Rationally designed glycosylated premithramycins: hybrid aromatic polyketides using genes from three different biosynthetic pathways

Heterologous expression of the urdGT2 gene from the urdamycin producer Streptomyces fradiae Tü2717, which encodes a C-glycosyltransferase, into mutants of the mithramycin producer Streptomyces argillaceus, in which either one or all glycosyltransferases were inactivated, yielded four novel C-glycosylated premithramycin-type molecules. Structure elucidation revealed these to be 9-C-olivosylpremithramycinone, 9-C-mycarosylpremithramycinone, and their respective 4-O-demethyl analogues. In another experiment, both the urdGT2 gene from S. fradiae and the lanGT1 gene from S. cyanogenus, were coexpressed into a S. argillaceus mutant lacking the MtmGIV glycosyltransferase. This experiment, in which genes from three different organisms were combined, resulted in the production of 9-C-(olivo-1-4-olivosyl)premithramycinone. These results prove the unique substrate flexibility of the C-glycosyltransferase UrdGT2, which tolerates not only a variety of sugar-donor substrates, but also various acceptor substrates. The five new hybrid products also represent the first compounds, in which sugars were attached to a position that is normally unglycosylated. The successful combination of two glycosyltransferases in the latter experiment proves that the design of saccharide side chains by combinatorial biosynthetic methods is possible.     

The changes in the electronic structure of B2 FeAl alloy with a Fe antisite and absorbed hydrogen

The electronic structure and bonding in a B2 FeAl alloy with and without hydrogen interaction with a Fe antisite were computed using a density functional theoretical method. The hydrogen absorption turns out to be a favorable process. The hydrogen was found close to an octahedral site where one of its Al capped is replaced by a Fe antisite. The Fe–H distance is of 1.45 Å same as the Al–H distance.The density of states (DOS) curves show several peaks below the d metal band which is made up mostly of hydrogen based states (>50% H_1s) while the metal contribution in this region includes mainly s and p orbitals.An electron transfer of nearby 0.21e⁻ comes from the metal to the H. The overlap population values reveal metal–metal bond breaking, the intermetallic bond being the most affected. The H bond mainly with the Al atom and the reported Fe–H overlap population is much lower than that corresponding to FePd alloys and BCC Fe. The changes in the overlap population show the Fe–Al bond is weakened nearly 41.5% after H absorption, while the Fe–Fe bond is only weakened 34.5%. H also develops a stronger bond with the Al atoms. The main bond is developed with Al being twice stronger than Fe–H.     

Time-resolved photo-electron spectroscopy on mass-selected metal clusters using a regenerative femtosecond amplifier up to 100 Hz

A femtosecond photo-electron experiment is described which provides excellent conditions for measuring time-resolved photo-electron spectra of free, monodispersed cluster anions using repetition rates up to 100 Hz. Cluster anions are synthesized in an electric arc and subsequently cooled in a helium carrier gas. A time-of-flight spectrometer is used for mass separation of the negatively charged clusters. The kinetic energy of the photo-electrons is analyzed by a magnetic-bottle time-of-flight spectrometer, which guarantees a maximum collection efficiency. Femtosecond laser pulses are generated by a seeded regenerative Ti:Sa amplifier, which is externally pumped with the second harmonic of a diode-pumped solid-state Nd:YAG laser. A retroreflector mounted on a computer-controlled translation stage serves as a reproducible time delay of the probe pulse. The high energy output of the laser pulses (∼3 mJ) in combination with the variable repetition rate and the high stability of the amplified pulses provide excellent conditions for recording pump-probe photo-electron spectra of mass-separated cluster anions even at the fairly low ion density of pulsed plasma cluster sources. First results on the electron dynamics of the Pt₃ ^- cluster demonstrate the reliability of the whole system. Further experimental investigations will concentrate on electron-relaxation processes in transition- and noble-metal clusters as well as on the nuclear and transition-state dynamics of chemically reacted adsorbate clusters. Received: 7 January 2000 / Published online: 7 August 2000     

Deposition of endohedral fullerenes from a laser evaporation cluster source

Mass spectra of fullerenes doped with transition and rare earth metals (Sc, Y, La, Ce, Gd) reveal a variety of singly and doubly doped metallofullerenes. Characteristic oscillations of the relative mass intensities of the La- and Y-metallofullerenes suggest the existence of two different isomers of the corresponding doubly doped fullerenes. The yield of the plasma-generated clusters is sufficient to be used for an in situ deposition of mass-selected metallofullerenes onto a surface. In particular, endohedral fullerenes with 60 or less atoms are accessible for deposition by means of a laser vaporization cluster source. Scanning tunneling microscopy has been used to image Ce@C₆₀ and La@C₆₀ on highly oriented pyrolytic graphite. Received: 24 May 2000 / Accepted: 6 November 2000 / Published online: 9 February 2001     

Biosynthesis of the orthosomycin antibiotic avilamycin A: deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces viridochromogenes Tü57 and production of new antibiotics.

Background: Streptomyces viridochromogenes Tü57 is the producer of avilamycin A. The antibiotic consists of a heptasaccharide side chain and a polyketide-derived dichloroisoeverninic acid as aglycone. Molecular cloning and characterization of the genes governing the avilamycin A biosynthesis is of major interest as this information might set the direction for the development of new antimicrobial agents.Results: A 60-kb section of the S. viridochromogenes Tü57 chromosome containing genes involved in avilamycin biosynthesis was sequenced. Analysis of the DNA sequence revealed 54 open reading frames. Based on the putative function of the gene products a model for avilamycin biosynthesis is proposed. Inactivation of aviG4 and aviH, encoding a methyltransferase and a halogenase, respectively, prevented the mutant strains from producing the complete dichloroisoeverninic acid moiety resulting in the accumulation of new antibiotics named gavibamycins.Conclusions: The avilamycin A biosynthetic gene cluster represents an interesting system to study the formation and attachment of unusual deoxysugars. Several enzymes putatively responsible for specific steps of this pathway could be assigned. Two genes encoding enzymes involved in post-PKS tailoring reactions were deleted allowing the production of new analogues of avilamycin A.     

Variable temperature photoacoustic spectroscopy. II. Temperature characteristic and applications

The temperature dependent photoacoustic signal of a carbon black sample is investigated with a previously described low temperature photoacoustic cell. Examples are given to demonstrate the utility of variable temperature photoacoustic spectroscopy: The photocycle of bacteriorhodopsin which is contained in the purple membrane fragments of halobacterium halobium is studied. In this experiment some photointermediates are shown to be preferentially populated and frozen out at low temperatures. The temperature dependence of the electronic ground state of solid Fe-(III)-diethyl-dithiocarbamate is investigated for the first time spectroscopically in the visible and ultraviolet spectral range. Excitonic levels in semiconductors are for the first time inspected by the photoacoustic technique.     

Ultrafast hot-electron dynamics observed in Pt( -)(3) using time-resolved photoelectron spectroscopy

Time-resolved two-photon photoelectron spectra have been measured for free Pt( -)(3) using femtosecond pulses of 1.5 eV photon energy in a pump-probe configuration. The time-dependent photoelectron distribution reveals a lifetime of optically excited states of less than 70 fs. Such an unexpected fast electron relaxation in Pt( -)(3) suggests the existence of inelastic electron-electron scattering processes in a triatomic cluster which result in a lifetime similar to those of bulk metals.     

Type II polyketide synthases: gaining a deeper insight into enzymatic teamwork

This review covers advances in understanding of the biosynthesis of polyketides produced by type II PKS systems at the genetic, biochemical and structural levels.     

A Cryptic Polyene Biosynthetic Gene Cluster in Streptomyces calvus Is Expressed upon Complementation with a Functional bldA Gene

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