Catalytic mechanism of aromatic prenylation by NphB

Abstract

NphB is an aromatic prenyltransferase that catalyzes the attachment of a 10-carbon geranyl group to aromatic substrates. Importantly, NphB exhibits a rich substrate selectivity and product regioselectivity. A systematic computational study has been conducted in order to address several question associated with NphB-catalyzed geranylation. The reaction mechanism of the prenylation step has been characterized as a S(N)1 type dissociative mechanism with a weakly stable carbocation intermediate. A novel π-chamber composed of Tyr121, Tyr216, and 1,6-DHN is found to be important in stabilizing the carbocation. The observed difference in the rates of product formation from 5- and 2-prenylation arises from the differing orientations of the aromatic substrate in the resting state. 4-Prenylation shares the same resting state with 5-prenylation, but the lower free energy barrier for carbocation formation makes the latter reaction more facile. The high free energy barrier associated with 7-prenylation is caused by the unfavorable orientation of 1,6-DHN in active site pocket, along with the difficulty of proton elimination after the prenylation step. A water-mediated proton transfer facilitates the loss of hydrogen at the prenylation site to form the final prenylated product. Interestingly, the same crystallographically observed water molecule has been found to be responsible for proton loss in all three experimentally identified products. After proton transfer, the relaxation of the final product from a sp(3) carbon center to a sp(2) center triggers a "spring-loaded" product release mechanism which pushes the final product out of the binding pocket toward the edge of the active site. The hydrogen bond interactions between the two hydroxyl groups of the aromatic product and the side chains of Ser214 and Tyr288 help to "steer" the movement of the product. In addition, mutagenesis studies identify these same two side chains as being responsible for the observed regioselectivity, particularly 2-prenylation. These observations provide valuable insights into NphB chemistry, offering an opportunity to better engineer the active site and to control the reactivity in order to obtain high yields of the desired product(s). Furthermore, the S(N)1 reaction mechanism observed for NphB differs from the prenylation reaction found in, for example, the farnesyltransferase, which proceeds via an S(N)2-like reaction pathway. The spring-loaded release mechanism highlighted herein also offers novel insights into how enzymes facilitate product release.

Figure 1

Schematic representation of geranylation catalyzed by NphB complexed with GPP and 1,6-DHN.

Figure 2

Distance variations observed during last 450ps of QM/MM MD simulation of the P5 reaction channel.

Figure 3

Free energy profiles for prenylation at four sites in 1,6-DHN. P2 starts at 2.3 kcal/mol, which is the relative free energy difference of the P2 state to that of P5.

Figure 4

Dissociative mechanism of NphB catalyzed geranylation (P5 channel).

Figure 5

Schematic representation of π-chamber. (a) Truncated representation of the π-chamber found at the intermediate state of (a) 2-prenylation, (b) 4-prenylation, (c) 5-prenylation and (d) 7-prenylation. (b) π-chamber in the NphB binding pocket consisting of Tyr121, Tyr216 and 1,6-DHN found at the resting state (left) and intermediate state (right) of 5-prenylation.

Figure 6

Schematic representation of point mutation effects of Y121L on 5-prenylation (top), and S214A & Y288F on 2-prenylation (bottom). Note: reaction direction is from LEFT to RIGHT.

Figure 7

Key snapshots from NphB catalysis taken during the reaction course of (From top to bottom) 2-, 4-, 5- and 7-prenylation. From left to right: resting state, intermediate state, prenylation intermediate state, final product state and product released state. Note: (1) GPP; (2) 1,6-DHN; (3) Mg; (4) D62; (5) K119; (6) Y121; (7) T171; (8) Y216; (9) R228; (10) K284; (11) S214; (12) Y288; (13) OPP; (14) Geranyl carbocation; (15) Prenylated-1,6-DHN; (16) Crystal water (shuttle); (17) Final product; (18) Hydronium.

Figure 8

(a) Computed free energy chart for the entire reaction course and (b) computed free energy profile for the proton transfer step for the three experimentally observed products (Note: reaction direction is from LEFT to RIGHT.). Note: (a) TS1 – carbocation formation step; TS2 – prenylation reaction step; TS3 – proton transfer step. (b) the starting points are chosen based on their corresponding prenylation product states and free energies shifted accordingly.

Figure 9

Schematic representation of the correlation between the 5P-1,6-DHN geometry relaxation and product release: (1) variation of distance (between Mg(II) and the aromatic ring of the product, (top, showing product release), (2) angle of C_5(1,6-DHN)-C_1(GPP)-C_2(GPP) (medium, showing geometry relaxation), and (3) correlation between them over 100ps QM/MM equilibration of P5 final product (bottom, showing correlation).

Figure 10

Top: (Dist 1) distance variation between the magnesium site and the aromatic ring of 5P-1,6-DHN. Middle: (Dist 2) distance variation between the C-terminus capping α-helix and the aromatic ring of 5P-1,6-DHN. Bottom: heat map based on Dist 1 and Dist 2.