Regioselectivity of hyoscyamine 6β-hydroxylase-catalysed hydroxylation as revealed by high-resolution structural information and QM/MM calculations

Kluza, Anna; Wojdyla, Zuzanna; Mrugala, B.; Kurpiewska, Katarzyna; Porebski, P.J.; Niedzialkowska, E.; Minor, Władek; Weiss, M.S.; Borowski, Tomasz

doi:10.1039/d0dt00302f

Cited by 20 publications

(31 citation statements)

References 120 publications

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“…H6H belongs to the family of non-heme 2-oxoglutarate/Fe(II)-dependent dioxygenases that share conserved double-stranded β-helix motif, so-called jelly-roll fold, composed of eight antiparallel β-strands. Here, we present crystal structures of H6H from Datura metel and its truncated version in complexes with 2-oxoglutarate, hyoscyamine and 6β-hydroxyhyoscyamine [3]. Through analysis of the substrate binding pocket, we point out crucial residues in hyoscyamine binding and explain results of previous studies on the substrate preference of H6H.…”

mentioning

confidence: 78%

On substrate-binding cavity of hyoscyamine 6β-hydroxylase from devil's trumpet

Kluza¹,

Mrugala²,

Kurpiewska³

et al. 2021

Acta Cryst Sect A

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mentioning

confidence: 78%

On substrate-binding cavity of hyoscyamine 6β-hydroxylase from devil's trumpet

Kluza¹,

Mrugala²,

Kurpiewska³

et al. 2021

Acta Cryst Sect A

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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: 91%

“…Recently Kluza et al (2020) [12] published a high-resolution crystal structure of the D. metel H6H and shed light on the molecular basis of H6H specificity towards hyoscyamine. The authors mentioned the possibility that a Gly220Cys substitution may disturb hyoscyamine binding through re-arrangement of the hydrophobic cavity of the protein, but the exact mechanism of this interference remained to be shown.…”

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

confidence: 99%

“…A rationale for this isomeric preference was not given. The crystallographic structure reported by Kluza et al (2020) [12] enabled us to check the role of the hydroxyl group bound to the 3' carbon (Figure 1). Our results, depicted in Figure 7, show that this hydroxyl group creates hydrogen bonds with Tyr-295 and Leu-290 through two crystallographic water molecules (689 and 843).…”

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

confidence: 99%

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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

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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“…. His motif, highly conserved in the oxoglutarate dependent oxygenases (ODD) family (Kluza et al, 2020). Protein models of H6H for D. metel and D. stramonium (Teo19488 and Tic8550) predict more interaction between residues in Tic8550 than on D. metel and Teo19488 (Fig.…”

Section: Protein Modeling and Molecular Dockingmentioning

confidence: 99%

Tropane alkaloids and terpenes synthase genes of Datura stramonium (Solanaceae)

Velázquez-Márquez¹,

De-la-Cruz²,

Tapia‐López³

et al. 2021

PeerJ

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Background Plants have evolved physical–chemical defense to prevent/diminish damage by their enemies. Chemical defense involves the synthesis’ pathways of specialized toxic, repellent, or anti-nutritive metabolites to herbivores. Molecular evolutionary studies have revealed the origin of new genes, acquisition and functional diversification along time in different plant lineages. Methods Using bioinformatic tools we analyze gene divergence of tropane alkaloids (TAs) and terpene synthases (TPSs) in Datura stramonium and other species of Solanaceae; compared gene and amino acids sequence of TAs and TPSs on genomes, cDNA and proteins sequences of Viridiplantae. We analyzed two recently assembled genomes of D. stramonium (Ticumán and Teotihuacán), transcriptomes of Datura metel and genomes of other Solanaceae. Hence, we analyzed variation of TAs and TPSs to infer genes involved in plant defense and plant responses before stress. We analyzed protein modeling and molecular docking to predict interactions between H6H and ligand; we translated the sequences (Teo19488, Tic8550 and Tic8549) obtained from the two genomes of D. stramonium by using Swiss-Model and Ramachandran plot and MolProbity structure validation of Teo19488 protein model. Results For TAs, we detected an expansion event in the tropinone reductase II (TRII) and the ratio synonymous/non-synonymous substitutions indicate positive selection. In contrast, a contraction event and negative selection was detected in tropinone reductase I (TRI). In Hy-oscyamine 6 b-hydroxylase (H6H), enzyme involved in the production of tropane alkaloids atropine and scopolamine, the synonymous/non-synonymous substitution ratio in its dominion indicates positive selection. For terpenes (TPS), we found 18 DsTPS in D. stramomiun and seven in D. metel; evolutionary analyses detected positive selection in TPS10.1 and TPS10.2 of D. stramonium and D. metel. Comparison of copies of TPSs in D. stramonium detected variation among them in the binding site. Duplication events and differentiation of TAs and TPSs of D. stramonium, as compared to other Solanaceae, suggest their possible involvement on adaptive evolution of defense to herbivores. Protein modeling and docking show that the three protein structures obtained of DsH6H from Teo19488, Tic-8550 and Tic8549 maintain the same interactions and the union site of 2OG-FeII_Oxy with the Hy-o ligand as in 6TTM of D. metel. Conclusion Our results indicate differences in the number of gene copies involved in the synthesis of tropane alkaloids, between the genomes of D. stramonium from two Mexican populations. More copies of genes related to the synthesis of tropane alkaloids (TRI, TRII, H6H, PMT) are found in D. stramonium as compared to Viridiplantae. Likewise, for terpene synthases (TPS), TPS-10 is duplicated in D. stramonium and D. metel. Further studies should be directed to experimentally assess gain (overexpression) or loss (silencing) of function of duplicated genes.

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Regioselectivity of hyoscyamine 6β-hydroxylase-catalysed hydroxylation as revealed by high-resolution structural information and QM/MM calculations

Abstract: Crystal structures and computational results reveal how Hyoscyamine 6β-hydroxylase targets its oxidative power at the C6 position of the tropane ring while sparing the nearby C7 site.

Cited by 20 publications

References 120 publications

On substrate-binding cavity of hyoscyamine 6β-hydroxylase from devil's trumpet

On substrate-binding cavity of hyoscyamine 6β-hydroxylase from devil's trumpet

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

Tropane alkaloids and terpenes synthase genes of Datura stramonium (Solanaceae)

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Regioselectivity of hyoscyamine 6β-hydroxylase-catalysed hydroxylation as revealed by high-resolution structural information and QM/MM calculations

Abstract: Crystal structures and computational results reveal how Hyoscyamine 6β-hydroxylase targets its oxidative power at the C6 position of the tropane ring while sparing the nearby C7 site.

Cited by 20 publications

References 120 publications

On substrate-binding cavity of hyoscyamine 6β-hydroxylase from devil's trumpet

On substrate-binding cavity of hyoscyamine 6β-hydroxylase from devil's trumpet

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

Tropane alkaloids and terpenes synthase genes of Datura stramonium (Solanaceae)

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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