1. Dr.
Mann
for
the
assistance,
guidance,
and
mentorship
WSU
Founda:on
for
providing
a
Research
and
Crea:ve
Scholarship
Grant
WSU
Chemistry
Department
for
use
of
facili:es
and
equipment
Acknowledgements
Use of Molecular Modeling to Direct the Functional Characterization of a Suspected
Short-Chain Prenyltransferase in Mycobacterium tuberculosis
Zachary
A.
Swanson,
Francis
M.
Mann
Winona
State
University,
Winona,
MN
Results
Conclusion
Abstract
Introduction
Discussion
Mycobacterium
tuberculosis
is
a
bacterium
that
is
spread
by
droplet
nuclei
from
the
respiratory
tract
of
an
infected
individual.
The
infec:on
of
M.
tuberculosis
is
one
of
the
leading
causes
of
death
among
bacterial
infec:ons
and
there
is
currently
no
cure.
M.
tuberculosis
u:lizes
menaquinone
as
an
electron
transporter
during
oxida:ve
phosphoryla:on,
making
inhibi:on
of
menaquinone
synthesis
a
target
for
development
of
therapeu:cs.
Rv2173
encodes
a
protein
that
has
been
iden:fied
as
a
possible
contributor
to
menaquinone
synthesis.
Menaquinone
synthesis
requires
isoprenoids,
which
are
likely
synthesized
by
the
enzyme
encoded
by
Rv2173.
Upon
sequence
analysis,
it
was
confirmed
that
the
gene
contains
a
region
of
conserved
aspartate
residues
characteris:c
of
prenyltransferases,
which
catalyze
forma:on
of
linear
isoprenoids
of
varying
lengths.
The
crystal
structure
of
Rv2173
indicates
that
the
enzyme
ac:ve
site
would
catalyze
produc:on
of
short
chain
isoprenoids
that
could
possibly
be
used
in
the
synthesis
of
menaquinone.
This
research
u:lizes
molecular
modeling
to
direct
the
func:onal
characteriza:on
of
the
enzyme
and
then
qualita:vely
inves:gate
the
products
of
Rv2173
based
on
constructed
models.
Synthesis
of
mul:ple
chain
length
isoprenoids
provides
the
cell
with
a
stock
to
be
used
for
menaquinone
synthesis
among
other
important
uses.
Menaquinone
is
essen:al
to
the
energy
produc:on
of
the
cell,
by
ac:ng
as
an
electron
shuPle
during
the
oxida:ve
phosphoryla:on
pathway3.
Without
menaquinone,
a
cell
would
be
deficient
in
energy
and
enter
apoptosis2.
Understanding
the
mechanism
of
how
prenyltransferases
work
is
essen:al
to
interpret
how
models
can
be
used
to
direct
characteriza:on
of
an
enzyme.
Ac:ve
sites
of
prenyltransferases
are
similar
and
contain
an
aspartate
rich
region.
Crystal
structures
show
these
aspartate
residues
can
chelate
with
Mg2+
ions
(Figure
1),
which
can
also
chelate
with
Prepara&on
of
enzyme
from
Rv2173
DNA:
The
Rv2173
gene
was
previously
cloned
from
M.
tuberculosis
CDC1551
genomic
DNA
and
placed
into
a
Gateway
pDEST17
expression
vector
(Life
Technologies,
Carlsbad,
CA).
2
μL
of
Rv2173/17
DNA
was
added
to
25μL
of
C41
competent
Escherichia
coli
cells
and
incubated
on
ice
for
30
minutes.
Cells
were
heat
shocked
at
42
C
for
30
seconds,
250
μL
of
NYZ
media
was
added,
and
the
cells
were
incubated
at
37
C
for
1
hour.
Cells
were
plated
on
35
ug/mL
carbenicillin
plates
and
incubated
at
37
C
for
24
hours.
Single
colonies
were
grown
in
500
mL
NYZ
media
to
an
op:mal
density
of
0.6-­‐0.8
nm.
The
cells
were
then
cooled
to
16
C
and
induced
with
0.5
mM
IPTG
for
16-­‐18
hours.
Cells
were
centrifuged,
supernatant
was
removed,
and
pellets
were
stored
at
-­‐80
C.
Prenyltransferase
assay:
Transformed
cells
were
thawed
slowly
in
ice
and
suspended
in
10mL
of
lysis
buffer.
Cells
were
lysed
in
an
ice
bath
sonicator
for
15
minutes.
One
replicate
of
the
assay
was
run
with
this
unclarified
lysate;
one
was
run
using
the
supernatent
ader
centrifuga:on
of
the
unclarified
lysate,
called
the
clarified
lysate.
Both
assays
were
prepared
the
same
way
as
shown
in
the
table
below.
All
components
were
added
to
test
tubes,
equilibrated
to
37
C
in
water
bath,
and
allowed
to
incubate
for
30
minutes.
Cells
were
treated
with
110uL
of
alkaline
phosphatase
buffer,
and
10uL
of
alkaline
phosphatase
and
allowed
to
react
overnight.
This
allowed
extrac:on
of
products
in
hexanes.
HPLC
analysis:
C41
E.
coli
cells
were
transformed
the
same
as
above
and
extracted
with
methanol.
HPLC
method:
0-­‐5min,
10%
MeOH;
5-­‐10min,
20%
MeOH;
10-­‐15min,
30%
MeOH;
15-­‐20min,
40%
MeOH;
20-­‐30min,
40%
MeOH.
HPLC
analysis
indicates
that
the
Rv2173
gene
is
causing
produc:on
of
a
new
product
consistent
with
a
chain
length
of
ten
carbons
or
shorter
when
transformed
into
E.
coli
cells.
GC-­‐MS
analysis
shows
the
forma:on
of
a
product
with
a
spectrum
indica:ve
of
a
25
carbon
isoprenoid,
but
because
no
peaks
were
observed
consistent
with
a
25
carbon
chain
on
the
HPLC,
it
is
hypothesized
that
the
25
carbon
isoprenoid
being
formed
is
a
result
of
another
enzyme
u:lizing
the
larger
stock
of
short
chain
isoprenoid
being
produced
by
the
Rv2173
enzyme.
Crystal
structure
models
show
that
a
much
deeper
hydrophobic
pocket
is
observed
in
a
known
FPP
synthase
when
compared
to
the
hydrophobic
pocket
of
the
Rv2173
enzyme,
also
suppor:ve
of
the
enzyme
product
being
shorter
than
a
15
carbon
chain.
Sequence
analysis
of
the
Rv2173
enzyme
reveals
numerous
similari:es
of
key
residues
indica:ve
of
a
short-­‐chain
prenyltransferase.
The
first
aspartate
rich
region
contains
3
residues
facing
the
inside
of
the
ac:ve
site,
directly
across
from
the
second
aspartate
rich
region.
This
second
region
is
interes:ng
because
instead
of
the
highly
conserved
DDxxD
sequence,
it
contains
a
DDxxG
sequence.
However,
the
close
proximity
of
two
more
aspartates,
Asp254
and
Asp255,
on
the
overhanging
helix
may
subs:tute
for
the
lacking
third
aspartate
in
the
second
conserved
region.
The
exact
reason
for
this
change
is
unknown,
but
it
has
been
hypothesized
by
Liang
et
al.
that
the
helix
in
which
Asp
254
and
Asp255
are
a
part
of
is
a
flexible
“cap”
that
encloses
the
ac:ve
site
upon
substrate
binding,
bringing
the
substrates
into
a
closer,
more
compact,
orienta:on
that
helps
the
reac:on
proceed.
The
importance
of
the
amino
acid
residues
four
and
five
residues
upstream
from
the
first
conserved
aspartate
was
demonstrated
by
Tarshis
et
al.
to
be
essen:al
in
determining
chain
length
in
a
known
farnesyl
pyrophosphate
synthase
because
they
acts
as
the
floor
of
the
hydrophobic
pocket,
inhibi:ng
further
chain
elonga:on.
In
the
case
of
the
Rv2173
enzyme,
the
large,
hydrophobic
phenylalanines
are
replaced
with
a
tryptophan
and
an
alanine,
which
possibly
could
allow
chain
elonga:on
to
propagate
further.
A
second
tryptophan,
Trp159,
is
located
on
a
separate
helix
but
extends
in
an
orienta:on
such
that
it
is
about
5.3
A
below
the
first
tryptophan.
This
extra
5.3
A
could
poten:ally
accommodate
the
length
of
another
5
carbon
chain,
making
the
product
GGPP.
Tarshish
et
al.
found
that
muta:ons
to
F112
and
113
resulted
in
products
that
were
consistent
with
a
25
carbon
chain.
GC-­‐MS
anaylsis
reveals
a
spectrum
that
also
correlates
to
a
25
carbon
product
in
GGPP
reac:ons.
A
specific
m/z
peak
is
not
observed,
but
a
fragment
peak
at
m/z
of
341.0
corresponds
with
the
molecular
weight
consistent
with
the
loss
of
water
from
GGOH.
The
only
2
assay
combina:ons
that
resulted
in
the
forma:on
of
this
peak
were
GGPP+IPP
and
GGPP+GGPP
which
would
be
congruent
with
a
25
carbon
prenyltransferase.
E.
coli
naturally
produces
small
amounts
of
IPP,
which
explains
why
the
25
carbon
peak
could
be
seen
in
the
GGPP+GGPP
reac:on.
However,
HPLC
analysis
revealed
the
forma:on
of
a
strong
peak
in
Rv2173-­‐transformed
cells
that
is
similar
to
a
10
carbon
chain
GOH
peak,
and
no
indica:on
of
any
long
25
carbon
chains.
A
model
of
the
hydrophobic
surface
in
the
ac:ve
site
showed
that
the
Rv2173
enzyme
had
a
significantly
more
shallow
pocket
than
an
FPP
synthase,
suppor:ng
that
the
Rv2173
enzyme
may
be
a
shorter
chain
synthase
such
as
GPP
synthase.
The
peaks
observed
on
the
GC-­‐MS
could
be
due
to
the
ac:vity
of
the
Rv2173
enzyme
supplying
large
amount
of
substrate
for
another
enzyme
to
synthesize
the
25
carbon
chain.
Substrate
1
Substrate
1
(mL)
Substrate
2
Substrate
2
(mL)
Lysate
(mL)
water
(mL)
Final
vol
(mL)
IPP
0.0149
DMAPP
0.0149
0.1
0.870
1
IPP
0.0149
GPP
0.0183
0.1
0.867
1
IPP
0.0149
FPP
0.0191
0.1
0.866
1
IPP
0.0149
GGPP
0.0225
0.1
0.863
1
DMAPP
0.0149
DMAPP
0.0149
0.1
0.870
1
GPP
0.0183
GPP
0.0183
0.1
0.863
1
FPP
0.0191
FPP
0.0191
0.1
0.862
1
GGPP
0.0225
GGPP
0.0225
0.1
0.855
1
NONE
0.0000
NONE
0.0000
0.1
0.900
1
GC-­‐MS
Analysis
of
Assay
Products
The
ac:ve
site
modeled
on
the
led
shows
a
tryptophan
(T79)
residue
that
is
similar
to
a
large,
conserved,
hydrophobic
amino
acid
among
other
prenytransferases
that
acts
as
a
key
element
in
chain
elonga:on
size.
The
ac:ve
site
modeled
on
the
right
shows
another
tryptophan
(T159)
residue
unique
to
the
enzyme
encoded
for
by
Rv2173
that
is
in
the
same
posi:oning,
but
is
extended
about
5.3
angstroms
further
towards
the
boPom
of
the
enzyme.
Models
of
AcCve
Site
Figure
1
the
nega:vely
charged
phosphates
of
isoprenoids6,7.
These
interac:ons
are
key
to
the
mechanis:c
steps
described
by
Burke
et
al.
that
catalyze
forma:on
of
short-­‐chain
isoprenoids.
Knowing
the
subtrate’s
orienta:on
in
the
ac:ve
site
allows
for
iden:fica:on
of
a
hydrophobic
pocket
in
which
the
tail
of
the
substrate
is
fed
into
a
hydrophobic
pocket.
The
depth
of
this
pocket
is
hypothesized
to
contribute
to
controlling
chain
length4,5.
Thus,
bioinforma:c
analysis
provide
important
hints
that
aid
in
the
design
of
experiments
to
gain
solid
evidence.
1st
Conserved
DDxxD
mo:f
Conserved
KT
mo:f
2nd
Conserved
DDxxD
mo:f
Chain
length
determining
residues
Comparison
of
the
Rv2173
amino
acid
sequence
with
sequences
of
several
other
known
enzymes
in
the
prenyltransferase
family
revealed
two
conserved
aspartate
rich
regions.
Also,
analysis
showed
conserva:on
of
Lys194,
which
is
thought
to
aid
the
subs:tu:on
reac:on
in
the
ac:ve
site
because
of
its
close
proximity
to
the
allylic
nucleophile
and
ca:onic
electrophile.
Contribu:onal
aspartates
References
(1)
Burke,
C.
C.;
Wildung,
M.
R.;
Croteau,
R.
Proc.
Natl.
Acad.
Sci.
1999,
96,
13062–
13067.
(2)
Schulbach,
M.
C.;
Brennan,
P.
J.;
Crick,
D.
C.
J.
Biol.
Chem.
2000.
(3)
Dhiman,
R.
K.;
Mahapatra,
S.
et
al.
Mol.
Microbiol.
2009,
72,
85–97.
(4)
Noike,
M.;
Ambo,
T.
et
al.
Biochem.
Biophys.
Res.
Commun.
2008,
377,
17–22.
(5)
Tarshis,
L.
C.;
Proteau,
P.
J.;
Kellogg,
B.
A.;
Saccheqni,
J.
C.;
Poulter,
C.
D.
Proc.
Natl.
Acad.
Sci.
1996,
93,
15018–15023.
(6)
Liang,
P.-­‐H.;
Ko,
T.-­‐P.;
Wang,
A.
H.-­‐J.
Eur.
J.
Biochem.
2002,
269,
3339–3354.
(7)
Wang,
W.;
Dong,
C.
et
al.
J.
Mol.
Biol.
2008,
381,
129–140.
HPLC
Analysis
of
Transformed
E.
coli
C41
control
cells
Rv2173
cells
GOH
standard
GGPP+IPP
GGPP+GGPP
GC-­‐MS
analysis
revealed
the
produc:on
of
a
new
product
in
Rv2173
transformed
lysates
in
the
reac:ons
of
GGPP+IPP
and
GGPP+GGPP.
The
peaks’
mass
spectra
of
these
newly
formed
peaks
represent
a
25
carbon
chain
with
an
important
fragmenta:on
peak
with
an
m/z
of
341.
HPLC
analysis
revealed
one
substan:al
product
formed
in
Rv2173
transformed
cells
when
compared
to
the
background
of
the
normal
C41
cells.
The
reten:on
:me
of
this
peak
is
indica:ve
of
a
short
chain
product,
so
a
geraniol
standard
was
run
to
try
to
iden:fy
the
peak.
Replicates
confirmed
that
the
new
peak
does
not
align
with
geraniol,
but
is
unique
to
the
Rv2173
cells.
Sequence
Analysis
Materials & Methods