Fungal indole alkaloid biogenesis through evolution of a bifunctional reductase/Diels-Alderase

Prenylated indole alkaloids such as the calmodulin-inhibitory malbrancheamides and anthelmintic paraherquamides possess great structural diversity and pharmaceutical utility. Here, we report complete elucidation of the malbrancheamide biosynthetic pathway accomplished through complementary approaches. These include a biomimetic total synthesis to access the natural alkaloid and biosynthetic intermediates in racemic form and in vitro enzymatic reconstitution to provide access to the natural antipode (+)-malbrancheamide. Reductive cleavage of an L-Pro–L-Trp dipeptide from the MalG non-ribosomal peptide synthetase (NRPS) followed by reverse prenylation and a cascade of post-NRPS reactions culminates in an intramolecular [4+2] hetero-Diels–Alder (IMDA) cyclization to furnish the bicyclo[2.2.2]diazaoctane scaffold. Enzymatic assembly of optically pure (+)-premalbrancheamide involves an unexpected zwitterionic intermediate where MalC catalyses enantioselective cycloaddition as a bifunctional NADPH-dependent reductase/Diels–Alderase. The crystal structures of substrate and product complexes together with site-directed mutagenesis and molecular dynamics simulations demonstrate how MalC and PhqE (its homologue from the paraherquamide pathway) catalyse diastereo- and enantioselective cyclization in the construction of this important class of secondary metabolites

UDP-sulfoquinovose formation by Sulfolobus acidocaldarius

The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP-d-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme superfamily, together with SQD1 from Arabidopsis thaliana, the only UDP-sulfoquinovose synthase with known crystal structure. By comparison of sequence and structure of Agl3 and SQD1, putative catalytic amino acids of Agl3 were selected for mutational analysis. The obtained data suggest for Agl3 a modified dehydratase reaction mechanism. We propose that in vitro biosynthesis of UDP-sulfoquinovose occurs through an NAD(+)-dependent oxidation/dehydration/enolization/sulfite addition process. In the absence of a sulfur donor, UDP-d-glucose is converted via UDP-4-keto-d-glucose to UDP-d-glucose-5,6-ene, the structure of which was determined by (1)H and (13)C-NMR spectroscopy. During the redox reaction the cofactor remains tightly bound to Agl3 and participates in the reaction in a concentration-dependent manner. For the first time, the rapid initial electron transfer between UDP-d-glucose and NAD(+) could be monitored in a UDP-sulfoquinovose synthase. Deuterium labeling confirmed that dehydration of UDP-d-glucose occurs only from the enol form of UDP-4-keto-glucose. The obtained functional data are compared with those from other UDP-sulfoquinovose synthases. A divergent evolution of Agl3 from S.acidocaldarius is suggested. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00792-015-0730-9) contains supplementary material, which is available to authorized users