Molecular basis for the P450-catalyzed C–N bond formation in indolactam biosynthesis

Exploiting a C–N Bond Forming Cytochrome P450 Monooxygenase for C–S Bond Formation

“…As aresult, the F287A mutant was determined to be inactive with 2 as asubstrate,suggesting the importance of the p-p interaction and radical-p interaction with the indole ring of the substrate.H owever,m utation of T286 and Q387, which interact with the amide-carbonyl and 14-OH of 2, respectively,t oa lanine,m aintained the significant activity toward 9 while these mutants dramatically decreased in activity (approximately 20 %) toward 2. [9] These results suggested that loss of the methyl group and the large atomic radius of sulfur atom changed the initial binding mode in the active site.B ecause of the difference in the binding mode,t he second conformational change of the radical intermediate and hydroxylation of the C3 indole ring compete against each other. Furthermore,w hen the 3b-hydroxyindolenine is generated, C-S bond formation between S13 and C2 is preferred due to the high nucleophilicity of sulfur to produce the 6/5/8 tricyclic 11 but not the 6/5/6 tricyclic product.…”

“…Forschungsartikel tural analysis. [9] TheT leB enzyme reactions with three substrates featuring different functional groups at the 13th position (namely,h ydroxy (3), methoxy (4), and primary amino (5)g roups) revealed that these substrates are converted into the 6/5/6 tricyclic products 6, 7,and 8,respectively, but not the 9-membered indolactam skeleton. (Scheme 1B).…”

“…We previously obtained two different conformations of substrates in the active site of TleB and its homologue HinD. [9] Thefirst binding mode should represent the initial conformation for the abstraction of the N1-proton, and we proposed that 2 undergoes re-orientation to the second conformation after the N1-hydrogen abstraction in the active site.I nt he second binding mode,the N13 (or S13) attacks the C4 atom from the si-face to generate the natural conformers.T hus,the enzyme strictly regulates the conformation of the substrate in the active site,i ndicating that we can rule out the possibility of group Bf ormation (for example,r -trans-FOLD,F igure 4) in nature.…”

“…Since our attempt at obtaining ac omplexed structure of TleB with 9 failed, the enzymatic reactions of TleB T286A, F287A, and Q387A mutants,r esidues that interact with substrate 2 in the crystal structure, [9] were performed to explore the interaction of 9 in the active site (Supporting Information, Figure S15). As aresult, the F287A mutant was determined to be inactive with 2 as asubstrate,suggesting the importance of the p-p interaction and radical-p interaction with the indole ring of the substrate.H owever,m utation of T286 and Q387, which interact with the amide-carbonyl and 14-OH of 2, respectively,t oa lanine,m aintained the significant activity toward 9 while these mutants dramatically decreased in activity (approximately 20 %) toward 2.…”

“…I nterestingly,t he indole ring of 11 was not b-hydroxylated, as observed in the C À Ob ondcontaining 6, 7,and 8 (Figure 1). [9] Thef ormation of 11 should proceed via a3 b-hydroxyindolenine (or 2,3-epoxide) intermediate,asinthe case of the formation of the 6/5/6 tricyclic products 6, 7,and 8.Thus,TleB catalyzes both direct C-S bond formation and C-S bond formation through the oxidation of the indole ring. However, interestingly,w hen we used the 2-analogues 3, 4,a nd 5 as substrates,t he C2-O13 or C2-N13 bond-containing 11-type products were not obtained.…”