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/ange.201910241

“…AKR (PDB ID: 4XK2, 44% sequence identity, 1.6Å RMSD), an Escherichia coli AKR, Tas (PDB ID: 1LQA, 35% sequence identity, 2.0Å RMSD), that has broad substrate speci city 35 , the beta subunit of the voltage-gated potassium channel from Rattus norvegicus (AKR6A2; PDB ID: 3EAU, 32% sequence identity, 2.1Å RMSD), E. coli AKR14A1 (PDB ID: 4AUB, 35% sequence identity, 2.4Å RMSD) that is speci c towards methylglyoxal and diketones such as isatin 36 and mithramycin side chain reductase from Streptomyces argillaceus (PDB ID: 6OW0, 33% sequence identity, 2.3Å 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: Substrate Speci City Pro Le Of Depb Rlegmentioning

confidence: 99%

Structure-Function characterization of an aldo-keto reductase involved in detoxification of the mycotoxin, deoxynivalenol

Abraham

¹

,

Schroeter

²

,

Zhu

³

et al. 2022

Preprint

0

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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 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 broad specificity towards a range of aldehydes and ketones, including the mycotoxin, patulin. DepBRleg produced 3-epi-DON and DON in diastereomeric ratios of 67.2% and 32.8% respectively. The modeled ternary complex 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.

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Structural Basis for Isomerization Reactions in Fungal Tetrahydroxanthone Biosynthesis and Diversification

Yang

¹

,

Mori

²

,

Wei

³

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.

show abstract

Structural Basis for Isomerization Reactions in Fungal Tetrahydroxanthone Biosynthesis and Diversification

Yang

¹

,

Mori

²

,

Wei

³

et al. 2021

View full text Add to dashboard Cite

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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Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis

Cited by 3 publications

References 33 publications

Structure-Function characterization of an aldo-keto reductase involved in detoxification of the mycotoxin, deoxynivalenol

Structure-Function characterization of an aldo-keto reductase involved in detoxification of the mycotoxin, deoxynivalenol

Structural Basis for Isomerization Reactions in Fungal Tetrahydroxanthone Biosynthesis and Diversification

Structural Basis for Isomerization Reactions in Fungal Tetrahydroxanthone Biosynthesis and Diversification

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