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Media type:
E-Article
Title:
Regulation of product chain length by isoprenyl diphosphate synthases
Contributor:
Tarshis, L. C.;
Proteau, Philip J.;
Kellogg, Brenda A.;
Sacchettini, James C.;
Poulter, C. Dale
imprint:
Proceedings of the National Academy of Sciences, 1996
Published in:Proceedings of the National Academy of Sciences
Language:
English
DOI:
10.1073/pnas.93.26.15018
ISSN:
0027-8424;
1091-6490
Origination:
Footnote:
Description:
<jats:p>
An analysis of the x-ray structure of homodimeric avian
farnesyl diphosphate synthase (geranyltransferase, EC
<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="ec" xlink:href="2.5.1.10">2.5.1.10</jats:ext-link>
) coupled
with information about conserved amino acids obtained from a sequence
alignment of 35 isoprenyl diphosphate synthases that synthesize
farnesyl (C
<jats:sub>15</jats:sub>
), geranylgeranyl (C
<jats:sub>20</jats:sub>
), and
higher chain length isoprenoid diphosphates suggested that the side
chains of residues corresponding to F112 and F113 in the avian enzyme
were important for determining the ultimate length of the hydrocarbon
chains. This hypothesis was supported by site-directed mutagenesis to
transform wild-type avian farnesyl diphosphate synthase (FPS) into
synthases capable of producing geranylgeranyl diphosphate (F112A),
geranylfarnesyl (C
<jats:sub>25</jats:sub>
) diphosphate (F113S), and longer chain
prenyl diphosphates (F112A/F113S). An x-ray analysis of the structure
of the F112A/F113S mutant in the apo state and with allylic
substrates bound produced the strongest evidence that these mutations
caused the observed change in product specificity by directly altering
the size of the binding pocket for the growing isoprenoid chain in the
active site of the enzyme. The proposed binding pocket in the apo
mutant structure was increased in depth by 5.8 Å as compared with that
for the wild-type enzyme. Allylic diphosphates were observed in the
holo structures, bound through magnesium ions to the aspartates of the
first of two conserved aspartate-rich sequences (D117–D121), with the
hydrocarbon tails of all the ligands growing down the hydrophobic
pocket toward the mutation site. A model was constructed to show how
the growth of a long chain prenyl product may proceed by creation of a
hydrophobic passageway from the FPS active site to the outside surface
of the enzyme.
</jats:p>