Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger

Matsuda, Jun; Okabe, S.; Hashimoto, Takashi; Yamada, Yasuyuki

doi:10.1016/s0021-9258(18)92843-7

Cited by 206 publications

(26 citation statements)

References 33 publications

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“…Therefore these observations likely apply for the rationalization of the enantiomeric specificity of H6H enzymes. Until the recently published crystal structure of DmH6H was available [12], the only recognized conserved regions regarding H6H were the conserved iron-binding sites and the conserved HXDX n H catalytic site related to the 2OGD family [4] [27] [28]. In the present study, we show amino acids sequence alignment of functionally tested H6H's from scopolamine producing Solanaceae (Figure 8).…”

Section: The 2's Position Of the Hydroxyl Group In Hyoscyamine And Anisodamine Is Crucial For Proper Protein Bindingmentioning

confidence: 92%

“…The conversion of hyoscyamine into scopolamine is catalyzed by hyoscyamine 6β-hydroxylase (EC 1.14.11.11, H6H), a key enzyme catalyzing the hydroxylation of L-hyoscyamine to 6β-hydroxyhyoscyamine (anisodamine) and its subsequent epoxidation to scopolamine (Figure 1) [4]. H6H is considered an important key enzyme in the biosynthetic pathway for scopolamine and its gene has been isolated from various scopolamine-producing species [5].…”

Section: Hyoscyamine 6β-hydroxylase Is a 2-oxoglutarate-dependent Dioxygenase (2ogd)mentioning

confidence: 99%

“…Reads were assembled de novo using Trinity software (version: v2.3.2; [13] with the trimmomatic option to remove adaptors [14] and 25 mer k-mer size. A tBLASTn search using the Hyoscyamus niger HnH6H (AAA33387.1) [4] was performed on the newly acquired transcriptome of D. innoxia. The candidate gene identified was identical to the previously reported D. innoxia DiH6H (MH392211) [15].…”

Section: Assembly and Candidates Searchmentioning

confidence: 99%

“…The nucleotide sequence of DiH6H (MH392211) [15] was not available, therefore we searched our root and leaf transcriptome using the functionally active H. niger H6H (HnH6H) (AAA33387.1) gene [4] as a template. A tBLASTn search revealed a highly similar sequence (1044 bp) encoding a protein (347 a.a) displaying 100% nucleotide sequence identity to the DiH6H (MH392211) [15] leaf.…”

Section: Datura Innoxiamentioning

confidence: 99%

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Further Insights on the Datura innoxia Hyoscyamine 6β-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling

Schlesinger¹,

Salama²,

Rikanati³

et al. 2021

AJPS

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Hyoscyamine, anisodamine and scopolamine are tropane alkaloids present in some Solanaceae species and used in modern medicine. L-Hyoscyamine is hydroxylated to 6β-hydroxyhyoscyamine (anisodamine) and then epoxidated to scopolamine by the dual action of hyoscyamine 6β-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. A natural mutation in the Gly-220 residue to Cys was previously shown to be associated with the loss of function of H6H in Mandragora officinarum, preventing the accumulation of anisodamine and scopolamine in these plants. We show here that a deliberate Gly220Cys mutation in the Datura innoxia DiH6H protein caused a loss of both its enzymatic abilities and rendered it unable to hydroxylate L-hyoscyamine into anisodamine and to epoxidate anisodamine into scopolamine. By using protein modeling based on an available crystal structure of H6H from Datura metel, we show how the Cys220 residue causes a steric interference in the active site cavity impairing the interaction of both substrates, hyoscyamine and anisodamine with the active site of the protein. We also address the enantiomeric preference of DiH6H based on molecular modeling.

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Section: The 2's Position Of the Hydroxyl Group In Hyoscyamine And Anisodamine Is Crucial For Proper Protein Bindingmentioning

confidence: 92%

Section: Hyoscyamine 6β-hydroxylase Is a 2-oxoglutarate-dependent Dioxygenase (2ogd)mentioning

confidence: 99%

Section: Assembly and Candidates Searchmentioning

confidence: 99%

Section: Datura Innoxiamentioning

confidence: 99%

See 2 more Smart Citations

Further Insights on the Datura innoxia Hyoscyamine 6β-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling

Schlesinger¹,

Salama²,

Rikanati³

et al. 2021

AJPS

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“…Scopolamine ( 23 , Scheme 6 ), also known as hyoscine, is a tropane alkaloid found in Solanaceae plants (Hashimoto & Yamada, 1986 ; Matsuda et al., 1991 ), It has been used to treat motion sickness, postoperative nausea, and vomiting for decades (Clissold & Heel, 1985 ). Hyoscyamine ( 21 ), the key biosynthetic precursor of scopolamine ( 23 ), is formed by the condensation of tropine and tropic acid, which are originated from l -ornithine and l -phenylalanine, respectively (Hashimoto & Yamada, 1986 , 1987 ; Zhang et al., 2004 ).…”

Section: Nonheme Iron Oxidase-catalyzed Radical Cyclization Reactionsmentioning

confidence: 99%

Activation modes in biocatalytic radical cyclization reactions

Hyster

2021

Journal of Industrial Microbiology and Biotechnology

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Radical cyclizations are essential reactions in the biosynthesis of secondary metabolites and the chemical synthesis of societally valuable molecules. In this review, we highlight the general mechanisms utilized in biocatalytic radical cyclizations. We specifically highlight cytochrome P450 monooxygenases (P450s) involved in the biosynthesis of mycocyclosin and vancomycin, non-heme iron- and α-ketoglutarate-dependent dioxygenases (Fe/αKGDs) used in the biosynthesis of kainic acid, scopolamine, and isopenicillin N, and radical S-adenosylmethionine (SAM) enzymes that facilitate the biosynthesis of oxetanocin A, menaquinone, and F420. Beyond natural mechanisms, we also examine repurposed flavin-dependent ‘ene’-reductases (ERED) for non-natural radical cyclization. Overall, these general mechanisms underscore the opportunity for enzymes to augment and enhance the synthesis of complex molecules using radical mechanisms.

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Natural Products and Metabolites

Yazaki

2004

Handbook of Plant Biotechnology

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Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger

Cited by 206 publications

References 33 publications

Further Insights on the <i>Datura innoxia</i> Hyoscyamine 6<i>β</i>-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling

Further Insights on the <i>Datura innoxia</i> Hyoscyamine 6<i>β</i>-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling

Activation modes in biocatalytic radical cyclization reactions

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Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger

Cited by 206 publications

References 33 publications

Further Insights on the &lt;i&gt;Datura innoxia&lt;/i&gt; Hyoscyamine 6&lt;i&gt;β&lt;/i&gt;-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling

Further Insights on the &lt;i&gt;Datura innoxia&lt;/i&gt; Hyoscyamine 6&lt;i&gt;β&lt;/i&gt;-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling

Activation modes in biocatalytic radical cyclization reactions

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Further Insights on the <i>Datura innoxia</i> Hyoscyamine 6<i>β</i>-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling

Further Insights on the <i>Datura innoxia</i> Hyoscyamine 6<i>β</i>-Hydroxylase (DiH6H) Based on Biochemical Characterization and Molecular Modeling