28 research outputs found
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
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 CāH 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 moieties 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, inā
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. The presented 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
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
Structure and Function of a Dual ReductaseāDehydratase Enzyme System Involved in p-Terphenyl Biosynthesis
We report the identification of the ter gene cluster responsible for the formation of the p-terphenyl derivatives terfestatins B and C and echoside B from the Appalachian Streptomyces strain RM-5-8. We characterize the function of TerB/C, catalysts that work together as a dual enzyme system in the biosynthesis of natural terphenyls. TerB acts as a reductase and TerC as a dehydratase to enable the conversion of polyporic acid to a terphenyl triol intermediate. X-ray crystallography of the apo and substrate-bound forms for both enzymes provides additional mechanistic insights. Validation of the TerC structural model via mutagenesis highlights a critical role of arginine 143 and aspartate 173 in catalysis. Cumulatively, this work highlights a set of enzymes acting in harmony to control and direct reactive intermediates and advances fundamental understanding of the previously unresolved early steps in terphenyl biosynthesis
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. 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
Regiospecific Synthesis of CalciumāIndependent Daptomycin Antibiotics Using a Chemoenzymatic Method
Daptomycin (DAP) is a calcium (Ca2+)ādependent FDAāapproved antibiotic drug for the treatment of Gramāpositive infections. It possesses a complex pharmacophore hampering derivatization and/or synthesis of analogs. In order to mimic the Ca2+ābinding effect we uses a chemoenzymatic approach to modify the tryptophan (Trp) residue of DAP and synthesize kinetically characterized and structurally elucidated regiospecific Trpāmodified DAP analogs. We demonstrated that the modified DAPs are severalāfolds active than the parent molecule against antibioticāsusceptible and antibioticāresistant Gramāpositive bacteria. Strikingly and in contrast to the parent molecule, the DAP derivatives do not rely on calcium or any additional elements for activity
Puromycins B-E, Naturally Occurring Amino-Nucleosides Produced by the Himalayan Isolate \u3cem\u3eStreptomyces\u3c/em\u3e sp. PU-14G.
The isolation and structure elucidation of four new naturally occurring amino-nucleoside [puromycins BāE (1ā4)] 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 2ā³-amine for anti-Gram-positive bacteria and antimycobacterial activity. Similarly, comparative cancer cell line cytotoxicity assays highlighted the importance of the puromycin-free 2ā³-amine and the impact of 3ā²-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 3ā²-N-amino acid substitution that differs from the 3ā²-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
Antibacterial and Cytotoxic Actinomycins Y6Y9 and Zp from Streptomyces sp. Strain Go-GS12
Four new Y-type actinomycin analogues named Y(6)āY(9) (1ā4) were isolated and characterized from the scale up fermentation of the Streptomyces sp. strain Gƶ-GS12, as well as actinomycin Zp (5) that 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 Ī²-ring of 1 uniquely undergoes both a rearrangement by a two-fold acyl shift and an additional ring closure with the amino group of the phenoxazinone chromophore, and the Ī±-rings of 4 and 5 contain a rare 5-methyl proline. Compounds 2ā5 showed potent antibacterial activities against Gram-positive bacteria that correlated with cytotoxicity against representative human cell lines. The combination of a Ī²-ring rearrangement and additional ring closure in 1 rendered this actinomycin significantly less potent relative to the non-rearranged comparator actinomycin Y(5) and other actinomycins
The native production of the sesquiterpene isopterocarpolone by Streptomyces
We report the production, isolation and structure elucidation of the sesquiterpene isopterocarpolone from an Appalachian isolate Streptomyces species RM-14-6. While isopterocarpolone was previously put forth as a putative plant metabolite, the current study highlights the first native bacterial production of isopterocarpolone and the first full characterization of isopterocarpolone using 1D and 2D NMR spectroscopy and HR-ESI mass spectrometry. Considering the biosynthesis of closely related metabolites (geosmin or 5-epiaristolochene), the structure of isopterocarpolone also suggests the potential participation of one or more unique enzymatic transformations. In this context, this work also sets the stage for the elucidation of potentially novel bacterial biosynthetic machinery
Herbimycins DāF, Ansamycin Analogues from <i>Streptomyce</i>s 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 (<i>Streptomyces</i> sp. RM-7-15)
revealed the presence of a unique set of metabolites ultimately determined
to be herbimycins DāF (<b>1</b>ā<b>3</b>). In addition, herbimycin A (<b>4</b>), dihydroherbimycin
A (TAN 420E) (<b>7</b>), and the structurally distinct antibiotic
bicycylomycin were isolated from the crude extract of <i>Streptomyces</i> sp. RM-7-15. Herbimycins A and DāF (<b>1</b>ā<b>3</b>) displayed comparable binding affinities to the Hsp90Ī±.
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