Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis

Wheeler, Ryan; Yu, Xia; Hou, Can; Mitra, Prithiba; Chen, Jhong Min; Herkules, Frank; Ivanov, Dmitri N.; Tsodikov, Oleg V.; Rohr, Jürgen

doi:10.1002/anie.201910241

Cited by 14 publications

(9 citation statements)

References 31 publications

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“…DynA1 shares sequence homology with a group of AclR/SnoaL-like cyclases/hydroxylases/aldolases. − Presumably by interacting with DynE13, DynA1 facilitates the release of Int-4 from DynE13 and may also play a role in catalyzing the oxidative C–N bond cleavage to generate Int-5. Although rare, similar enzyme systems composed of a flavoenzyme and a physically interacting downstream enzyme are known from other biosynthetic pathways …”

Section: Resultsmentioning

confidence: 99%

“…Although rare, similar enzyme systems composed of a flavoenzyme and a physically interacting downstream enzyme are known from other biosynthetic pathways. 40 The next step is the aryl−alkyl C−C coupling to generate the hallmark C8−C9 bond in Int-6. DynA2 is the most likely candidate for catalyzing the aryl−alkyl C−C coupling reaction.…”

Section: ■ Introductionmentioning

confidence: 99%

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Pathway Retrofitting Yields Insights into the Biosynthesis of Anthraquinone-Fused Enediynes

Tran

Low

et al. 2021

J. Am. Chem. Soc.

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Anthraquinone-fused enediynes (AQEs) are renowned for their distinctive molecular architecture, reactive enediyne warhead, and potent anticancer activity. Although the first members of AQEs, i.e., dynemicins, were discovered three decades ago, how their nitrogen-containing carbon skeleton is synthesized by microbial producers remains largely a mystery. In this study, we showed that the recently discovered sungeidine pathway is a “degenerative” AQE pathway that contains upstream enzymes for AQE biosynthesis. Retrofitting the sungeidine pathway with genes from the dynemicin pathway not only restored the biosynthesis of the AQE skeleton but also produced a series of novel compounds likely as the cycloaromatized derivatives of chemically unstable biosynthetic intermediates. The results suggest a cascade of highly surprising biosynthetic steps leading to the formation of the anthraquinone moiety, the hallmark C8–C9 linkage via alkyl–aryl cross-coupling, and the characteristic epoxide functionality. The findings provide unprecedented insights into the biosynthesis of AQEs and pave the way for examining these intriguing biosynthetic enzymes.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Pathway Retrofitting Yields Insights into the Biosynthesis of Anthraquinone-Fused Enediynes

Tran

Low

et al. 2021

J. Am. Chem. Soc.

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“…We also tested the interactions among these enzymes by using an electrophoretic mobility shift assay (EMSA;F igure S16) in the presence of substrates 3,F AD,a nd NADPH. [20] As ar esult, no new band was observed at any conditions,i ndicating that the three proteins do not form long-lived complexes.F urther investigations of the transient protein-protein interactions will clarify the means by which the enzymes efficiently catalyze the conversion of unstable compounds.…”

Section: Enzyme Reaction Mechanism Of Nsrqmentioning

confidence: 74%

Structural Basis for Isomerization Reactions in Fungal Tetrahydroxanthone Biosynthesis and Diversification

et al. 2021

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The novel isomerase NsrQ, from Aspergillus novofumigatus, is a key enzyme in the biosynthesis of fungal tetrahydroxanthones and is responsible for dearomatizing cyclization to provide a tetrahydroxanthone scaffold. NsrQ catalyzes a two‐step isomerization reaction, involving the isomerization of allylic alcohol and subsequent inversion of configuration at the methyl group. We report on the biochemical and structural characterizations of NsrQ, and its homologue Dcr3, from Diaporthe longicolla. The crystal structures of NsrQ and Dcr3 revealed their similar overall structures, with a cone‐shaped α+β barrel fold, to those of the nuclear transport factor 2‐like superfamily enzymes. Furthermore, the structures of Dcr3 and NsrQ variants complexed with substrate analogues and the site‐directed mutagenesis studies identified the catalytic residues and the important hydrophobic residues in shaping the active site pocket for substrate binding. These enzymes thus utilize Glu and His residues as acid‐base catalysts. Based on these observations, we proposed a detailed reaction mechanism for NsrQ‐catalyzed isomerization reactions.

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“…AKR (PDB ID: 4XK2, 44% sequence identity, 1.6 Å RMSD), an Escherichia coli AKR, Tas (PDB ID: 1LQA, 35% sequence identity, 2.0 Å RMSD), that has broad substrate specificity 35 , the beta subunit of the voltage-gated potassium channel from Rattus norvegicus (AKR6A2; PDB ID: 3EAU, 32% sequence identity, 2.1 Å RMSD), E. coli AKR14A1 (PDB ID: 4AUB, 35% sequence identity, 2.4 Å RMSD) that is specific towards methylglyoxal and diketones such as isatin 36 and mithramycin side chain reductase from Streptomyces argillaceus (PDB ID: 6OW0, 33% sequence identity, 2.3 Å RMSD). Mithramycin side chain reductase is a ketoreductase that reduces the 4’ ketone side chain of mithramycin DK, an intermediate in the biosynthesis of the tricyclic antitumor polyketide, mithramycin 37 .…”

Section: Resultsmentioning

confidence: 99%

Structure–function characterization of an aldo–keto reductase involved in detoxification of the mycotoxin, deoxynivalenol

Abraham

Schroeter

Zhu

et al. 2022

Sci Rep

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Deoxynivalenol (DON) is a mycotoxin, produced by filamentous fungi such as Fusarium graminearum, that causes significant yield losses of cereal grain crops worldwide. One of the most promising methods to detoxify this mycotoxin involves its enzymatic epimerization to 3-epi-DON. DepB plays a critical role in this process by reducing 3-keto-DON, an intermediate in the epimerization process, to 3-epi-DON. DepBRleg from Rhizobium leguminosarum is a member of the new aldo–keto reductase family, AKR18, and it has the unusual ability to utilize both NADH and NADPH as coenzymes, albeit with a 40-fold higher catalytic efficiency with NADPH compared to NADH. Structural analysis of DepBRleg revealed the putative roles of Lys-217, Arg-290, and Gln-294 in NADPH specificity. Replacement of these residues by site-specific mutagenesis to negatively charged amino acids compromised NADPH binding with minimal effects on NADH binding. The substrate-binding site of DepBRleg is larger than its closest structural homolog, AKR6A2, likely contributing to its ability to utilize a wide range of aldehydes and ketones, including the mycotoxin, patulin, as substrates. The structure of DepBRleg also suggests that 3-keto-DON can adopt two binding modes to facilitate 4-pro-R hydride transfer to either the re- or si-face of the C3 ketone providing a possible explanation for the enzyme’s ability to convert 3-keto-DON to 3-epi-DON and DON in diastereomeric ratios of 67.2% and 32.8% respectively.

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Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis

Cited by 14 publications

References 31 publications

Pathway Retrofitting Yields Insights into the Biosynthesis of Anthraquinone-Fused Enediynes

Pathway Retrofitting Yields Insights into the Biosynthesis of Anthraquinone-Fused Enediynes

Structural Basis for Isomerization Reactions in Fungal Tetrahydroxanthone Biosynthesis and Diversification

Structure–function characterization of an aldo–keto reductase involved in detoxification of the mycotoxin, deoxynivalenol

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