Covering: 1997 to 2010. The angucycline group is the largest group of type II PKS-engineered natural products, rich in biological activities and chemical scaffolds. This stimulated synthetic creativity and biosynthetic inquisitiveness. The synthetic studies used five different strategies, involving Diels-Alder reactions, nucleophilic additions, electrophilic additions, transition-metal mediated cross-couplings and intramolecular cyclizations to generate the angucycline frames. Biosynthetic studies were particularly intriguing when unusual framework rearrangements by post-PKS tailoring oxidoreductases occurred, or when unusual glycosylation reactions were involved in decorating the benz[a]anthracene-derived cores. This review follows our previous reviews, which were published in 1992 and 1997, and covers new angucycline group antibiotics published between 1997 and 2010. However, in contrast to the previous reviews, the main focus of this article is on new synthetic approaches and biosynthetic investigations, most of which were published between 1997 and 2010, but go beyond, e.g. for some biosyntheses all the way back to the 1980s, to provide the necessary context of information.

GilOII has been unambiguously identified as the key enzyme performing the crucial C-C bond cleavage reaction responsible for the unique rearrangement of a benz[a]anthracene skeleton to the benzo[d]naphthopyranone backbone typical for the gilvocarcin type natural anticancer antibiotics. Further investigations of this enzyme led to the isolation of a hydroxy-oxepinone intermediate which allowed important conclusions regarding the cleavage mechanism.

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo-and NADP + -bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation. directed evolution | crystallographic structures | molecular dynamics | theozyme | enantioselectivity B iocatalysis is a common method of stereoselective ketone reduction (1). This approach often replaces multistep syntheses and uses renewable, biodegradable, and nontoxic reagents and mild conditions (2). Ketoreductases (KREDs), the most commonly used enzymes in industrial pharmaceutical synthesis (3), reduce a wide range of ketones to alcohols with high chemoselectivity and stereoselectivity. These enzymes have been engineered to synthesize alcohols as intermediates for the production of atorvastatin (Lipitor), montelukast (Singulair), and atazanavir (Reyetaz) (4).Small and almost symmetrical ketones, such as prochiral cyclopentanones, are attractive substrates that are difficult to reduce asymmetrically by chemical methods (5, 6). In particular, the enantiopure chiral alcohols derived from 3-oxacyclopentanone (1) and 3-thiacyclopentanone (2) are used in the synthesis of the pharmaceutical agents fosamprenavir and sulopenem, respectively (Fig. 1). Through a directed evolution (DE) program, Codexis, Inc. engineered a KRED obtained from Lactobacillus kefir for the reduction of 3-thiacyclopentanone (2) for the large-scale production of the antibiotic sulopenem. L. kefir KRED (WT) belongs to the short-chain dehydrogenase/reductase (SDR) family (7,8). Via DE, a variant containing 10 mutati...

The lipopeptidyl nucleoside antibiotics reperesented by A-90289, caprazamycin, and muraymycin, are structurally highlighted by a nucleoside core that contains a nonproteinogenic β-hydroxy-α-amino acid named 5′-C-glycyluridine (GlyU). Bioinformatic analysis of the biosynthetic gene clusters revealed a shared open reading frame encoding a protein with sequence similarity to serine hydroxymethyltransferases, resulting in the proposal that this shared enzyme catalyzes an aldol-type condensation with glycine and uridine-5′-aldehyde to furnish GlyU. Using LipK involved in A-90289 biosynthesis as a model, we now functionally assign and characterize the enzyme responsible for the C-C bond-forming event during GlyU biosynthesis as an l-threonine:uridine-5′-aldehyde transaldolase. Biochemical analysis revealed this transformation is dependent upon pyridoxal-5′-phosphate, the enzyme has no activity with alternative amino acids such as glycine or serine as aldol donors, and acetaldehyde is a co-product. Structural characterization of the enzyme product is consistent with stereochemical assignment as the threo diastereomer (5′S,6′S)-GlyU. Thus this enzyme orchestrates C-C bond breaking and formation with concomitant installation of two stereocenters to make a new l-α-amino acid with a nucleoside side chain.

Background: Therapeutic values of Valeriana officinalis have been associated with sesquiterpenes whose biosynthetic origins have remained enigmatic. Results: A cyclobutenyl intermediate in the catalytic cascade of valerena-1,10-diene synthase is reported. Conclusion: A new class of sesquiterpene synthases for the biosynthesis of sesquiterpenes harboring isobutenyl functional groups is proposed. Significance: Similar catalytic mechanisms from evolutionarily diverse organisms are proposed and portend sources for sesquiterpene diversity.