Product distributions of EIZS mutants were determined by GC-MS as previously
described (Li, 2014). Briefly, 40 μM FPP was incubated with 1 μM EIZS mutant in size
exclusion buffer [20 mM Tris (pH 7.5), 300 mM NaCl, 10 mM MgCl2, 10% glycerol, 2
mM TCEP] in 6 mL total reaction volume. The reaction mixture was overlaid with 5 mL
HPLC grade n-pentane in a glass test tube and incubated at 30 °C for 16-18 h. The reaction
mixture was extracted with an additional 3 x 5 mL pentane (total 20 mL extracts). The
pentane extracts were dried with anhydrous MgSO4, filtered, and concentrated on an
ice/water bath under reduced pressure to approximately 100 μL. Reaction mixture extracts
were analyzed with an Agilent mass spectrometer with a 30 m x 0.25 mm HP5MS capillary
column using a temperature program with an initial 2 min hold at 60 °C, a 20 °C/min
temperature gradient from 60 °C to 280 °C, and a final 2 min hold at 280 °C. Sesquiterpene
products were identified by comparison of their mass spectra and GC retention indices with
those of authentic compounds using the MassFinder 4.0 Program and Database (Harangi,
2003).
10.2 Results
We determined the sesquiterpene product distributions of eight mutants of EIZS:
Y69F, Y69A, F95N, F95Q, F95C, F96S, F96T, and F96H (Table 10.1). Mainly, these
mutations have produced enzymes that catalyze the formation of previously observed
products from this enzyme. Thirteen products not previously observed were produced by
128 Table 10.1. GC-MS product analysis of EIZS mutants
EIZS mutantd
RT [min]a
RIexpb RIdbc Name Y69F Y69A F95N F95Q F95C F96S F96T F96H
8.43 1386 1385 α-funebrene 1 1 <1 1 8.48 1395 --- Unknown <1 8.57 1408 1406 Helifolene <1 8.60 1413 --- Unknown 1 8.65 1420 --- Unknown 1 4 8.65 1420 1418 α-cedrene <1 3 8.65 1421 1423 β-duprezianene <1 8.70 1428 --- Unknown 4 8.71 1429 1424 β-cedrene 4 5 5 8.78 1430 --- Unknown 3 8.71 1429 1433 Selina-4(15),5- diene <1 8.75 1435 --- Unknown 2 8.75 1435 1435 Sesquisabinene A 1 1 78 8.77 1439 1446 (E)-β-farnesene 2 1 1 1 30 18 8.86 1452 1444 epi-isozizaene 84 66 5 6 6 1 8.92 1455 --- Unknown 3 8.93 1463 1452 Prezizaene 2 5 9.00 1467 --- Unknown 1 9.00 1468 --- Unknown 26 8.98 1470 --- Unknown 5 8.98 1470 1456 Zizaene 6 7 8.98 1471 1475 γ-curcumene 25 22 9.03 1478 1475 α- neocallitropsene 2 1 1 23 5 9.09 1481 --- Unknown 3 9.06 1483 --- Unknown <1 9.07 1485 --- Unknown <1 9.10 1489 1498 (E,E)-α-farnesene 1 5 9.11 1490 --- Unknown <1 9.16 1498 1503 β-bisabolene <1 6 <1 9.18 1501 1503 β-curcumene <1 28 29 2 9.22 1508 1505 (Z)-γ-bisabolene 3 3 5 3 8 35 9.26 1515 1516 β- sesquiphellandrene <1 <1 9.31 1523 --- Unknown <1 9.35 1530 1530 (E)-α-bisabolene 1 9.38 1535 --- Unknown <1 9.46 1548 1553 (E)-nerolidol 2 6 3 9 5 73 9.87 1616 1603 Cedrol 1 9.89 1619 1620 12-epi-cedrol <1 1 10.17 1667 1667 Acorenol 2 3 10.17 1667 --- Unknown 2 10.22 1675 --- Unknown 1 10.22 1676 1673 α-bisabolol 18 13 4 10.26 1682 --- Unknown 1 10.29 1687 --- Unknown <1 10.38 1696 1694 (E,E)-Farnesol 5 10.38 1703 --- Unknown 13 14 21
129
aRetention time (minutes); bRetention index, experimental; cRetention index, MassFinder
130 Figure 10.1. Products of EIZS mutants
131
The most conservative mutations to EIZS involve those to residue Y69, which does
not contribute any surface area to the active site contour but lies behind the F96 side chain
(Figure 10.2). Y69 forms a hydrogen bond with R338, one of the residues that stabilizes
PPi bound in the active site. We hypothesized that deleting this hydrogen bond with a
conservative mutation, Y69F, and a small aliphatic mutation, Y69A, might affect product
distribution by allowing flexibility for F96, thereby changing the shape of the active site
contour. The major product of Y69F and Y69A mutants is epi-isozizaene, as in the wild
type enzyme, and the relative percentage of epi-isozizaene produced for these three
enzymes is comparable (79% for wild type, 84% for Y69F, 66% for Y69A). EIZS Y69A
also produces a significant number of other sesquiterpene products in small amounts, in
addition to the reduced relative amount of epi-isozizaene when compared to wild type.
These results indicate that deletion of the hydrogen bond from the side chain of residue 69
(Y69F) does not significantly affect product distribution. However, a more drastic
mutation, Y69A, does produce greater flexibility in the active site template as evidenced
by the greater number of minor products made by the EIZS Y69A mutant in comparison
to wild type, even though this mutant still produces the same major sesquiterpene product.
It was observed previously that mutation of a residue that does not contribute surface area
to the active site contour can change product distribution in EIZS. Two mutations at residue
A236 (A236G and A236F) display mostly wild type product distribution, producing epi- isozizaene and zizaene exclusively; the A236F mutant also produces a small amount of β-
cedrene. A third mutant at this position, A236M, is inactive and does not produce cyclic
132
Figure 10.2. Stereo view of EIZS active site and residues lying behind the active site
contour. F96 lies at the active site and has been shown to be a key residue for manipulation
133
observance of mutation to a residue behind the active site contour to a smaller side chain
that results in the generation of new sesquiterpene products in this system.
Mutation of residue F95 to two different polar side chains (N and Q) resulted in conversion of EIZS to a β-curcumene synthase, although this product accounts for only
28% and 29% of the total terpene product yield in EIZS F95N and F95Q, respectively. These mutants produce lower relative percentages of β-curcumene than EIZS F95H, which
produces β-curcumene as its major product in 50% yield (Li, 2014). Along with other
minor sesquiterpene products, these two mutants produce the cyclic sesquiterpene alcohol α-bisabolol and the linear sesquiterpene alcohol (E)-nerolidol. These are the first mutants
of EIZS observed to produce alcohol sesquiterpene products; previous mutants have
produced only hydrocarbon terpene products (Li, 2014). Another mutant at position 95, F95C, is an α-neocallitropsene synthase, which is a product not previously observed for
any EIZS mutants. EIZS F95C produces γ-curcumene in almost equal amounts to α-
neocallitropsene (22% and 23%, respectively), as well as some sesquiterpene alcohol
products.
The F96H mutant has largely lost cyclization activity and produces almost
exclusively the linear terpenoid products (E)-nerolidol (73%) and (E)-β-farnesene (18%).
This is the first EIZS mutant observed to produce a sesquiterpene alcohol, (E)-nerolidol,
as a major product. The F96T mutant is a (Z)-γ-bisabolene synthase (35% product yield),
and also produces (E)-β-farnesene in almost the same amount (30%). Interestingly, EIZS
F96V, which is approximately isosteric with F96T, also produces (Z)-γ-bisabolene as its
134
(Z)-γ-bisabolene is directed by the methyl group of the Val and Thr side chains at position
96, rather than the hydroxyl group of the mutant T96 side chain.
EIZS F96S provides the most striking product distribution, as this mutant produces
in 78% total product yield sesquisabinene A, a product made in small amounts by the wild
type enzyme. This is the first EIZS mutant enzyme to produce a sesquiterpene product
other than epi-isozizaene as its major product with such high fidelity; the only other
mutants that approach the fidelity of the wild type enzyme for epi-isozizaene, but produce
other sesquiterpene major products, are F95M (β-acoradiene synthase, 68% yield), F96W (zizaene, 65% yield), and F198L (β-cedrene, 61% yield) (Li, 2014).
The product distributions of EIZS polar mutants described here demonstrate that
polar side chains can be tolerated at the active site of this cyclase with retention of
cyclization activity. Consistent with previous aromatic and aliphatic mutations to EIZS,
introduction of polar side chains often remodels the shape of the active site, leading to
production of alternative cyclization products. From this set of EIZS polar mutants, thirteen
sesquiterpenoid products were observed that had not been previously produced in this
system; these novel products include sesquiterpenoid alcohols, which have not been
produced by any EIZS mutants containing aromatic or aliphatic mutations, but were
produced by six EIZS mutants with polar residues at the active site (F95N, F95Q, F95C,
F96S, F96T, and F96H). These results demonstrate that polar side chains are tolerated both
structurally (Chapter 9) and functionally in the highly hydrophobic active site of this
135
Chapter 11: Kinetic characterization of EIZS polar mutants