Unusual cysteine modifications in natural product biosynthesis.

l-Cysteine is a highly reactive amino acid that is modified into a variety of chemical structures, including cysteine sulfinic acid in human metabolic pathways, and sulfur-containing scaffolds of amino acids, alkaloids, and peptides in natural product biosynthesis. Among the modification enzymes responsible for these cysteine-derived compounds, metalloenzymes constitute an important family of enzymes that catalyze a wide variety of reactions. Therefore, understanding their reaction mechanisms is important for the biosynthetic production of cysteine-derived natural products. This review mainly summarizes recent mechanistic investigations of metalloenzymes, with a particular focus on recently discovered mononuclear non-heme iron (NHI) enzymes, dinuclear NHI enzymes, and radical-SAM enzymes involved in unusual cysteine modifications in natural product biosynthesis.

Recent developments in the enzymatic modifications of steroid scaffolds.

Steroids are an important family of bioactive compounds. Steroid drugs are renowned for their multifaceted pharmacological activities and are the second-largest category in the global pharmaceutical market. Recent developments in biocatalysis and biosynthesis have led to the increased use of enzymes to enhance the selectivity, efficiency, and sustainability for diverse modifications of steroids. This review discusses the advancements achieved over the past five years in the enzymatic modifications of steroid scaffolds, focusing on enzymatic hydroxylation, reduction, dehydrogenation, cascade reactions, and other modifications for future research on the synthesis of novel steroid compounds and related drugs, and new therapeutic possibilities.

Discovery of (±)-Penindolenes Reveals an Unusual Indole Ring Cleavage Pathway Catalyzed by P450 Monooxygenase.

(±)-Penindolenes A-D (1-4), the first representatives of indole terpenoids featuring a γ-lactam skeleton, were isolated from the mangrove-derived endophytic fungus Penicillium brocae MA-231. Our bioactivity tests revealed their potent antimicrobial and acetylcholinesterase inhibitory activities. The biosynthetic reactions by the five enzymes PbaABCDE leading to γ-lactam ring formation were identified with heterologous expression and in vitro enzymatic assays. Remarkably, the cytochrome P450 monooxygenase PbaB and its homolog in Aspergillus oryzae  catalyzed the 2,3-cleavage of the indole ring to generate two keto groups in 1, in different manners from well-known tryptophan dioxygenases. This is the first example of the oxidative cleavage of indole by a P450 monooxygenase. In addition, rare secondary amide bond formation by the glutamine synthetase-like enzyme PbaD was reported. These findings will contribute to the engineered biosynthesis of unnatural, bioactive indole terpenoids.

Biosynthesis of Enfumafungin-type Antibiotic Reveals an Unusual Enzymatic Fusion Pattern and Unprecedented C-C Bond Cleavage.

Enfumafungin-type antibiotics, represented by enfumafungin and fuscoatroside, belong to a distinct group of triterpenoids derived from fungi. These compounds exhibit significant antifungal properties with ibrexafungerp, a semisynthetic derivative of enfumafungin, recently gaining FDA's approval as the first oral antifungal drug for treating invasive vulvar candidiasis. Enfumafungin-type antibiotics possess a cleaved E-ring with an oxidized carboxyl group and a reduced methyl group at the break site, suggesting unprecedented C-C bond cleavage chemistry involved in their biosynthesis. Here, we show that a 4-gene (fsoA, fsoD, fsoE, fsoF) biosynthetic gene cluster is sufficient to yield fuscoatroside by heterologous expression in Aspergillus oryzae. Notably, FsoA is an unheard-of terpene cyclase-glycosyltransferase fusion enzyme, affording a triterpene glycoside product that relies on enzymatic fusion. FsoE is a P450 enzyme that catalyzes successive oxidation reactions at C19 to facilitate a C-C bond cleavage, producing an oxidized carboxyl group and a reduced methyl group that have never been observed in known P450 enzymes. Our study thus sets the important foundation for the manufacture of enfumafungin-type antibiotics using biosynthetic approaches.

Lincosamide Antibiotics: Structure, Activity, and Biosynthesis.

Lincosamides are naturally occurring antibiotics isolated from Streptomyces sp. Currently, lincomycin A and its semisynthetic analogue clindamycin are used as clinical drugs. Due to their unique structures and remarkable biological activities, derivatizations of lincosamides via semi-synthesis and biosynthetic studies have been reported. This review summarizes the structures and biological activities of lincosamides, and the recent studies of lincosamide biosynthetic enzymes.

Characterization of Aziridine-Forming α-Ketoglutarate-Dependent Oxygenase in l-Isovaline Biosynthesis.

l-Isovaline biosynthesis by TqaLFM-ti from Tolypocladium inflatum was demonstrated in vitro. The biochemical analysis of the α-ketoglutarate-dependent oxygenase TqaL-ti revealed that it produces (2S,3S)-3-ethyl-3-methylaziridine-2-carboxylic acid from l-isoleucine, thus exhibiting a stereoselectivity different from those of the reported homologues. Remarkably, a single mutation on I295 in TqaL-ti completely exchanged its stereoselectivity to produce the C-3 stereoisomer. TqaFM-ti generates d-isovaline from (2S,3R)-aziridine-2-carboxylic acid, suggesting that the stereochemistry of the TqaL product defines that of isovaline.

Catalytic Mechanism of Nonglycosidic C-N Bond Formation by the Pseudoglycosyltransferase Enzyme VldE.

The pseudoglycosyltransferase (PsGT) enzyme VldE is a homologue of the retaining glycosyltransferase (GT) trehalose 6-phosphate synthase (OtsA) that catalyzes a coupling reaction between two pseudo-sugar units, GDP-valienol and validamine 7-phosphate, to give a product with α,α-N-pseudo-glycosidic linkage. Despite its biological importance and unique catalytic function, the molecular bases for its substrate specificity and reaction mechanism are still obscure. Here, we report a comparative mechanistic study of VldE and OtsA using various engineered chimeric proteins and point mutants of the enzymes, X-ray crystallography, docking studies, and kinetic isotope effects. We found that the distinct substrate specificities between VldE and OtsA are most likely due to topological differences within the hot spot amino acid regions of their N-terminal domains. We also found that the Asp158 and His182 residues, which are in the active site, play a significant role in the PsGT function of VldE. They do not seem to be directly involved in the catalysis but may be important for substrate recognition or contribute to the overall architecture of the active site pocket. Moreover, results of the kinetic isotope effect experiments suggest that VldE catalyzes a C-N bond formation between GDP-valienol and validamine 7-phosphate via an SNi-like mechanism. The study provides new insights into the substrate specificity and catalytic mechanism of a member of the growing family of PsGT enzymes, which may be used as a basis for developing new PsGTs from GTs.

Functional analysis of a fungal P450 enzyme.

Fungal cytochrome P450s participate in various physiological reactions, including the synthesis of internal cellular components, metabolic detoxification of xenobiotic compounds, and oxidative modification of natural products. Although functional analysis reports of fungal P450s continue to grow, there are still some difficulties as compared to prokaryotic P450s, because most of these fungal enzymes are transmembrane proteins. In this chapter, we will describe the methods for heterologous expression, in vivo analysis, enzyme preparation, and in vitro enzyme assays of the fungal P450 enzyme Trt6 and isomerase Trt14, which play important roles in the divergence of the biosynthetic pathway of terretonins, as a model for the functional analysis of fungal P450 enzymes.

Mechanistic Analysis of Stereodivergent Nitroalkane Cyclopropanation Catalyzed by Nonheme Iron Enzymes.

BelL and HrmJ are α-ketoglutarate-dependent nonheme iron enzymes that catalyze the oxidative cyclization of 6-nitronorleucine, resulting in the formation of two diastereomeric 3-(2-nitrocyclopropyl)alanine (Ncpa) products containing trans-cyclopropane rings with (1'R,2'R) and (1'S,2'S) configurations, respectively. Herein, we investigate the catalytic mechanism and stereodivergency of the cyclopropanases. The results suggest that the nitroalkane moiety of the substrate is first deprotonated to produce the nitronate form. Spectroscopic analyses and biochemical assays with substrates and analogues indicate that an iron(IV)-oxo species abstracts proS-H from C4 to initiate intramolecular C-C bond formation. A hydroxylation intermediate is unlikely to be involved in the cyclopropanation reaction. Additionally, a genome mining approach is employed to discover new homologues that perform the cyclopropanation of 6-nitronorleucine to generate cis-configured Ncpa products with (1'R,2'S) or (1'S,2'R) stereochemistries. Sequence and structure comparisons of these cyclopropanases enable us to determine the amino acid residues critical for controlling the stereoselectivity of cyclopropanation.

Structural and Mechanistic Insights into the C-C Bond-Forming Rearrangement Reaction Catalyzed by Heterodimeric Hinokiresinol Synthase.

Hinokiresinol synthase (HRS) from Asparagus officinalis consists of two subunits, α and ß, and catalyzes an unusual decarboxylative rearrangement reaction of 4-coumaryl 4-coumarate to generate (Z)-hinokiresinol with complete stereoselectivity. Herein, we describe the mechanism of rearrangement catalysis and the role played by the heterodimeric HRS, through structural and computational analyses. Our results suggest that the HRS reaction is unlikely to proceed via the previously hypothesized Claisen rearrangement mechanism. Instead, we propose that the 4-coumaryl 4-coumarate substrate is first cleaved into coumarate and an extended p-quinone methide, which then recombine to generate a new C-C bond. These processes are facilitated by proton transfers mediated by the basic residues (α-Lys164, α-Arg169, ß-Lys168, and ß-Arg173) in the cavity at the heterodimer interface. The active site residues, α-Asp165, ß-Asp169, ß-Trp17, ß-Met136, and ß-Ala171, play crucial roles in controlling the regioselectivity of the coupling between the fragmented intermediates as well as the stereoselectivity of the decarboxylation step, leading to the formation of the (Z)-hinokiresinol product.

Photoinduced Reductive Dehalogenation of Phenacyl Bromides with Pyridoxal 5'-Phosphate.

We describe the photoinduced reductive debromination of phenacyl bromides using pyridoxal 5'-phosphate (PLP). The reaction requires irradiation with cyan or blue light in an anaerobic atmosphere. Mechanistic analysis reveals the formation of the phenacyl radical as an intermediate in the reaction, implying a single electron transfer to phenacyl bromides from a PLP-derived species resulting from excitation by illumination.

Diverse enzymatic chemistry for propionate side chain cleavages in tetrapyrrole biosynthesis.

Tetrapyrroles represent a unique class of natural products that possess diverse chemical architectures and exhibit a broad range of biological functions. Accordingly, they attract keen attention from the natural product community. Many metal-chelating tetrapyrroles serve as enzyme cofactors essential for life, while certain organisms produce metal-free porphyrin metabolites with biological activities potentially beneficial for the producing organisms and for human use. The unique properties of tetrapyrrole natural products derive from their extensively modified and highly conjugated macrocyclic core structures. Most of these various tetrapyrrole natural products biosynthetically originate from a branching point precursor, uroporphyrinogen III, which contains propionate and acetate side chains on its macrocycle. Over the past few decades, many modification enzymes with unique catalytic activities, and the diverse enzymatic chemistries employed to cleave the propionate side chains from the macrocycles, have been identified. In this review, we highlight the tetrapyrrole biosynthetic enzymes required for the propionate side chain removal processes and discuss their various chemical mechanisms. ONE-SENTENCE SUMMARY: This mini-review describes various enzymes involved in the propionate side chain cleavages during the biosynthesis of tetrapyrrole cofactors and secondary metabolites.

Structure-Function Analysis of the S-Glycosylation Reaction in the Biosynthesis of Lincosamide Antibiotics.

The S-glycosyltransferase LmbT, involved in the biosynthesis of lincomycin A, is the only known enzyme that catalyzes the enzymatic incorporation of rare amino acid L-ergothioneine (EGT) into secondary metabolites. Here, we show the structure and function analyses of LmbT. Our in vitro analysis of LmbT revealed that the enzyme shows promiscuous substrate specificity toward nitrogenous base moieties in the generation of unnatural nucleotide diphosphate (NDP)-D-α-D-lincosamides. Furthermore, the X-ray crystal structures of LmbT in its apo form and in complex with substrates indicated that the large conformational changes of the active site occur upon binding of the substrates, and that EGT is strictly recognized by salt-bridge and cation-π interactions with Arg260 and Trp101, respectively. The structure of LmbT in complex with its substrates, the docking model with the EGT-S-conjugated lincosamide, and the structure-based site-directed mutagenesis analysis revealed the structural details of the LmbT-catalyzed SN 2-like S-glycosylation reaction with EGT.

Multiple C-C Bond Cleavage Reactions Catalyzed by Tolyporphin Tetrapyrrole Biosynthetic Enzymes.

Tolyporphin A is an unusual tetrapyrrole secondary metabolite containing pendant deoxysugars and unsubstituted pyrrole ß sites. Herein, we describe the biosynthesis of the tolyporphin aglycon core. HemF1 catalyzes the oxidative decarboxylation of two propionate side chains of coproporphyrinogen III, an intermediate in heme biosynthesis. HemF2 then processes the two remaining propionate groups to generate a tetravinyl intermediate. All four vinyl groups from the macrocycle are truncated by TolI via repeated C-C bond cleavages to generate the unsubstituted pyrrole ß sites of tolyporphins. This study illustrates how the unprecedented C-C bond cleavage reactions branch from canonical heme biosynthesis to produce tolyporphins.

Genomic and Spectroscopic Signature-Based Discovery of Natural Macrolactams.

The logical and effective discovery of macrolactams, structurally unique natural molecules with diverse biological activities, has been limited by a lack of targeted search methods. Herein, a targeted discovery method for natural macrolactams was devised by coupling genomic signature-based PCR screening of a bacterial DNA library with spectroscopic signature-based early identification of macrolactams. DNA library screening facilitated the efficient selection of 43 potential macrolactam-producing strains (3.6% of 1,188 strains screened). The PCR amplicons of the amine-deprotecting enzyme-coding genes were analyzed to predict the macrolactam type (α-methyl, α-alkyl, or ß-methyl) produced by the hit strains. 1H-15N HSQC-TOCSY NMR analysis of 15N-labeled culture extracts enabled macrolactam detection and structural type assignment without any purification steps. This method identified a high-titer Micromonospora strain producing salinilactam (1), a previously reported α-methyl macrolactam, and two Streptomyces strains producing new α-alkyl and ß-methyl macrolactams. Subsequent purification and spectroscopic analysis led to the structural revision of 1 and the discovery of muanlactam (2), an α-alkyl macrolactam with diene amide and tetraene chromophores, and concolactam (3), a ß-methyl macrolactam with a [16,6,6]-tricyclic skeleton. Detailed genomic analysis of the strains producing 1-3 identified putative biosynthetic gene clusters and pathways. Compound 2 displayed significant cytotoxicity against various cancer cell lines (IC50 = 1.58 µM against HCT116), whereas 3 showed inhibitory activity against Staphylococcus aureus sortase A. This genomic and spectroscopic signature-based method provides an efficient search strategy for new natural macrolactams and will be generally applicable for the discovery of nitrogen-bearing natural products.

Orthoester formation in fungal meroterpenoid austalide F biosynthesis.

Fungal meroterpenoids are important bioactive natural products. Their biosynthetic machineries are highly diverse, and reconstitutions lead to the production of unnatural meroterpenoids. In this study, heterologous gene expression in Aspergillus oryzae and in vitro assays elucidated the biosynthetic pathway of the orthoester-containing fungal meroterpenoid austalide F. Remarkably, the α-ketoglutarate-dependent oxygenase AstB produces the hemiacetal intermediate, and the methyltransferase AstL transfers a methyl group on it to construct the orthoester functionality. This study presents the extraordinary orthoester biosynthetic machinery and provides valuable insights into the creation of unnatural novel bioactive meroterpenoids through engineered biosynthesis. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Structure-based engineering of α-ketoglutarate dependent oxygenases in fungal meroterpenoid biosynthesis.

Non-heme iron- and α-ketoglutarate-dependent oxygenases (αKG OXs) are key enzymes that play a major role in diversifying the structure of fungal meroterpenoids. They activate a specific C-H bond of the substrate to first generate radical species, which is usually followed by oxygen rebound to produce cannonical hydroxylated products. However, in some cases remarkable chemistry induces dramatic structural changes in the molecular scaffolds, depending on the stereoelectronic characters of the substrate/intermediates and the resulting conformational changes/movements of the active site of the enzyme. Their molecular bases have been extensively investigated by crystallographic structural analyses and structure-based mutagenesis, which revealed intimate structural details of the enzyme reactions. This information facilitates the manipulation of the enzyme reactions to create unnatural, novel molecules for drug discovery. This review summarizes recent progress in the structure-based engineering of αKG OX enzymes, involved in the biosynthesis of polyketide-derived fungal meroterpenoids. The literature published from 2016 through February 2022 is reviewed.

Cordycicadins A-D, Antifeedant Polyketides from the Entomopathogenic Fungus Cordyceps cicadae JXCH1.

Cordycicadins A-D (1-4) are four novel polyketides that were isolated from the liquid fermentation of the insect-pathogenic fungus Cordyceps cicadae JXCH1. The structures were determined by a combination of spectroscopic analysis, single-crystal X-ray diffraction, and computational methods. Compounds 1, 3, and 4 harbor an unusual exocyclic enol ether bridge that connects the separated ring systems. Hypothetical biosynthetic pathways for 1-4 were proposed. Cordycicadins A (1) and B (2) showed antifeedant activity against silkworm larvae (Bombyx mori) with EC50 values of 65.4 and 57.0 µg/cm2, respectively.