116002-67-6Relevant academic research and scientific papers
The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism
Duan, Lian,Jogl, Gerwald,Cane, David E.
, p. 12678 - 12689 (2016)
The final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1) is the highly unusual cytochrome P450-catalyzed, oxidative rearrangement of pentalenolactone F (2), involving the transient generation and rearrangement of a neopentyl cation. In Streptomyces arenae this reaction is catalyzed by CYP161C2 (PntM), with highly conserved orthologs being present in at least 10 other Actinomycetes. Crystal structures of substrate-free PntM, as well as PntM with bound substrate 2, product 1, and substrate analogue 6,7-dihydropentalenolactone F (7) revealed interactions of bound ligand with three residues, F232, M77, and M81 that are unique to PntM and its orthologs and absent from essentially all other P450s. Site-directed mutagenesis, ligand-binding measurements, steady-state kinetics, and reaction product profiles established there is no special stabilization of reactive cationic intermediates by these side chains. Reduced substrate analogue 7 did not undergo either oxidative rearrangement or simple hydroxylation, suggesting that the C1 carbocation is not anchimerically stabilized by the 6,7-double bond of 2. The crystal structures also revealed plausible proton relay networks likely involved in the generation of the key characteristic P450 oxidizing species, Compound I, and in mediating stereospecific deprotonation of H-3re of the substrate. We conclude that the unusual carbocation intermediate results from outer shell electron transfer from the transiently generated C1 radical to the tightly paired heme-a€￠Fe3+-OH radical species. The oxidative electron transfer is kinetically dominant as a result of the unusually strong steric barrier to oxygen rebound to the neopentyl center C-1si, which is flanked on each neighboring carbon by syn-axial substituents.
Genome mining in Streptomyces. Discovery of an unprecedented P450-catalyzed oxidative rearrangement that is the final step in the biosynthesis of pentalenolactone
Zhu, Dongqing,Seo, Myung-Ji,Ikeda, Haruo,Cane, David E.
supporting information; experimental part, p. 2128 - 2131 (2011/04/23)
The penM and pntM genes from the pentalenolactone biosynthetic gene clusters of Streptomyces exfoliatus UC5319 and Streptomyces arenae TUe469 were predicted to encode orthologous cytochrome P450s, CYP161C3 and CYP161C2, responsible for the final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1). Synthetic genes optimized for expression in Escherichia coli were used to obtain recombinant PenM and PntM, each carrying an N-terminal His6-tag. Both proteins showed typical reduced-CO UV maxima at 450 nm, and each bound the predicted substrate, pentalenolactone F (4), with KD values of 153 ± 14 and 126 ± 11 μM for PenM and PntM, respectively, as determined by UV shift titrations. PenM and PntM both catalyzed the oxidative rearrangement of 4 to 1 when incubated in the presence of NADPH, spinach ferredoxin, ferredoxin reductase, and O2. The steady-state kinetic parameters were kcat = 10.5 ± 1.7 min-1 and Km = 340 ± 100 μM 4 for PenM and kcat = 8.8 ± 0.9 min-1 and Km = 430 ± 100 μM 4 for PntM. The in vivo function of both gene products was confirmed by the finding that the corresponding deletion mutants S. exfoliatus/ΔpenM ZD22 and S. arenae/ΔpntM ZD23 no longer produced pentalenolactone but accumulated the precursor pentalenolactone F. Complementation of each deletion mutant with either penM or pntM restored production of antibiotic 1. Pentalenolactone was also produced by an engineered strain of Streptomyces avermitilis that had been complemented with pntE, pntD, and either pntM or penM, as well as the S. avermitilis electron-transport genes for ferredoxin and ferrodoxin reductase, fdxD and fprD.
