- Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases
-
Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6′-deoxyallose substituent occurs in a stepwise manner first at the 2′- and then the 3′-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3′-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2′-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4′-specific homologue, active site residues were identified that correlate with the 3′ or 4′ specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.
- Bernard, Steffen M.,Akey, David L.,Tripathi, Ashootosh,Park, Sung Ryeol,Konwerski, Jamie R.,Anzai, Yojiro,Li, Shengying,Kato, Fumio,Sherman, David H.,Smith, Janet L.
-
p. 1340 - 1351
(2015/05/27)
-
- New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners
-
Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.
- Zhang, Wei,Liu, Yi,Yan, Jinyong,Cao, Shaona,Bai, Fali,Yang, Ying,Huang, Shaohua,Yao, Lishan,Anzai, Yojiro,Kato, Fumio,Podust, Larissa M.,Sherman, David H.,Li, Shengying
-
supporting information
p. 3640 - 3646
(2014/03/21)
-