Substrate Recognition by the Multifunctional Cytochrome P450 MycG in Mycinamicin Hydroxylation and Epoxidation Reactions

Li, Shengying; Tietz, Drew; Rutaganira, Florentine U.; Kells, P.M.; Anzai, Yojiro; Kato, Fumio; Pochapsky, Thomas C.; Sherman, David H.; Podust, L.M.

doi:10.1074/jbc.m112.410340

Cited by 59 publications

(90 citation statements)

References 60 publications

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“…Other P450 enzymes with high structural similarity to StaF are those from secondary metabolism and involve oxidative functionalisation of large substrates, such as pravastatin (CYP105AS, PDB ID: 4OQS) [42], oleandomycin (OleP, PDB ID: 4XE3) [43], mycinamicin (MycG, PDB ID: 2YCA) [44] and filipin (CYP105P1, PDB ID: 3E5L) [45] (Table 3). StaF also shows moderate levels of structural similarity to other P450s that oxidise carrier protein-bound substrates, including the fattyacyl-ACP oxidase P450 BioI (PDB ID: 3EJD) [46–48] and the aminoacyl-PCP hydroxylases OxyD (PDB ID: 3MGX) and P450 sky (PDB ID: 4PXH) [49–50] (Table 3).…”

Section: Resultsmentioning

confidence: 99%

Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934

Ulrich¹,

Brieke²,

Cryle³

2016

Beilstein J. Org. Chem.

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The chemical complexity and biological activity of the glycopeptide antibiotics (GPAs) stems from their unique crosslinked structure, which is generated by the actions of cytochrome P450 (Oxy) enzymes that affect the crosslinking of aromatic side chains of amino acid residues contained within the GPA heptapeptide precursor. Given the crucial role peptide cyclisation plays in GPA activity, the characterisation of this process is of great importance in understanding the biosynthesis of these important antibiotics. Here, we report the cyclisation activity and crystal structure of StaF, the D-O-E ring forming Oxy enzyme from A47934 biosynthesis. Our results show that the specificity of StaF is reduced when compared to Oxy enzymes catalysing C-O-D ring formation and that this activity relies on interactions with the non-ribosomal peptide synthetase via the X-domain. Despite the interaction of StaF with the A47934 X-domain being weaker than for the preceding Oxy enzyme StaH, StaF retains higher levels of in vitro activity: we postulate that this is due to the ability of the StaF/X-domain complex to allow substrate reorganisation after initial complex formation has occurred. These results highlight the importance of testing different peptide/protein carrier constructs for in vitro GPA cyclisation assays and show that different Oxy homologues can display significantly different catalytic propensities despite their overall similarities.

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Section: Resultsmentioning

confidence: 99%

Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934

Ulrich¹,

Brieke²,

Cryle³

2016

Beilstein J. Org. Chem.

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“…This result has important implications for metabolic engineering of crop plants for enhanced disease resistance and also for the generation of antimicrobial triterpenes for other applications. Multifunctional P450 enzymes from other P450 families that catalyze both hydroxylation and epoxidation reactions have been described recently from bacteria (47)(48)(49)(50)(51) and fungi (52,53). Examples of both possible reaction orders (hydroxylation followed by epoxidation and epoxidation followed by hydroxylation) have been reported (Fig.…”

Section: Resultsmentioning

confidence: 99%

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

Geisler

Hughes

Sainsbury

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

145

134

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Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family-as a multifunctional stereo-and regio-specific hydroxylase in plant specialized metabolism.

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“…20,22 Consequently, much of our recent work has focused on the discovery and biochemical/structural characterization of new P450s involved in the biosynthesis of bacterial natural products. 31–35 As we have demonstrated with PikC, 36–38 these fundamental studies serve as a key starting point to guide future efforts to modulate the substrate scope and selectivity properties of P450 enzymes. Engineered biosynthetic P450s may ultimately be employed in vitro or in vivo as biocatalysts for the efficient production of novel biologically active compounds.…”

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confidence: 95%

Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway

et al. 2016

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Cytochrome P450 monooxygenases (P450s) are some of nature’s most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize inert C-H bonds has led to their increasing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. In the present study, we investigated the reactivity of the P450 MycCI from the mycinamicin biosynthetic pathway toward a variety of macrocyclic compounds and discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding experiments, steady-state kinetics studies, and x-ray crystallographic analysis of MycCI bound to its native substrate mycinamicin VIII. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared to MycCI. Thus, the ability of the latter to hydroxylate both macrocyclic aglycones and macrolides sets it apart from related biosynthetic P450s and highlights its potential for developing novel P450 biocatalysts with broad substrate scope and high regioselectivity.

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Substrate Recognition by the Multifunctional Cytochrome P450 MycG in Mycinamicin Hydroxylation and Epoxidation Reactions

Cited by 59 publications

References 60 publications

Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934

Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway

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