A Dehydratase Domain in Ambruticin Biosynthesis Displays Additional Activity as a Pyran‐Forming Cyclase

Hydropyran rings are a common structural motif in reduced polyketides. Information on their biosynthetic formation and particularly the biochemical characterization of the responsible enzymes has only been reported in few cases. The dehydratase domain AmbDH3 from the ambruticin polyketide synthase was investigated. Through in vitro assay of the recombinant domain with synthetically‐derived substrate surrogates, it was shown that it has a second catalytic activity as a cyclase that performs oxa‐conjugate addition. Probing AmbDH3 with synthetic substrate analogues revealed stereoselectivity and substrate tolerance in both substeps. This is the first characterization of a pyran‐forming cyclase from a cis‐AT PKS system and the first report of a polyketide synthase domain with this kind of dual activity. Finally, it was revealed that this domain shows potential for application in chemoenzymatic synthesis.     

Enzymology of Pyran Ring A Formation in Salinomycin Biosynthesis

Tetrahydropyran rings are a common feature of complex polyketide natural products, but much remains to be learned about the enzymology of their formation. The enzyme SalBIII from the salinomycin biosynthetic pathway resembles other polyether epoxide hydrolases/cyclases of the MonB family, but SalBIII plays no role in the conventional cascade of ring opening/closing. Mutation in the salBIII gene gave a metabolite in which ring A is not formed. Using this metabolite in vitro as a substrate analogue, SalBIII has been shown to form pyran ring A. We have determined the X‐ray crystal structure of SalBIII, and structure‐guided mutagenesis of putative active‐site residues has identified Asp38 and Asp104 as an essential catalytic dyad. The demonstrated pyran synthase activity of SalBIII further extends the impressive catalytic versatility of α+β barrel fold proteins.     

Acyl‐Chain Elongation Drives Ketosynthase Substrate Selectivity in trans‐Acyltransferase Polyketide Synthases

Type I modular polyketide synthases (PKSs), which are responsible for the biosynthesis of many biologically active agents, possess a ketosynthase (KS) domain within each module to catalyze chain elongation. Acylation of the KS active site Cys residue is followed by transfer to malonyl‐ACP to yield an extended β‐ketoacyl chain (ACP=acyl carrier protein). To date, the precise contribution of KS selectivity in controlling product fidelity has been unclear. Six KS domains from trans‐acyltransferase (trans‐AT) PKSs were subjected to a mass spectrometry based elongation assay, and higher substrate selectivity was identified for the elongating step than in preceding acylation. A close correspondence between the observed KS selectivity and that predicted by phylogenetic analysis was seen. These findings provide insights into the mechanism of KS selectivity in this important group of PKSs, can serve as guidance for engineering, and show that targeted mutagenesis can be used to expand the repertoire of acceptable substrates.     

The Interplay between a Multifunctional Dehydratase Domain and a C‐Methyltransferase Effects Olefin Shift in Ambruticin Biosynthesis

The olefin shift is an important modification during polyketide biosynthesis. Particularly for type I cis‐AT PKS, little information has been gained on the enzymatic mechanisms involved. We present our in vitro investigations on the olefin shift occurring during ambruticin biosynthesis. The unique, multifunctional domain AmbDH4 catalyzes consecutive dehydration, epimerization, and enoyl isomerization. The resulting 3‐enethioate is removed from the equilibrium by α‐methylation catalyzed by the highly specific C‐methyltransferase AmbM. This thermodynamically unfavorable overall process is enabled by the high, concerted substrate specificity of the involved enzymes. AmbDH4 shows close relationship to DH domains and initial mechanistic studies suggest that the olefin shift occurs via a similar proton‐shuttling mechanism as previously described for EI domains from trans‐AT‐PKS.     

Insights into the Pamamycin Biosynthesis

Pamamycins are macrodiolides of polyketide origin with antibacterial activities. Their biosynthesis has been proposed to utilize succinate as a building block. However, the mechanism of succinate incorporation into a polyketide was unclear. Here, we report identification of a pamamycin biosynthesis gene cluster by aligning genomes of two pamamycin‐producing strains. This unique cluster contains polyketide synthase (PKS) genes encoding seven discrete ketosynthase (KS) enzymes and one acyl‐carrier protein (ACP)‐encoding gene. A cosmid containing the entire set of genes required for pamamycin biosynthesis was successfully expressed in a heterologous host. Genetic and biochemical studies allowed complete delineation of pamamycin biosynthesis. The pathway proceeds through 3‐oxoadipyl‐CoA, a key intermediate in the primary metabolism of the degradation of aromatic compounds. 3‐Oxoadipyl‐CoA could be used as an extender unit in polyketide assembly to facilitate the incorporation of succinate.     

Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation

While type II polyketide synthases (PKSs) are known for producing aromatic compounds, a phylogenetically new subfamily of type II PKSs have been recently proposed to synthesize polyene structures. Here we report in vitro analysis of such a type II PKS, IgaPKS for ishigamide biosynthesis. The ketoreductase (Iga13) and dehydratase (Iga16) were shown to catalyze the reduction of a β‐keto group and dehydration of a β‐hydroxy group, respectively, to form a trans double bond. Incubation of the acyl carrier protein (Iga10), the ketosynthase/chain length factor complex (Iga11–Iga12), Iga13 and Iga16 with malonyl and hexanoyl‐CoAs and NADPH followed by KOH hydrolysis resulted in the formation of four unsaturated carboxylic acids (C₈, C₁₀, C₁₂, and C₁₄), indicating that IgaPKS catalyzes tetraene formation by repeating the cycle of condensation, keto‐reduction and dehydration with strict stereo‐specificity. We propose “highly reducing type II PKS subfamily” for the polyene‐producing type II PKSs.     

Insights into 6‐Methylsalicylic Acid Bio‐assembly by Using Chemical Probes

Chemical probes capable of reacting with KS (ketosynthase)‐bound biosynthetic intermediates were utilized for the investigation of the model type I iterative polyketide synthase 6‐methylsalicylic acid synthase (6‐MSAS) in vivo and in vitro. From the fermentation of fungal and bacterial 6‐MSAS hosts in the presence of chain termination probes, a full range of biosynthetic intermediates was isolated and characterized for the first time. Meanwhile, in vitro studies of recombinant 6‐MSA synthases with both nonhydrolyzable and hydrolyzable substrate mimics have provided additional insights into substrate recognition, providing the basis for further exploration of the enzyme catalytic activities.     

Solving the Puzzle of One‐Carbon Loss in Ripostatin Biosynthesis

Ripostatin is a promising antibiotic that inhibits RNA polymerase by binding to a novel binding site. In this study, the characterization of the biosynthetic gene cluster of ripostatin, which is a peculiar polyketide synthase (PKS) hybrid cluster encoding cis‐ and trans‐acyltransferase PKS genes, is reported. Moreover, an unprecedented mechanism for phenyl acetic acid formation and loading as a starter unit was discovered. This phenyl‐C2 unit is derived from phenylpyruvate (phenyl‐C3) and the mechanism described herein explains the mysterious loss of one carbon atom in ripostatin biosynthesis from the phenyl‐C3 precursor. Through in vitro reconstitution of the whole loading process, a pyruvate dehydrogenase like protein complex was revealed that performs thiamine pyrophosphate dependent decarboxylation of phenylpyruvate to form a phenylacetyl‐S ‐acyl carrier protein species, which is supplied to the subsequent biosynthetic assembly line for chain extension to finally yield ripostatin.     

Mechanistic Insights into the Mode of Action of Bifunctional Pyrrolidine‐Squaramide‐Derived Organocatalysts

The catalytic modes of action of three squaramide‐derived bifunctional organocatalysts have been investigated using DFT methods. The [5+2] cycloaddition between oxidopyrylium ylides and enals was used as the model reaction. Two primary modes were possible for the different catalysts studied. The preference for one mode over the other was due to the possibility of additional favorable π–π interactions between the hydrogen‐bond activated pyrylium ylide and an electron‐deficient aromatic ring bonded to the squaramide NH group. The model can be extended to other reactions catalyzed by the same catalysts, such as formal [2+2] cycloadditions between nitroalkenes and α,β‐unsaturated aldehydes. The computational results were in excellent concurrence with the available experimental reports on the observed total enantioselectivity and differences in diastereoselectivity depending on the substrate and the reaction.     

Rational Design of Modular Polyketide Synthases: Morphing the Aureothin Pathway into a Luteoreticulin Assembly Line

The unusual nitro‐substituted polyketides aureothin, neoaureothin (spectinabilin), and luteoreticulin, which are produced by diverse Streptomyces species, point to a joint evolution. Through rational genetic recombination and domain exchanges we have successfully reprogrammed the modular (type I) aur polyketide synthase (PKS) into a synthase that generates luteoreticulin. This is the first rational transformation of a modular PKS to produce a complex polyketide that was initially isolated from a different bacterium. A unique aspect of this synthetic biology approach is that we exclusively used genes from a single biosynthesis gene cluster to design the artificial pathway, an avenue that likely emulates natural evolutionary processes. Furthermore, an unexpected, context‐dependent switch in the regiospecificity of a pyrone methyl transferase was observed. We also describe an unprecedented scenario where an AT domain iteratively loads an extender unit onto the cognate ACP and the downstream ACP. This aberrant function is a novel case of non‐colinear behavior of PKS domains.     

Heteroaromatic Belts through Fold‐in Synthesis: Mechanistic Insights into a Macrocycle‐Templated Friedel–Crafts Alkylation

Direct alkylation of 9,9′,9′′‐triethyl[2.2.2](2,7)carbazolophane with dimethoxymethane or paraformaldehyde affords a belt‐like heteroaromatic structure, which forms as a kinetic product in acid‐catalyzed condensations. In a competing, thermodynamically favored process, polymeric structures are formed by a largely regioselective condensation of stereochemically rigid “semi‐belts”. The relationship between these reactivity routes is rationalized in terms of strain release and differential reversibility of consecutive condensation steps.     

Stereochemical Studies of the Karlotoxin Class Using NMR Spectroscopy and DP4 Chemical‐Shift Analysis: Insights into their Mechanism of Action

After publication of karlotoxin 2 (KmTx2; 1 ), the harmful algal bloom dinoflagellate Karlodinium sp. was collected and scrutinized to identify additional biologically active complex polyketides. The structure of 1 was validated and revised at C49 using computational NMR tools including J‐based configurational analysis and chemical‐shift calculations. The characterization of two new compounds [KmTx8 ( 2 ) and KmTx9 ( 3 )] was achieved through overlaid 2D HSQC NMR techniques, while the relative configurations were determined by comparison to 1 and computational chemical‐shift calculations. The detailed evaluation of 2 using the NCI‐60 cell lines, NMR binding studies, and an assessment of the literature supports a mode of action (MoA) for targeting cancer‐cell membranes, especially of cytostatic tumors. This MoA is uniquely different from that of current agents employed in the control of cancers for which 2 shows sensitivity.     

Aldo‐X Bifunctional Building Blocks for the Synthesis of Heterocycles

Compounds containing oxygen, nitrogen, or sulfur atoms inside the rings are attracting much attention and interest due to their biological importance. In recent years, several methods for the synthesis of such molecules have been reported by using aldo‐X bifunctional building blocks (AXB3 s) as substrates; these are a wide class of organic molecules that contain at least two reactive sites, among them, one aldehyde, acetal, or semiacetal group was involved. Because of the multiple reactivities, AXB3 s are widely used in the one‐pot synthesis of biologically important heterocycles. This review summarizes the synthesis of important heterocycles by using AXB3 s as pivotal components in establishing multicomponent reactions, tandem reactions, and so forth. In many cases, the established reaction systems with AXB3 s were characterized by some green properties, such as easy access to the substrate, mild and environmentally benign conditions, and wide scope of the substrate.     

Molecular‐Level Insights into Intrinsic Peroxidase‐Like Activity of Nanocarbon Oxides

Nanocarbon oxides have been proved to possess great peroxidase‐like activity, catalyzing the oxidation of many peroxidase substrates, such as 3,3′,5,5′‐tetramethylbenzidine (TMB) and o‐phenylenediamine dihydrochloride (OPD), accompanied by a significant color change. This chromogenic reaction is widely used to detect glucose and occult blood. The chromogenic reaction was intensively investigated with density functional theory and molecular‐level insights into the nature of peroxidase‐like activity were gained. A radical mechanism was unraveled and the carboxyl groups of nanocarbon oxides were identified as the reactive sites. Aromatic domains connected with the carboxyl groups were critical to the peroxidase‐like activity.     

Palladium(0)‐Catalyzed Iminohalogenation of Alkenes: Synthesis of 2‐Halomethyl Dihydropyrroles and Mechanistic Insights into the Alkyl Halide Bond Formation

Although the advances on carbon halide reductive elimination have been made, the alkyl bromide and chloride analogues remain a challenge. Here, a palladium(0)‐catalyzed iminohalogenation of γ,δ‐unsaturated oxime esters is described, and the use of electron‐poor phosphine ligands proved to be crucial to promoting alkyl bromide and chloride reductive elimination. Furthermore, S_N2‐type alkyl bromide and chloride reductive elimination has also been established.