Skipping in a hybrid polyketide synthase. Evidence for ACP-to-ACP chain transfer

A tetraketide synthase containing a loading module (LM), the extension modules erythromycin module 1, rapamycin module 2, and erythromycin module 2 (LM-Ery1-Rap2-Ery2-TE), when expressed in Saccharopolyspora erythraea strain JC2, produced as previously reported a mixture of tetraketide lactones (minor products) and triketide lactones (major products). Several alternative plausible mechanisms by which this "skipping" phenomenon might occur may be proposed. Site-directed mutagenesis of the ketosynthase (KS) and acylcarrier protein (ACP) domains in the interpolated module has shown that skipping within the hybrid PKS involves passage of the growing polyketide through the interpolated module, by direct ACP-to-ACP transfer of the polyketide chain.     

Loss of co-linearity by modular polyketide synthases: a mechanism for the evolution of chemical diversity

Modular polyketide synthases biosynthesise natural products through successive Claisen-type condensations, where one module is responsible for one round of chain extension. This review describes recent findings where this rule of co-linearity is broken, either by one module being bypassed (skipping) or through one module being used for multiple chain extension events (stuttering).     

Structure,biosynthetic origin,and engineered biosynthesis of calcium-dependent antibiotics from Streptomyces coelicolor

The calcium-dependent antibiotic (CDA), from Streptomyces coelicolor, is an acidic lipopeptide comprising an N-terminal 2,3-epoxyhexanoyl fatty acid side chain and several nonproteinogenic amino acid residues. S. coelicolor grown on solid media was shown to produce several previously uncharacterized peptides with C-terminal Z-dehydrotryptophan residues. The CDA biosynthetic gene cluster contains open reading frames encoding nonribosomal peptide synthetases, fatty acid synthases, and enzymes involved in precursor supply and tailoring of the nascent peptide. On the basis of protein sequence similarity and chemical reasoning, the biosynthesis of CDA is rationalized. Deletion of SCO3229 (hmaS), a putative 4-hydroxymandelic acid synthase-encoding gene, abolishes CDA production. The exogenous supply of 4-hydroxymandelate, 4-hydroxyphenylglyoxylate, or 4-hydroxyphenylglycine re-establishes CDA production by the DeltahmaS mutant. Feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues.     

Asymmetric alkylation of diarylmethane derivatives. Improved results using methoxyethoxy substituent

Alkylation of 2-methoxyethoxyphenyl phenyl methane using sec-BuLi and (−)-sparteine has been carried out in excellent yields and up to 94% ee. The best results were obtained in allylation reactions but methylation, ethylation, benzylation and trimethylsilylation have all been carried out with acceptable ee.     

Two polymorphs of 20‐desmethyl‐β‐carotene

Two polymorphs of 20‐desmethyl‐β‐carotene (systematic name: 20‐nor‐β,β‐carotene), C₃₉H₅₄, in monoclinic and triclinic space groups, were formed in the same vial by recrystallization from pyridine and water. Each polymorph crystallizes with the complete molecule as the asymmetric unit, and the two polymorphs show differing patterns of disorder. The β end rings of both polymorphs have the 6‐s‐cis conformation, and are twisted out of the plane of the polyene chain by angles of −53.2 (8) and 47.3 (8)° for the monoclinic polymorph, and −43.6 (3) and 56.1 (3)° for the triclinic polymorph. The cyclohexene end groups are in the half‐chair conformation, but the triclinic polymorph shows disorder of one ring. Overlay of the molecules shows that they differ in the degree of nonplanarity of the polyene chains and the angles of twist of the end rings. The packing arrangements of the two polymorphs are quite different, with the monoclinic polymorph showing short intermolecular contacts of the disordered methyl groups with adjacent polyene chain atoms, and the triclinic polymorph showing π–π stacking interactions of the almost parallel polyene chains. The determination of the crystal structures of the two title polymorphs of 20‐desmethyl‐β‐carotene allows information to be gained regarding the structural effects on the polyene chain, as well as on the end groups, versus that of the parent compound β‐carotene. The absence of the methyl group is known to have an impact on various functions of the title compound.     

Substrate specificity of the acyl transferase domains of EpoC from the epothilone polyketide synthase

The production of epothilone mixtures is a direct consequence of the substrate tolerance of the module 3 acyltransferase (AT) domain of the epothilone polyketide synthase (PKS) which utilises both malonyl- and methylmalonyl-CoA extender units. Particular amino acid motifs in the active site of AT domains influence substrate selection for methylmalonyl-CoA (YASH) or malonyl-CoA (HAFH). This motif appears in hybrid form (HASH) in epoAT3 and may represent the molecular basis for the relaxed specificity of the domain. To investigate this possibility the AT domains from modules 2 and 3 of the epothilone PKS were examined in the heterologous DEBS1-TE model PKS. Substitution of AT1 of DEBS1-TE by epoAT2 and epoAT3 both resulted in functional PKSs, although lower yields of total products were observed when compared to DEBS1-TE (2% and 11.5% respectively). As expected, epoAT3 was significantly more promiscuous in keeping with its nature during epothilone biosynthesis. When the mixed motif (HASH) of epoAT3 within the hybrid PKS was mutated to HAFH (indicative of malonyl-CoA selection) it resulted in a non-productive PKS. When this mixed motif was converted to YASH (indicative of methylmalonyl-CoA selection) the selectivity of the hybrid PKS for methylmalonyl-CoA showed no statistically significant increase, and was associated with a loss of productivity.     

Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon Receptor as a Molecular Target of the Utrophin Modulator Ezutromid

Duchenne muscular dystrophy (DMD) is a fatal muscle‐wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first‐in‐class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent K_D of 50 nm  and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies.     

Time-dependent photoionization of azulene: competition between ionization and relaxation in highly excited states

Pump-probe photoionization has been used to map the relaxation processes taking place from highly vibrationally excited levels of the S(2) state of azulene, populated directly or via internal conversion from the S(4) state. Photoelectron spectra obtained by 1+2(') two-color time-resolved photoelectron imaging are invariant (apart from in intensity) to the pump-probe time delay and to the pump wavelength. This reveals a photoionization process which is driven by an unstable electronic state (e.g., doubly excited state) lying below the ionization potential. This state is postulated to be populated by a probe transition from S(2) and to rapidly relax via an Auger-like process onto highly vibrationally excited Rydberg states. This accounts for the time invariance of the photoelectron spectrum. The intensity of the photoelectron spectrum is proportional to the population in S(2). An exponential energy gap law is used to describe the internal conversion rate from S(2) to S(0). The vibronic coupling strength is found to be larger than 60+/-5 microeV.