Control of Stereoselectivity in Diverse Hapalindole Metabolites is Mediated by Cofactor‐Induced Combinatorial Pairing of Stig Cyclases

Stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by Stig cyclases through a three‐step cascade involving Cope rearrangement, 6‐exo‐trig cyclization, and a final electrophilic aromatic substitution. Reported here is a comprehensive study of all currently annotated Stig cyclases, revealing that these proteins can assemble into heteromeric complexes, induced by Ca2+, to cooperatively control the stereochemistry of hapalindole natural products.Die stereospezifische Bildung des polycyclischen Kerns der Hapalindole und Fischerindole wird durch Stig‐Cyclasen gesteuert, die eine dreistufige Kaskade aus Cope‐Umlagerung, 6‐exo‐trig‐Cyclisierung und elektrophiler aromatischer Substitution vermitteln. Die Proteine können sich induziert durch Ca2+ zu heterotrimeren Komplexen zusammenlagern, um auf kooperative Weise die Stereochemie zu steuern.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155506/1/ange201913686.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155506/2/ange201913686-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155506/3/ange201913686_am.pd

Structural Insights into the Function of the Nicotinate Mononucleotide:phenol/<i>p</i>‑cresol Phosphoribosyltransferase (ArsAB) Enzyme from <i>Sporomusa ovata</i>

Cobamides (Cbas) are cobalt (Co) containing tetrapyrrole-derivatives involved in enzyme-catalyzed carbon skeleton rearrangements, methyl-group transfers, and reductive dehalogenation. The biosynthesis of cobamides is complex and is only performed by some bacteria and achaea. Cobamides have an upper (<i>Coβ</i>) ligand (5′-deoxyadenosyl or methyl) and a lower (<i>Coα</i>) ligand base that contribute to the axial Co coordinations. The identity of the lower <i>Coα</i> ligand varies depending on the organism synthesizing the Cbas. The homoacetogenic bacterium <i>Sporomusa ovata</i> synthesizes two unique phenolic cobamides (i.e., Coα-(phenolyl/<i>p</i>-cresolyl)­cobamide), which are used in the catabolism of methanol and 3,4-dimethoxybenzoate by this bacterium. The <i>S. ovata</i> ArsAB enzyme activates a phenolic lower ligand prior to its incorporation into the cobamide. ArsAB consists of two subunits, both of which are homologous (∼35% identity) to the well-characterized <i>Salmonella enterica</i> CobT enzyme, which transfers nitrogenous bases such as 5,6-dimethylbenzimidazole (DMB) and adenine, but cannot utilize phenolics. Here we report the three-dimensional structure of ArsAB, which shows that the enzyme forms a pseudosymmetric heterodimer, provide evidence that only the ArsA subunit has base:phosphoribosyl-transferase activity, and propose a mechanism by which phenolic transfer is facilitated by an activated water molecule

Bioprospecting for Trichothecene 3-O-Acetyltransferases in the Fungal Genus Fusarium Yields Functional Enzymes with Different Abilities To Modify the Mycotoxin Deoxynivalenol▿ †

The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that are able to modify DON and reduce its toxicity. Recent work has highlighted differences in the activities of TRI101 from two different species of Fusarium (F. graminearum and F. sporotrichioides), but little is known about the relative activities of TRI101/TRI201 enzymes produced by other species of Fusarium. We cloned TRI101 or TRI201 genes from seven different species of Fusarium and found genetic identity between sequences ranging from 66% to 98%. In vitro feeding studies using transformed yeast showed that all of the TRI101/TRI201 enzymes tested were able to acetylate DON; conversion of DON to 3-acetyl-deoxynivalenol (3ADON) ranged from 50.5% to 100.0%, depending on the Fusarium species from which the gene originated. A time course assay showed that the rate of acetylation varied from species to species, with the gene from F. sporotrichioides having the lowest rate. Steady-state kinetic assays using seven purified enzymes produced catalytic efficiencies for DON acetylation ranging from 6.8 × 104 M−1·s−1 to 4.7 × 106 M−1·s−1. Thermostability measurements for the seven orthologs ranged from 37.1°C to 43.2°C. Extended sequence analysis of portions of TRI101/TRI201 from 31 species of Fusarium (including known trichothecene producers and nonproducers) suggested that other members of the genus may contain functional TRI101/TRI201 genes, some with the potential to outperform those evaluated in the present study

Flavin-Dependent Monooxygenases NotI and NotI′ Mediate Spiro-Oxindole Formation in Biosynthesis of the Notoamides

The fungal indole alkaloids are a unique class of complex molecules that have a characteristic bicyclo[2.2.2]diazaoctane ring and frequently contain a spiro-oxindole moiety. While various strains produce these compounds, an intriguing case involves the formation of individual antipodes by two unique species of fungi in the generation of the potent anticancer agents (+)- and (-)-notoamide A. NotI and NotI′ have been characterized as flavin-dependent monooxygenases that catalyze epoxidation and semi-Pinacol rearrangement to form the spiro-oxindole center within these molecules. This work elucidates a key step in the biosynthesis of the notoamides and provides an evolutionary hypothesis regarding a common ancestor for production of enantiopure notoamides. </div