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Research has been conducted on the epothilones, hybrid polyketide/nonribosomal peptide antitumor agents. The expression and purification of Escherichia coli enzymes involved in the epothilone production have been carried out and the results are reported.

Research has been conducted on the amino acids found in proteins and forming peptide antibiotics by specific oxidation at the beta carbon. The strategy in the peptide antibiotic biosynthesis has been developed and discussed.

Mutational analysis was performed to gain insight into the switch activity mechanistic of ergot alkaloid biosynthesis which are diverse class of fungal-derived indole alkaloid natural products with potent pharmacological activities. The results showed the old yellow enzyme homologue EasA from the ergot biosynthetic gene cluster of Aspergillus fumigatus to act on chanoclavine-I aldehyde to yield festuclavine while EasA from Neotyphodium lolii produces agroclavine.

A homologue of Old Yellow Enzyme encoded in he Aspergillus fumigatus ergot gene cluster has catalyzed reduction of the [laph],[beta] unsaturated alkene of chanoclavine-1 aldehyde. The reduction, which has produced dihydrochanoclavine aldehyde, has facilitated an intramolecular reaction between a secondary amine and aldehyde to form the D ring of the ergot alkaloid structural framework.

The specific roles for acid-base catalysis is strictosidine synthase are described by using the data derived from kinetic isotope effects (KIE), rate dependence of pH, and comparisons to a nonenzymatic Pictet-Spengler reaction. The studies have shown that a spiroindolenine intermediate, which is often invoked in the Pictet-Spengler mechanism, does not occur.

Triptamine analogs were fed to Catharanthus roseus to demonstrate that the enzymes of this pathway can turnover unnatural substrates to make new alkaloids. It is also shown that the electronic and steric properties of the non-natural substrates impact how these substrates partition among the branches of the terpene indole alkaloids (TIA) pathway.

Unnatural substrates with EpoA and EpoB are examined to investigate the enzymatic construction of alternate heterocyclic structures, and the subsequent elongation of these products by EpoC. These studies with the early enzymes of the epothilone biosynthesis cluster suggest that combinatorial biosynthesis may be a viable means for producing a variety of epothilone analogues that incorporate diversity into the heterocycle starter unit.

A new study investigates the effect of the outer saccharide residues of N-linked glycans on peptide conformation.

Two-dimensional 1H nuclear magnetic resonance spectrometry was done to characterize the structures of nonglycosylated and glycosylated peptides in water. The results indicated that asparagine-linked glycosylation induces a conformational change in the backbone structure of small peptides in aqueous solution. While the nonglycosylated peptide was found to adopt an Asx-turn conformation which is important for N-linked glycosylation, the glycosylated peptide exhibited a compact type I beta-turn conformation normally seen in the final native protein structure.

Catharanthus roseusmonoterpene indole alkaloid analogs have been produced via a combination of biosynthetic and chemical strategies. Specifically, introduction of a chemical handlea chlorine or a bromineinto the target molecule by mutasynthesis, followed by postbiosynthetic chemical derivatization using Pd-catalyzed Suzuki-Miyaura cross-coupling reactions robustly afforded aryl and heteroaryl analogs of these alkaloids. [ABSTRACT FROM AUTHOR]

The Pictet-Spengler reaction, which yields either a β-carboline or a tetrahydroquinoline product from an aromatic amine and an aldehyde, is widely utilized in plant alkaloid biosynthesis. Here we deconvolute the role that the biosynthetic enzyme strictosidine synthase plays in catalyzing the stereoselective synthesis of a β-carboline product. Notably, the rate-controlling step of the enzyme mechanism, as identified by the appearance of a primary kinetic isotope effect (KIE), is the rearomatization of a positively charged intermediate. The KIE of a nonenzymatic Pictet-Spengler reaction indicates that rearomatization is also rate-controlling in solution, suggesting that the enzyme does not significantly change the mechanism of the reaction. Additionally, the pH dependence of the solution and enzymatic reactions provides evidence for a sequence of acid-base catalysis steps that catalyze the Pictet-Spengler reaction. An additional acid-catalyzed step, most likely protonation of a carbinolamine intermediate, is also significantly rate controlling. We propose that this step is efficiently catalyzed by the enzyme. Structural analysis of a bisubstrate inhibitor bound to the enzyme suggests that the active site is exquisitely tuned to correctly orient the iminium intermediate for productive cyclization to form the diastereoselective product. Furthermore, ab initio calculations suggest the structures of possible productive transition states involved in the mechanism. Importantly, these calculations suggest that a spiroindolenine intermediate, often invoked in the Pictet-Spengler mechanism, does not occur. A detailed mechanism for enzymatic catalysis of the β-carboline product is proposed from these data. [ABSTRACT FROM AUTHOR]

Here we report the one-pot, cell-free enzymatic synthesis of the plant monoterpene nepetalactol starting from the readily available geraniol. A pair of orthogonal cofactor regeneration systems permitted NAD + -dependent geraniol oxidation followed by NADPH-dependent reductive cyclization without isolation of intermediates. The orthogonal cofactor regeneration system maintained a high ratio of NAD + to NADH and a low ratio of NADP + to NADPH. The overall reaction contains four biosynthetic enzymes, including a soluble P450; and five accessory and cofactor regeneration enzymes. Furthermore, addition of a NAD + -dependent dehydrogenase to the one-pot mixture led to ~1 g/L of nepetalactone, the active cat- attractant in catnip.

The enzymatic basis for quinine 1 biosynthesis was investigated. Transcriptomic data from the producing plant led to the discovery of three enzymes involved in the early and late steps of the pathway. A medium-chain alcohol dehydrogenase (CpDCS) and an esterase (CpDCE) yielded the biosynthetic intermediate dihydrocorynantheal 2 from strictosidine aglycone 3 . Additionally, the discovery of an O -methyltransferase specific for 6'-hydroxycinchoninone 4 suggested the final step order to be cinchoninone 16/17 hydroxylation, methylation, and keto-reduction.