Catalytic Mechanism of
Aromatic Prenylation by NphB
- Publication date
- Publisher
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<sub>N</sub>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<sup>3</sup> carbon center to a sp<sup>2</sup> 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<sub>N</sub>1 reaction mechanism observed for NphB
differs from the prenylation reaction found in, for example, the farnesyltransferase,
which proceeds via an S<sub>N</sub>2-like reaction pathway. The spring-loaded
release mechanism highlighted herein also offers novel insights into
how enzymes facilitate product release