Promiscuous Enzymes for Residue-Specific Peptide and Protein Late-Stage Functionalization.

The late-stage functionalization of peptides and proteins holds significant promise for drug discovery and facilitates bioorthogonal chemistry. This selective functionalization leads to innovative advances in in vitro and in vivo biological research. However, it is a challenging endeavor to selectively target a certain amino acid or position in the presence of other residues containing reactive groups. Biocatalysis has emerged as a powerful tool for selective, efficient, and economical modifications of molecules. Enzymes that have the ability to modify multiple complex substrates or selectively install nonnative handles have wide applications. Herein, we highlight enzymes with broad substrate tolerance that have been demonstrated to modify a specific amino acid residue in simple or complex peptides and/or proteins at late-stage. The different substrates accepted by these enzymes are mentioned together with the reported downstream bioorthogonal reactions that have benefited from the enzymatic selective modifications.

Late-Stage Chemoenzymatic Installation of Hydroxy-Bearing Allyl Moiety on the Indole Ring of Tryptophan-Containing Peptides.

The late-stage functionalization of indole- and tryptophan-containing compounds with reactive moieties facilitates downstream diversification and leads to changes in their biological properties. Here, the synthesis of two hydroxy-bearing allyl pyrophosphates is described. A chemoenzymatic method is demonstrated which uses a promiscuous indole prenyltransferase enzyme to install a dual reactive hydroxy-bearing allyl moiety directly on the indole ring of tryptophan-containing peptides. This is the first report of late-stage indole modifications with this reactive group.

Regiospecific Synthesis of Calcium-Independent Daptomycin Antibiotics using a Chemoenzymatic Method.

Daptomycin (DAP) is a calcium (Ca2+ )-dependent FDA-approved antibiotic drug for the treatment of Gram-positive infections. It possesses a complex pharmacophore hampering derivatization and/or synthesis of analogues. To mimic the Ca2+ -binding effect, we used a chemoenzymatic approach to modify the tryptophan (Trp) residue of DAP and synthesize kinetically characterized and structurally elucidated regiospecific Trp-modified DAP analogues. We demonstrated that the modified DAPs are several times more active than the parent molecule against antibiotic-susceptible and antibiotic-resistant Gram-positive bacteria. Strikingly, and in contrast to the parent molecule, the DAP derivatives do not rely on calcium or any additional elements for activity.

Structure and specificity of a permissive bacterial C-prenyltransferase.

This study highlights the biochemical and structural characterization of the L-tryptophan C6 C-prenyltransferase (C-PT) PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to a diverse array of prenyl donors and acceptors including daptomycin. Two additional PTs also produced novel prenylated daptomycins with improved antibacterial activities over the parent drug.

Baraphenazines A-G, Divergent Fused Phenazine-Based Metabolites from a Himalayan Streptomyces.

The structures and bioactivities of three unprecedented fused 5-hydroxyquinoxaline/alpha-keto acid amino acid metabolites (baraphenazines A-C, 1-3), two unique diastaphenazine-type metabolites (baraphenazines D and E, 4 and 5) and two new phenazinolin-type (baraphenazines F and G, 6 and 7) metabolites from the Himalayan isolate Streptomyces sp. PU-10A are reported. This study highlights the first reported bacterial strain capable of producing diastaphenazine-type, phenazinolin-type, and izumiphenazine A-type metabolites and presents a unique opportunity for the future biosynthetic interrogation of late-stage phenazine-based metabolite maturation.

Structure Determination, Functional Characterization, and Biosynthetic Implications of Nybomycin Metabolites from a Mining Reclamation Site-Associated Streptomyces.

We report the isolation and characterization of three new nybomycins (nybomycins B-D, 1-3) and six known compounds (nybomycin, 4; deoxynyboquinone, 5; α-rubromycin, 6; ß-rubromycin, 7; γ-rubromycin, 8; and [2α(1E,3E),4ß]-2-(1,3-pentadienyl)-4-piperidinol, 9) from the Rock Creek (McCreary County, KY) underground coal mine acid reclamation site isolate Streptomyces sp. AD-3-6. Nybomycin D (3) and deoxynyboquinone (5) displayed moderate (3) to potent (5) cancer cell line cytotoxicity and displayed weak to moderate anti-Gram-(+) bacterial activity, whereas rubromycins 6-8 displayed little to no cancer cell line cytotoxicity but moderate to potent anti-Gram-(+) bacterial and antifungal activity. Assessment of the impact of 3 or 5 cancer cell line treatment on 4E-BP1 phosphorylation, a predictive marker of ROS-mediated control of cap-dependent translation, also revealed deoxynyboquinone (5)-mediated downstream inhibition of 4E-BP1p. Evaluation of 1-9 in a recently established axolotl embryo tail regeneration assay also highlighted the prototypical telomerase inhibitor γ-rubromycin (8) as a new inhibitor of tail regeneration. Cumulatively, this work highlights an alternative nybomycin production strain, a small set of new nybomycin metabolites, and previously unknown functions of rubromycins (antifungal activity and inhibition of tail regeneration) and also provides a basis for revision of the previously proposed nybomycin biosynthetic pathway.

Puromycins B-E, Naturally Occurring Amino-Nucleosides Produced by the Himalayan Isolate Streptomyces sp. PU-14G.

The isolation and structure elucidation of four new naturally occurring amino-nucleoside [puromycins B-E (1-4)] metabolites from a Himalayan isolate ( Streptomyces sp. PU-14-G, isolated from the Bara Gali region of northern Pakistan) is reported. Consistent with prior reports, comparative antimicrobial assays revealed the need for the free 2″-amine for anti-Gram-positive bacteria and antimycobacterial activity. Similarly, comparative cancer cell line cytotoxicity assays highlighted the importance of the puromycin-free 2″-amine and the impact of 3'-nucleoside substitution. These studies extend the repertoire of known naturally occurring puromycins and their corresponding SAR. Notably, 1 represents the first reported naturally occurring bacterial puromycin-related metabolite with a 3'- N-amino acid substitution that differs from the 3'- N-tyrosinyl of classical puromycin-type natural products. This discovery suggests the biosynthesis of 1 in Streptomyces sp. PU-14G may invoke a uniquely permissive amino-nucleoside synthetase and/or multiple synthetases and sets the stage for further studies to elucidate, and potentially exploit, new biocatalysts for puromycin chemoenzymatic diversification.

Spoxazomicin D and Oxachelin C, Potent Neuroprotective Carboxamides from the Appalachian Coal Fire-Associated Isolate Streptomyces sp. RM-14-6.

The isolation and structure elucidation of six new bacterial metabolites [spoxazomicin D (2), oxachelins B and C (4, 5), and carboxamides 6-8] and 11 previously reported bacterial metabolites (1, 3, 9-12a, and 14-18) from Streptomyces sp. RM-14-6 is reported. Structures were elucidated on the basis of comprehensive 1D and 2D NMR and mass spectrometry data analysis, along with direct comparison to synthetic standards for 2, 11, and 12a,b. Complete 2D NMR assignments for the known metabolites lenoremycin (9) and lenoremycin sodium salt (10) were also provided for the first time. Comparative analysis also provided the basis for structural revision of several previously reported putative aziridine-containing compounds [exemplified by madurastatins A1, B1, C1 (also known as MBJ-0034), and MBJ-0035] as phenol-dihydrooxazoles. Bioactivity analysis [including antibacterial, antifungal, cancer cell line cytotoxicity, unfolded protein response (UPR) modulation, and EtOH damage neuroprotection] revealed 2 and 5 as potent neuroprotectives and lenoremycin (9) and its sodium salt (10) as potent UPR modulators, highlighting new functions for phenol-oxazolines/salicylates and polyether pharmacophores.

Identification of Neuroprotective Spoxazomicin and Oxachelin Glycosides via Chemoenzymatic Glycosyl-Scanning.

The assessment of glycosyl-scanning to expand the molecular and functional diversity of metabolites from the underground coal mine fire-associated Streptomyces sp. RM-14-6 is reported. Using the engineered glycosyltransferase OleD Loki and a 2-chloro-4-nitrophenylglycoside-based screen, six metabolites were identified as substrates of OleD Loki, from which 12 corresponding metabolite glycosides were produced and characterized. This study highlights the first application of the 2-chloro-4-nitrophenylglycoside-based screen toward an unbiased set of unique microbial natural products and the first reported application of the 2-chloro-4-nitrophenylglycoside-based transglycosylation reaction for the corresponding preparative synthesis of target glycosides. Bioactivity analysis (including antibacterial, antifungal, anticancer, and EtOH damage neuroprotection assays) revealed glycosylation to attenuate the neuroprotective potency of 4, while glycosylation of the structurally related inactive spoxazomicin C (3) remarkably invoked neuroprotective activity.

Bi- and Tetracyclic Spirotetronates from the Coal Mine Fire Isolate Streptomyces sp. LC-6-2.

The structures of 12 new "enantiomeric"-like abyssomicin metabolites (abyssomicins M-X) from Streptomyces sp. LC-6-2 are reported. Of this set, the abyssomicin W (11) contains an unprecedented 8/6/6/6 tetracyclic core, while the bicyclic abyssomicin X (12) represents the first reported naturally occurring linear spirotetronate. Metabolite structures were determined based on spectroscopic data and X-ray crystallography, and Streptomyces sp. LC-6-2 genome sequencing also revealed the corresponding putative biosynthetic gene cluster.

Mccrearamycins A-D, Geldanamycin-Derived Cyclopentenone Macrolactams from an Eastern Kentucky Abandoned Coal Mine Microbe.

Four cyclopentenone-containing ansamycin polyketides (mccrearamycins A-D), and six new geldanamycins (Gdms B-G, including new linear and mycothiol conjugates), were characterized as metabolites of Streptomyces sp. AD-23-14 isolated from the Rock Creek underground coal mine acid drainage site. Biomimetic chemical conversion studies using both simple synthetic models and Gdm D confirmed that the mccrearamycin cyclopentenone derives from benzilic acid rearrangement of 19-hydroxy Gdm, and thereby provides a new synthetic derivatization strategy and implicates a potential unique biocatalyst in mccrearamycin cyclopentenone formation. In addition to standard Hsp90α binding and cell line cytotoxicity assays, this study also highlights the first assessment of Hsp90α modulators in a new axolotl embryo tail regeneration (ETR) assay as a potential new whole animal assay for Hsp90 modulator discovery.

Antibacterial and Cytotoxic Actinomycins Y6-Y9 and Zp from Streptomyces sp. Strain Gö-GS12.

Four new Y-type actinomycin analogues named Y6-Y9 (1-4) were isolated and characterized from the scale-up fermentation of the Streptomyces sp. strain Gö-GS12, as well as actinomycin Zp (5), which was, for the first time, isolated as a natural product. Structures of the new compounds were elucidated by the cumulative analyses of NMR spectroscopy and HRMS. The 4-hydroxythreonine on the ß-ring of 1 uniquely undergoes both a rearrangement by a 2-fold acyl shift and an additional ring closure with the amino group of the phenoxazinone chromophore, and the α-rings of 4 and 5 contain a rare 5-methyl proline. Compounds 2-5 showed potent antibacterial activities against Gram-positive bacteria that correlated with cytotoxicity against representative human cell lines. The combination of a ß-ring rearrangement and additional ring closure in 1 rendered this actinomycin significantly less potent relative to the nonrearranged comparator actinomycin Y5 and other actinomycins.

Boronated tartrolon antibiotic produced by symbiotic cellulose-degrading bacteria in shipworm gills.

Shipworms are marine wood-boring bivalve mollusks (family Teredinidae) that harbor a community of closely related Gammaproteobacteria as intracellular endosymbionts in their gills. These symbionts have been proposed to assist the shipworm host in cellulose digestion and have been shown to play a role in nitrogen fixation. The genome of one strain of Teredinibacter turnerae, the first shipworm symbiont to be cultivated, was sequenced, revealing potential as a rich source of polyketides and nonribosomal peptides. Bioassay-guided fractionation led to the isolation and identification of two macrodioloide polyketides belonging to the tartrolon class. Both compounds were found to possess antibacterial properties, and the major compound was found to inhibit other shipworm symbiont strains and various pathogenic bacteria. The gene cluster responsible for the synthesis of these compounds was identified and characterized, and the ketosynthase domains were analyzed phylogenetically. Reverse-transcription PCR in addition to liquid chromatography and high-resolution mass spectrometry and tandem mass spectrometry revealed the transcription of these genes and the presence of the compounds in the shipworm, suggesting that the gene cluster is expressed in vivo and that the compounds may fulfill a specific function for the shipworm host. This study reports tartrolon polyketides from a shipworm symbiont and unveils the biosynthetic gene cluster of a member of this class of compounds, which might reveal the mechanism by which these bioactive metabolites are biosynthesized.

A comprehensive review of glycosylated bacterial natural products.

A systematic analysis of all naturally-occurring glycosylated bacterial secondary metabolites reported in the scientific literature up through early 2013 is presented. This comprehensive analysis of 15 940 bacterial natural products revealed 3426 glycosides containing 344 distinct appended carbohydrates and highlights a range of unique opportunities for future biosynthetic study and glycodiversification efforts.

Cytotoxic Indolocarbazoles from Actinomadura melliaura ATCC 39691.

Actinomadura melliaura ATCC 39691, a strain isolated from a soil sample collected in Bristol Cove, California, is a known producer of the disaccharide-substituted AT2433 indolocarbazoles (6-9). Reinvestigation of this strain using new media conditions led to >40-fold improvement in the production of previously reported AT2433 metabolites and the isolation and structure elucidation of the four new analogues, AT2433-A3, A4, A5, and B3 (1-4). The availability of this broader set of compounds enabled a subsequent small antibacterial/fungal/cancer SAR study that revealed disaccharyl substitution, N-6 methylation, and C-11 chlorination as key modulators of bioactivity. The slightly improved anticancer potency of the newly reported N-6-desmethyl 1 (compared to 6) contrasts extensive SAR of monoglycosylated rebeccamycin-type topoisomerase I inhibitors where N-6 alkylation has contributed to improved potency and ADME. Complete 2D NMR assignments for the known metabolite BMY-41219 (5) and (13)C NMR spectroscopic data for the known analogue AT2433-B1 (7) are also provided for the first time.

Complex microbiome underlying secondary and primary metabolism in the tunicate-Prochloron symbiosis.

The relationship between tunicates and the uncultivated cyanobacterium Prochloron didemni has long provided a model symbiosis. P. didemni is required for survival of animals such as Lissoclinum patella and also makes secondary metabolites of pharmaceutical interest. Here, we present the metagenomes, chemistry, and microbiomes of four related L. patella tunicate samples from a wide geographical range of the tropical Pacific. The remarkably similar P. didemni genomes are the most complex so far assembled from uncultivated organisms. Although P. didemni has not been stably cultivated and comprises a single strain in each sample, a complete set of metabolic genes indicates that the bacteria are likely capable of reproducing outside the host. The sequences reveal notable peculiarities of the photosynthetic apparatus and explain the basis of nutrient exchange underlying the symbiosis. P. didemni likely profoundly influences the lipid composition of the animals by synthesizing sterols and an unusual lipid with biofuel potential. In addition, L. patella also harbors a great variety of other bacterial groups that contribute nutritional and secondary metabolic products to the symbiosis. These bacteria possess an enormous genetic potential to synthesize new secondary metabolites. For example, an antitumor candidate molecule, patellazole, is not encoded in the genome of Prochloron and was linked to other bacteria from the microbiome. This study unveils the complex L. patella microbiome and its impact on primary and secondary metabolism, revealing a remarkable versatility in creating and exchanging small molecules.

Selective Late-Stage Functionalization of Tryptophan-Containing Peptides To Facilitate Bioorthogonal Tetrazine Ligation.

Site-selective modification of complex peptides and the functionalization of their C-H bonds hold great promise for expanding their use in therapeutics and biomedical research. Herein, we leverage the power of late-stage chemoenzymatic catalysis using an indole prenyltransferase (IPT) enzyme and alkyl diphosphates to specifically modify the indole ring of tryptophan in clinically relevant peptides. Furthermore, the installed handle enables bioorthogonal click chemistry through an inverse electron-demand Diels-Alder (IEDDA) reaction with a biotin-conjugated tetrazine probe.

Herbimycins D-F, ansamycin analogues from Streptomyces sp. RM-7-15.

Bacterial strains belonging to the class actinomycetes were isolated from the soil near a thermal vent of the Ruth Mullins coal fire (Appalachian Mountains of eastern Kentucky). High-resolution electrospray ionization mass spectrometry and ultraviolet absorption profiles of metabolites from one of the isolates (Streptomyces sp. RM-7-15) revealed the presence of a unique set of metabolites ultimately determined to be herbimycins D-F (1-3). In addition, herbimycin A (4), dihydroherbimycin A (TAN 420E) (7), and the structurally distinct antibiotic bicycylomycin were isolated from the crude extract of Streptomyces sp. RM-7-15. Herbimycins A and D-F (1-3) displayed comparable binding affinities to the Hsp90α. While the new analogues were found to be inactive in cancer cell cytotoxicity and antimicrobial assays, they may offer new insights in the context of nontoxic ansamycin-based Hsp90 inhibitors for the treatment of neurodegenerative disease.

Frenolicins C-G, pyranonaphthoquinones from Streptomyces sp. RM-4-15.

Appalachian active coal fire sites were selected for the isolation of bacterial strains belonging to the class actinobacteria. A comparison of high-resolution electrospray ionization mass spectrometry (HRESIMS) and ultraviolet (UV) absorption profiles from isolate extracts to natural product databases suggested Streptomyces sp. RM-4-15 to produce unique metabolites. Four new pyranonaphthoquinones, frenolicins C-F (1-4), along with three known analogues, frenolicin (6), frenolicin B (7), and UCF76-A (8), were isolated from the fermentation of this strain. An additional new analogue, frenolicin G (5), along with two known compounds, deoxyfrenolicin (9) and UCF 13 (10), were isolated from the fermentation supplied with 18 mg/L of scandium chloride, the first example, to the best of our knowledge, wherein scandium chloride supplementation led to the confirmed production of new bacterial secondary metabolites. Structures 1-5 were elucidated on the basis of spectral analysis and chemical modification. While frenolicins are best known for their anticoccidial activity, the current study revealed compounds 6-9 to exhibit moderate cytotoxicity against the human lung carcinoma cell line (A549) and thereby extends the anticancer SAR for this privileged scaffold.

Structure-guided Mutagenesis Reveals the Catalytic Residue that Controls the Regiospecificity of C6-Indole Prenyltransferases.

Indole is a significant structural moiety and functionalization of the C-H bond in indole-containing molecules expands their chemical space, and modifies their properties and/or activities. Indole prenyltransferases (IPTs) catalyze the direct regiospecific installation of prenyl, C5 carbon units, on indole-derived compounds. IPTs have shown relaxed substrate flexibility enabling them to be used as tools for indole functionalization. However, the mechanism by which certain IPTs target a specific carbon position is not fully understood. Herein, we use structure-guided site-directed mutagenesis, in vitro enzymatic reactions, kinetics and structural-elucidation of analogs to verify the key catalytic residues that control the regiospecificity of all characterized regiospecific C6 IPTs. Our results also demonstrate that substitution of PriB_His312 to Tyr leads to the synthesis of analogs prenylated at different positions than C6. This work contributes to understanding of how certain IPTs can access a challenging position in indole-derived compounds.