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Directing group-controlled regioselectivity in an enzymatic C-H bond oxygenation.

Negretti S, Narayan AR, Chiou KC, Kells PM, Stachowski JL, Hansen DA, Podust LM, Montgomery J, Sherman DH - J. Am. Chem. Soc. (2014)

Bottom Line: We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate.The natural anchoring group desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4.The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C-H functionalization in biocatalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, §Life Sciences Institute, ⊥Department of Chemistry, and ∥Department of Microbiology & Immunology, University of Michigan , Ann Arbor, Michigan 48109, United States.

ABSTRACT
Highly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikCD50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the desosamine N,N-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent C-H bond functionalization. In the present study, a substrate-engineering approach was conducted that involves replacing desosamine with varied synthetic N,N-dimethylamino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry, and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation. The natural anchoring group desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4. The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C-H functionalization in biocatalysis.

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Active siteof PikCD50N with 8a (A) and 8b (B) bound. Substrates are in yellow, heme in gray, andamino acid residues in pink sticks; fragments of protein structureare shown as green ribbon. Distances are in angstroms.
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fig2: Active siteof PikCD50N with 8a (A) and 8b (B) bound. Substrates are in yellow, heme in gray, andamino acid residues in pink sticks; fragments of protein structureare shown as green ribbon. Distances are in angstroms.

Mentions: To establish thesubstrate binding orientation within the activesite, X-ray structures of catalytically competent compounds 8a and 8b bound within the active site of PikCD50N were obtained. The salt-bridge contact between the N,N-dimethylamino group of the synthetic anchor and E94was preserved in these complexes as in natural substrate binding (Figure 2). Additionally, a new hydrogen-bonding interactionbetween H238 and the ester carbonyl group of the anchor emerged inthe active site for both 8a and 8b.


Directing group-controlled regioselectivity in an enzymatic C-H bond oxygenation.

Negretti S, Narayan AR, Chiou KC, Kells PM, Stachowski JL, Hansen DA, Podust LM, Montgomery J, Sherman DH - J. Am. Chem. Soc. (2014)

Active siteof PikCD50N with 8a (A) and 8b (B) bound. Substrates are in yellow, heme in gray, andamino acid residues in pink sticks; fragments of protein structureare shown as green ribbon. Distances are in angstroms.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4012894&req=5

fig2: Active siteof PikCD50N with 8a (A) and 8b (B) bound. Substrates are in yellow, heme in gray, andamino acid residues in pink sticks; fragments of protein structureare shown as green ribbon. Distances are in angstroms.
Mentions: To establish thesubstrate binding orientation within the activesite, X-ray structures of catalytically competent compounds 8a and 8b bound within the active site of PikCD50N were obtained. The salt-bridge contact between the N,N-dimethylamino group of the synthetic anchor and E94was preserved in these complexes as in natural substrate binding (Figure 2). Additionally, a new hydrogen-bonding interactionbetween H238 and the ester carbonyl group of the anchor emerged inthe active site for both 8a and 8b.

Bottom Line: We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate.The natural anchoring group desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4.The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C-H functionalization in biocatalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, §Life Sciences Institute, ⊥Department of Chemistry, and ∥Department of Microbiology & Immunology, University of Michigan , Ann Arbor, Michigan 48109, United States.

ABSTRACT
Highly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikCD50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the desosamine N,N-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent C-H bond functionalization. In the present study, a substrate-engineering approach was conducted that involves replacing desosamine with varied synthetic N,N-dimethylamino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry, and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation. The natural anchoring group desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4. The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C-H functionalization in biocatalysis.

Show MeSH
Related in: MedlinePlus