Jordan Nafie

The mechanism of the bacterial diterpene synthase ChtS from Chitinophaga japonensis was investigated. ChtS was found to produce two major diterpenes, 5/11-bicyclic chitino-2,5,9-triene (11), and the previously uncharacterized 6/10-bicyclic chitinoeunicellene (12). Deuterium labeling experiments, site-directed mutagenesis, and isolation of minor products cembrene C (13), cembrene A (14), and chitinol (15) support initial 1,14-cyclization, two sequential 1,2-hydride shifts, prior to bifurcation to 11 or 12, ultimately retaining the E-configured C-2/C-3 alkene.

Enzyme engineering serves as a powerful tool in biocatalysis, enabling the development of enzymes with improved stability, activity, and specificity for a range of academic, industrial, and pharmaceutical applications. However, a limited understanding of sequence–structure–function relationships in terpene synthases, the enzymes that form the complex polycyclic hydrocarbon skeletons of terpenoid natural products, presents a major challenge in predicting and engineering the products of terpene synthases. In this study, we investigated the product profiles of two bacterial variediene synthases, OdVS from Olivibacter domesticus and PsVS from Prauserella shujinwangii, and found that they share some products with the bacterial phomopsene synthase, PmS from Allokutzneria albata, despite low sequence similarity. Thirteen diterpenes were isolated and structurally elucidated including two previously unreported compounds. A series of variants of OdVS, PsVS, and PmS were constructed by targeting conserved residues around the active site and aided in the identification of key residues that control the cyclization pathway. Ultimately, mutation of a single residue, PsVSY86L, was found to switch the major product of PsVS from the tricyclic variediene to the tetracyclic phomopsene, although this switch came at the cost of significantly reduced overall yield indicating a tradeoff between activity and product diversification.

Fungal natural products, also called secondary metabolites (SMs), continue to inspire chemists and drug development. Around 30,000 fungal SMs are known, mainly from filamentous fungi of the phylum Ascomycota. Only 5% of all fungal species are described today, and even fewer have been cultured or chemically screened, which makes fungi an untapped reservoir for chemical discovery. The phylum Basidiomycota harbors chemically talented fungi such as the psilocybin producer Psilocybe cubensis (Agaricales, Hymenogastraceae), but other members like the genus Anthracophyllum (Agaricales, Omphalotaceae) remain largely genetically and chemically underexplored although new species are being discovered frequently. Here, we present the chemical and bioactivity exploration of a Chilean Anthracophyllum discolor fungus. We discovered anthracaurisin A, the first fungal nor-sesquiterpene homodimer with potent cytotoxic activity in vitro and in vivo, isolated one new and several known illudins, and established their absolute configurations.

N‐acetyl‐L‐leucine (levacetylleucine, ALL) is the neuroprotective enantiomer of the racemic antivertigo drug, Tanganil (acetyl‐DL‐leucine, ADLL). ALL has recently been clinically repurposed for the treatment of Niemann Pick disease type C1, a disorder characterised by lysosomal accumulation of glycolipids and cholesterol. Isotopically labelled ALL was required, as well as its negative control, D‐enantiomer (ADL), to monitor the in vivo metabolic fate of the drug and to distinguish it from endogenous leucine and associated metabolites. Here, we describe the synthesis of N‐acetyl‐D‐ and ‐L‐leucine‐ ¹³ C ₆ , as well as N‐acetyl‐L‐leucine‐ ¹³ C ₁ , by N‐acetylation of their amino acid precursors, their spectroscopic characterisation and stereochemical and stability studies. These investigations found that formation of the products, as well as their water‐soluble sodium salt formulation, occurs without compromising stereochemical integrity.

Odyverdienes are a rare class of bacterial diterpenes possessing 6/8/4- or 6/7/5-tricyclic skeletons and the cyclization mechanism underlying their formation remains unknown. Based on their skeletons, we hypothesized that their tricyclic skeletons pass through a neutral or cationic 6/10-eunicellane intermediate. Here, we investigated the mechanism of odyverdiene B2 synthase, SalkS from Streptomyces alkaliterrae, using site-directed mutagenesis, isotopic labeling experiments, and quantum chemical calculations. Two key shunt products, the 10-membered monocyclic 13-deoxolobophytumin A and a trans-eunicellane named alkacellene, isolated from SalkS variants and quantum chemical calculations supported the proposed mechanism to odyverdiene B2 via initial 1,10-cyclization and a cationic eunicellanyl intermediate. Deuterium labeling studies also supported the occurrence of two sequential 1,2-hydride shifts that set distinctive stereocenters during eunicellanyl formation. Alkacellene was also shown to undergo acid-catalyzed cyclization to the 6/6/6/6-tetracyclic isohydropyrene, an isomer of the bacterial diterpene synthase product hydropyrene. This mechanistic study of SalkS highlights the frequent formation and use of eunicellanyl intermediates in diversifying bacterial diterpene biosynthesis and provides a model system to address fundamental questions involving 6/10-bicyclic ring systems.

Life is predicated on chirality, a molecular asymmetry akin to the left and right versions of human hands. Here we show that privileged protein residues are predisposed for chiral regulation. We developed enantiomeric oxaziridine reagents that systematically identify pro-( S ) and pro-( R ) methionine oxidation sites across proteomes that can be erased by stereospecific methionine sulfoxide reductase enzymes A and B, respectively. These probes reveal that chiral regulation of methionine oxidation-reduction processes can allosterically regulate protein function, as shown in cell and murine models of oxidative stress where selective ( R )-methionine sulfoxide formation on M69 of biphenyl hydrolase-like protein leads to hydrolase inhibition and amplification of proteome N -homocysteinylation modifications. This work introduces a platform for characterizing sites of asymmetric methionine oxidation and the functional consequences concomitant with an individual chiral single-atom modification.

Myxobacterial terpenoids are rare and only a few myxobacterial terpene synthases are known. A terpene synthase, IbdS, from Polyangium aurulentum sp. SDU3-1 was found to produce the Δ^9,10 isomer (1) of the spirocyclic 6/7/5-tricyclic bonnadiene, along with several 6/10-eunicellanes and cembrenoids as minor products. Sequence comparison and site-directed mutagenesis of IbdS and the actinobacterial bonnadiene synthase revealed a single residue switch at position 57 controlling isomer selectivity.

Myxobacterial terpenoids are rare and only a few myxobacterial terpene synthases are known. A terpene synthase, IbdS, from Polyangium aurulentum sp. SDU3-1 was found to produce the [delta]9,10 isomer (1) of the spirocyclic 6/7/5-tricyclic bonnadiene, along with several 6/10-eunicellanes and cembrenoids as minor products. Sequence comparison and site-directed mutagenesis of IbdS and the actinobacterial bonnadiene synthase revealed a single residue switch at position 57 controlling isomer selectivity.

Odyverdienes are a unique class of bacterial diterpenes possessing 6/8/4- or 6/7/5-tricyclic skeletons and the cyclization mechanism underlying their formation remains unknown. Based on their skeletons, we hypothesized that their tricyclic skel-etons pass through a neutral or cationic 6/10-eunicellane intermediate. Here, we investigated the mechanism of odyverdiene B2 synthase, SalkS from Streptomyces alkaliterrae, using site-directed mutagenesis, isotopic labeling experiments, and quantum chemical calculations. Two key shunt products, the 10-membered monocyclic 13-deoxolobophytumin A and a trans-eunicellane named alkacellene, isolated from SalkS variants and quantum chemical calculations supported the proposed mechanism to odyverdiene B2 via initial 1,10-cyclization and a cationic eunicellanyl intermediate. Deuterium labeling stud-ies also supported the occurrence of two sequential 1,2-hydride shifts that set distinctive stereocenters during eunicellanyl formation. Alkacellene was also shown to undergo acid-catalyzed cyclization to the 6/6/6/6-tetracyclic isohydropyrene, an isomer of the bacterial diterpene synthase product hydropyrene. This mechanistic study of SalkS highlights the frequent for-mation and use of eunicellanyl intermediates in diversifying bacterial diterpene biosynthesis and provides a model system to address fundamental questions involving 6/10-bicyclic ring systems.

UbiA-type terpene synthases, traditionally annotated as prenyltransferases, have been shown to catalyze terpene cyclization in recent years, expanding their catalytic repertoire beyond primary metabolism. Here, we report on the genome-guided discovery and functional characterization of bacterial UbiA diterpene synthases (diTSs). Using a geranylgeranyl diphosphate (GGPP)-overproducing E. coli system, we screened 32 candidate enzymes and identified five that generate structurally diverse diterpenes, two of which represent the first examples of cyathane synthases from bacteria. Site-directed mutagenesis uncovered active- site residues that influence product formation, directing cyclization towards mono- or tricyclic products. This study expands the known catalytic repertoire of UbiA enzymes and highlights their untapped potential in bacterial terpenoid biosynthesis. Our findings suggest that bacteria may produce diverse and bioactive diterpenoids using UbiA TSs for the first committed biosynthetic step, warranting further exploration of UbiA TSs for natural product discovery.