Interactive Regulation between Aliphatic Hydroxylation and Aromatic Hydroxylation of Thaxtomin D in TxtC: A Theoretical Investigation

Abstract

TxtC is an unusual bifunctional cytochrome P450 that is able to perform sequential aliphatic and aromatic hydroxylation of the diketopiperazine substrate thaxtomin D in two distinct sites to produce thaxtomin A. Though the X-ray structure of TxtC complexed with thaxtomin D revealed a binding mode for its aromatic hydroxylation, the preferential hydroxylation site is aliphatic C14. It is thus intriguing to unravel how TxtC accomplishes such two-step catalytic hydroxylation on distinct aliphatic and aromatic carbons and why the aliphatic site is preferred in the hydroxylation step. In this work, by employing molecular docking and molecular dynamics (MD) simulation, we revealed that thaxtomin D could adopt two different conformations in the TxtC active site, which were equal in energy with either the aromatic C20–H or aliphatic C14–H pointing toward the active Cpd I oxyferryl moiety. Further ONIOM calculations indicated that the energy barrier for the rate-limiting hydroxylation step on the aliphatic C14 site was 9.6 kcal/mol more favorable than that on the aromatic C20 site. The hydroxyl group on the monohydroxylated intermediate thaxtomin B C14 site formed hydrogen bonds with Ser280 and Thr385, which induced the l-Phe moiety to rotate around the Cβ–Cγ bond of the 4-nitrotryptophan moiety. Thus, it adopted an energetically favorable conformation with aromatic C20 adjacent to the oxyferryl moiety. In addition, the hydroxyl group induced solvent water molecules to enter the active site, which propelled thaxtomin B toward the heme plane and resulted in heme distortion. Based on this geometrical layout, the rate-limiting aromatic hydroxylation energy barrier decreased to 15.4 kcal/mol, which was comparable to that of the thaxtomin D aliphatic hydroxylation process. Our calculations indicated that heme distortion lowered the energy level of the lowest Cpd I α-vacant orbital, which promoted electron transfer in the rate-limiting thaxtomin B aromatic hydroxylation step in TxtC.

Mechanism of Melatonin Metabolism by CYP1A1: What Determines the Bifurcation Pathways of Hydroxylation versus Deformylation?

“…After IM1 HA , a second electron is transferred to form P Hy , resulting in a close-shell porphyrin group (doubly occupied a 2u orbital) and an iron­(III) state with occupation π* xz 2 π* yz 1 in the doublet spin state and π* xz 1 π* yz 1 σ* z 2 1 in the quartet spin state. Overall, the optimized geometries match previous calculations of substrate hydroxylation reactions by P450 CpdI models well and find analogous distances, charge distributions, and structures. − …”

“…The imaginary frequencies, therefore, are not dramatically different between the model A II and model A III structures and would implicate similar kinetic isotope effects for replacing hydrogen by deuterium due to a similar shape of the potential energy profile around the transition state. These values are typical for hydrogen atom abstraction transition state and seen before for P450 model reactions. − The large imaginary frequency in the hydrogen atom abstraction transition states will result in a large kinetic isotope effect and a significant amount of quantum chemical tunneling for the reaction. , After the radical intermediate, a small rebound barrier via TS2 leads to the hydroxylated methoxy group products 4,2 P Hy,A with large exothermicity. From the hydroxylated methoxy group, a subsequent proton relay from the alcohol group to the methoxy oxygen atom leads to deformylation.…”

“…In the model A III transition-state structures, the C–H–O angle is somewhat bent (at 167–168°), whereas it is closer to linearity for the model A II structures (176°). Under ideal circumstances, these hydrogen atom abstraction barriers give linear C–H–O angles that lower their barriers. ,− As such, the model A II structures manage to position the substrate better than the model A III structures for better electron transfer, and therefore, the barriers for model A II are significantly lower in energy. Probably, this is caused by the tight substrate-binding pocket that latches the substrate in a specific orientation and prevents the ideal linear hydrogen atom transfer angle for model A III .…”

Biotransformation of Bisphenol by Human Cytochrome P450 2C9 Enzymes: A Density Functional Theory Study

“…Modeling and computational analysis of enzymatic reactions such as P450 oxygenation have given understanding into the electronic features that govern reactivity while also complementing experimental information. , Density functional theory (DFT)-based calculations were employed to thoroughly understand energy profiles and the intrinsic electronic features and its contribution to selectivity and reactivity. − In this work, we focus on realistic enzymatic structures of the liver P450 2C9 and ask ourselves what products can be expected from a reaction between P450 CpdI and BPA. We find a novel pathway starting with hydrogen atom abstraction from the phenol group followed by a bifurcation pathway of OH attack on either the ortho - or para -position with respect to the phenol group.…”

How Do Preorganized Electric Fields Function in Catalytic Cycles? The Case of the Enzyme Tyrosine Hydroxylase

“…In this article, we demonstrate that the preorganized electric field exerts opposing effects on the two steps of the catalytic cycles of metalloenzymes. We focus on a recently characterized heme enzyme of l -tyrosine hydroxylases (TyrH), which catalyzes the aromatic hydroxylation of the substrate l -tyrosine (Tyr) to form the natural product intermediates l -3,4-dihydroxyphenylalanine (DOPA), , which is the rate-limiting step responsible for the biosynthesis of natural product catecholamines dopamine, norepinephrine, and epinephrine. − This kind of aromatic hydroxylation is common in the metabolism of xenobiotic compounds and in biosyntheses of natural products. − As shown in Scheme , the catalytic reactions of TyrH consist of two stages. The first stage involves the activation of H 2 O 2 to form the active species of Cpd I, while the second stage involves the Cpd I-mediated hydroxylation of l -Tyr to afford l -DOPA.…”