm for thinking about problems of gene expression, such as growth regulation and differentiation. From this experiment emerged entities such as repressors, regulatory genes, the operon as a group of jointly controlled genes, and messenger RNA.     

Biochemistry on a Leash: The Roles of Tether Length and Geometry in Signal Integration Proteins

We use statistical mechanics and simple ideas from polymer physics to develop a quantitative model of proteins whose activity is controlled by flexibly tethered ligands and receptors. We predict how the properties of tethers influence the function of these proteins and demonstrate how their tether length dependence can be exploited to construct proteins whose integration of multiple signals can be tuned. One case study to which we apply these ideas is that of the Wiskott-Aldrich Syndrome Proteins as activators of actin polymerization. More generally, tethered ligands competing with those free in solution are common phenomena in biology, making this an important specific example of a widespread biological idea.