Sharpless and co-workers [1] used the term on-water to describe the substantial rate acceleration that is observed when some insoluble organic reactants are stirred in aqueous suspension. We now propose a mechanism that accounts for the phenomenon of on-water catalysis. Three of the observations of Sharpless and co-workers are directly pertinent to the mechanism we propose below. Firstly, the reaction mixture must be heterogeneous, that is, there must be an interface between the organic reactants and water. The presence of some methanol in the aqueous phase made little difference to the rate of the reaction, "but the rate slowed considerably when enough methanol was used to make the reaction homogeneous." Secondly, the interface must be with an aqueous phase. An emulsion formed with the organic reactants in perfluorohexane gave an only slightly enhanced rate. Thirdly, there was a significant solvent isotope effect, with a noticeably slower rate in D 2 O.Our proposed mechanism is supported by two additional considerations. One is that all of the reactions that have been described as accelerated by the on-water effect (with the possible exception of those of metal complexes) are also known to be subject to acid catalysis. [1,2] This suggests that acid-base chemistry at the interface is responsible. Hence the second consideration is our recently developed model that explains the intrinsic charge that develops at the interface of water with low relative permittivity (low dielectric constant) materials.[3] This model accounts for the numerous observations made over many decades that the surface of water at these interfaces, whether they are with gas, liquid or solid, becomes negatively charged by the strong adsorption of hydroxide ions. [4,5] The adsorption equilibrium constant can be estimated from the pH dependence of the zeta potentials of oil drops in water to be at least 10 8 .[6]At first this seems unlikely to account for acid catalysis at the same interface, but a little reflection reveals that this counterintuitive result is to be expected. Consider a substrate that is activated by protonation. Reaction with water at the interface results in the protonated activated substrate and a hydroxide ion that is stabilised by its strong adsorption at the interface. This drives the protonation equilibrium of the substrate [Eq. (1)] strongly to the right and accounts for acid catalysis even in neutral solution.This mechanism accounts for all of the available evidence described above. It requires water, at the interface with the organic reactants, which provides the conditions for the protonation of the substrate, driven by the adsorption of the hydroxide ion product, with the associated deuterium isotope effect, leading to acid catalysis and the enhanced rate. We now describe additional experimental evidence consistent with this mechanism. We chose to examine another Diels-Alder reaction, that between cyclopentadiene and dimethylfumarate (Scheme 1). In addition to having a rate that is convenient to measure, this reactio...

Novofumigatonin (1), isolated from the fungus Aspergillus novofumigatus, is a heavily oxygenated meroterpenoid containing a unique orthoester moiety. Despite the wide distribution of orthoesters in nature and their biological importance, little is known about the biogenesis of orthoesters. Here we show the elucidation of the biosynthetic pathway of 1 and the identification of key enzymes for the orthoester formation by a series of CRISPR-Cas9-based gene-deletion experiments and in vivo and in vitro reconstitutions of the biosynthesis. The novofumigatonin pathway involves endoperoxy compounds as key precursors for the orthoester synthesis, in which the Fe(II)/α-ketoglutarate-dependent enzyme NvfI performs the endoperoxidation. NvfE, the enzyme catalyzing the orthoester synthesis, is an Fe(II)-dependent, but cosubstrate-free, endoperoxide isomerase, despite the fact that NvfE shares sequence homology with the known Fe(II)/α-ketoglutarate-dependent dioxygenases. NvfE thus belongs to a class of enzymes that gained an isomerase activity by losing the α-ketoglutarate-binding ability.

The bacterial family Vibrionaceae (vibrios) is considered a major player in the degradation of chitin, the most abundant polymer in the marine environment; however, the majority of studies on the topic have focused on a small number of Vibrio species. In this study, we analyzed the genomes of two vibrios to assess their genetic potential for the degradation of chitin. We then used transcriptomics and metabolomics to demonstrate that chitin strongly affects these vibrios at both the transcriptional and metabolic levels. We observed a strong increase in production of secondary metabolites, suggesting an ecological role for these molecules during chitin colonization in the marine environment.

Tropodithietic acid (TDA) is an antibacterial compound produced by some Phaeobacter and Ruegeria spp. of the Roseobacter clade. TDA production is studied in marine broth or agar since antibacterial activity in other media is not observed. The purpose of this study was to determine how TDA production is influenced by substrate components. High concentrations of ferric citrate, as present in marine broth, or other iron sources were required for production of antibacterially active TDA. However, when supernatants of noninhibitory, low-iron cultures of Phaeobacter inhibens were acidified, antibacterial activity was detected in a bioassay. The absence of TDA in nonacidified cultures and the presence of TDA in acidified cultures were verified by liquid chromatography-high-resolution mass spectrometry. A noninhibitory TDA analog (pre-TDA) was produced by P. inhibens, Ruegeria mobilis F1926, and Phaeobacter sp. strain 27-4 under low-iron concentrations and was instantaneously converted to TDA when pH was lowered. Production of TDA in the presence of Fe 3؉ coincides with formation of a dark brown substance, which could be precipitated by acid addition. From this brown pigment TDA could be liberated slowly with aqueous ammonia, and both direct-infusion mass spectrometry and elemental analysis indicated a [Fe III (TDA) 2 ] x complex. The pigment could also be produced by precipitation of pure TDA with FeCl 3 . Our results raise questions about how biologically active TDA is produced in natural marine settings where iron is typically limited and whether the affinity of TDA to iron points to a physiological or ecological function of TDA other than as an antibacterial compound.T ropodithietic acid (TDA) is an antibacterial compound produced by members of the genera Ruegeria and Phaeobacter of the Roseobacter clade as well as by Pseudovibrio strains (Alphaproteobacteria) (1-4). Several studies have focused on TDA-producing bacteria, especially as colonizers and symbionts of micro-and macroalgae (5-9) and as potential probiotics for marine aquaculture (10-14). Thiotropocin, a tautomer of tropodithietic acid, was described as the antibiotic produced by a Pseudomonas species isolate from a Japanese soil sample (15, 16). A computational study has revealed that thiotropocin and tropodithietic acid exist as a pair of interconverting tautomers (17), and it has been speculated that thiotropocin was formed as an artifact from TDA during the p-bromobenzyl thioether derivatization used for its X-ray structure elucidation. To simplify matters, in the present study we refer to both compounds as TDA. The complete biosynthetic pathway of TDA has not been described; however, biosynthesis of TDA from glucose was investigated in a Pseudomonas sp., and shikimate and phenylpyruvate were identified as intermediate compounds (18). Twelve genes that are necessary for TDA production were found by random transposon mutagenesis in a Ruegeria (Silicibacter) sp. strain TM1040 (6), and a possible biosynthetic pathway has been suggested (19); however, the ...

Nunamycin and nunapeptin are two antimicrobial cyclic lipopeptides (CLPs) produced by Pseudomonas fluorescens In5 and synthesized by nonribosomal synthetases (NRPS) located on two gene clusters designated the nun–nup regulon. Organization of the regulon is similar to clusters found in other CLP‐producing pseudomonads except for the border regions where putative LuxR‐type regulators are located. This study focuses on understanding the regulatory role of the LuxR‐type‐encoding gene nunF in CLP production of P. fluorescens In5. Functional analysis of nunF coupled with liquid chromatography–high‐resolution mass spectrometry (LC‐HRMS) showed that CLP biosynthesis is regulated by nunF. Quantitative real‐time PCR analysis indicated that transcription of the NRPS genes catalyzing CLP production is strongly reduced when nunF is mutated indicating that nunF is part of the nun–nup regulon. Swarming and biofilm formation was reduced in a nunF knockout mutant suggesting that these CLPs may also play a role in these phenomena as observed in other pseudomonads. Fusion of the nunF promoter region to mCherry showed that nunF is strongly upregulated in response to carbon sources indicating the presence of a fungus suggesting that environmental elicitors may also influence nunF expression which upon activation regulates nunamycin and nunapeptin production required for the growth inhibition of phytopathogens.