Transcript A-TIM - University of Helsinki

Towards new enzymes:
From Triosephosphate Isomerase to Kealases
Peter Neubauer
University of Oulu
31.08.2006 (PDB2006)
Introduction
BPEL
Peter Neubauer
• Enzyme of the glycolysis
pathway
• Wild type enzyme is a
dimer that consists of two
identical ()8-fold subunits
with the size of 250
residues
• Loop-1 and 4 (subunit 1)
and loop-3 (subunit 2) form
the dimer interfase
• Highly specific binding
pocket
(phosphate)
is
formed by loops 6, 7 and 8
• Catalyses
the
interconversion of dihydroxy
acetone phosphate (DHAP)
and
D-glyceraldehyde
3-phosphate (DGAP)
Norledge et al; Proteins: structure, function and genetics (2001)
Introduction
Loop6
Loop7
BPEL
Catalytic residues:
Lysine 13
Histidine 95
Glutamate 167
Peter Neubauer
Ligand is represented as green density
Loop-6 closes upon ligand binding
Reaction mechanism as proposed by Kursula et al. Eur.
J. Biochem (2001)
Dimer to monomer
BPEL
• Dimeric
stabilization
is
important for the catalytic
machinery in wild type TIM
• MonoTIM and ml1 TIM are
active
enzymes
and
catalyze the substrates in a
similar fashion
• The
affinity
of
the
monomeric TIMs for the
transition state analogues is
lower
• The turnover number for the
monomeric TIMs is lower
• Ml8b TIM does no longer
convert
any
known
substrates. However the
active site remains the
same.
e.g. Wierenga
(2001)
Borchert et al
(1993; 1994)
Thanki et al.
(1997)
ml8b TIM
Norledge etl. (2001)
Peter Neubauer
Monomeric TIM activity
BPEL
Peter Neubauer
• The turnover number is
1000 lower in monomeric
TIM compared to wild type
This is correlated with a
different environment, such
as an increased solvent
accessibility
• The reduction in affinity for
2PG is less than for the
phosphate moiety
• Lysine 13 and Histidine 95
show more conformational
flexibility compared to wild
type. Monomeric TIMs are
more “floppy“
• The more accessible active
site is an interesting starting
point
for
enzyme
engineering
Loop-6
Glu 167
2PG
His 95
Lys 13
Mode of binding of 2PG in wild type
and monomeric TIM (ml1 TIM).
Tools for new enzymes
Importance of
chirally active
aldehydes
What?
• Design of new artificial
enzymes
• Design and organic synthesis
of new substrates
• Creating active enzymes
Multidisciplinary approach of the biocatalyst consortium
Wild type
studies
BPEL
NMR
crystallography
Pool of
enzymes
Our Start:
TIM
(variants)
[inactive]
Peter Neubauer
X-Ray
Mass
Spectrometry
Screen for
activity
Input
Our goal:
Output
(Random)
mutagenesis
Selection of
best mutants
•iterative process
Chemistry
Kealases
Recent wild type studies
• Investigations on the properties of the conserved active site
proline identified a molecular switch to bring the enzyme in a
competent state for catalysis upon ligand binding.
• Enzymology studies on P168A mutant versus wild type show a
dramatic drop in Kcat/Km
• The affinity for 2PG is reduced
• The mode of binding of 2PG in wild type differs from P168A
mutant.
• Conclusions:
BPEL
►
Proline 168 is needed for conformational strain around the
catalytic glutamate. This strain is transferred to the
catalytic glutamate.
►
Ligand binding triggers the conformational switch of the
catalytic machinery needed for catalysis.
Poster:
”The functional role of the conserved active site proline of
triosephosphate isomerase”
Peter Neubauer
Why monomeric TIM?
Monomeric TIM is a very
suitable protein for
biocatalysis:
• small size (suitable for
NMR; easy to crystallize): so
far all constructs of monomeric TIMs
could be crystallised)
• easily expressed in high
amounts of soluble protein
in E.coli
• Monomeric protein has
advantages (Vanvaca et al. PNAS
2004)
BPEL
Peter Neubauer
• No cofactors needed
• In the closed conformation
the binding site is an
extended groove: ml8b
TIM (*)
• Its wild type precursor is the
extensively studied TIM.
L
Tailored ligands
The Concept
• Catalytic head group
stays the same
(cleavable) Anchor
Catalytic head group
• Achor part contributes
most to the binding
affinity
• The anchor is
optimized for
maximum affinity, but
can be cleavable
BPEL
A possible Substrate
A Possible Inhibitor
Peter Neubauer
Extended binding pocket
Original binding site
Enzyme surface
-Site
C
a
t
a
l
y
t
i
c
A-TIM
BPEL
Peter Neubauer
• Over 20 constructs based
on ml8b TIM have been
created
• Several mutations have
been investigated: pocket,
rim, active site and loop
mutants.
Many
have
structures
have
been
solved
• A variant with a deeper and
wider binding groove have
been created: A-TIM
• A-TIM is as stable as ml1
TIM in solution
• The active site is identical
to wild type TIM
Active site
Extended
groove
A-TIM
BPEL
Peter Neubauer
• Over 35 novel tailored
ligands have been created
• Binding studies with NMR,
Surface Plasma Resonance
and X-ray crystallography
have
identified
lead
compounds which can be
called “binders”
• Site directed evolution is
the next logical step to
convert
“binders“
to
substrates
• Site directed evolution has
worked
previously
to
improve activity
• If A-TIM variants converts
α-hydroxy
ketones
to
α-hydroxy aldehydes, they
can be called Kealases
Poster:
Non-natural enzymes: Directed evolution of
a monomeric triosephosphate isomerase
variant – a case study
Saab-Rincon et al. Protein eng. (2001):
Recent findings
BPEL
Peter Neubauer
• In wild type Proline 168 acts
as a molecular switch upon
ligand binding. Manuscript
is submitted
• A study on C-terminal hinge
residue A178 in wild type
and
monomeric
TIM
provides interesting details
about the “floppy“ nature of
monomeric TIM. Manuscript
is under preparation
• An
atomic
resolution
structure of wild type TIM
with PGH (0.82 Å) will give
new insights in the reaction
mechanism (data under
investigation)
1.06 Å detail of A-TIM variant
Recent findings
Substrate wild type
A
Transition state
analogue wild type
O
HO
P
O
OH
Anchor
BPEL
Peter Neubauer
O
C
C
C
O
O
Catalytic
end
HO
P
O
C
O
OH
C
V
O
C
O
O
R
A
R
A
Citric acid
binding in
A-TIM
O
C
Loop 7
C
C
ing
O
O
ind
b
t
New ocke
p
Loop 8
• In X-ray structures the three
following compounds are
seen as “binders“
• In an X-Ray structure BHAP
(bromo
hydroxyactone
phosphate;
a
suicide
inhibitor) has been found to
react in the active site
similar
to
wild
type.
This finding indicates that
A-TIM can still convert the
original substrate (Kcat is too
low to measure):
• A-TIM is an active molecule
R
C
O
C
O
O
Citric acid
OH
O
OH
HO
O
OH
O
Malic acid
OH
HO
OH
O
MW-1 H C
3
S
O
Mare, de La et al. Biochem J (1972)
O
O
OH
A possible application for A-TIM
• New A-TIM enzymes towards the
transformation of
new
modified
nucleosides
O
Y
Z
OH
X
O
AV-TIM
transformation
H2
C
W
O
Y
Z
H
O
X
H2
C
W
O
H
=
H
OH
O
1
OH
OH
2
O
H
• New result: citric acid binds to the active
site of A-TIM (structure 1.5 Å)
• Also malic acid and compound MV-1 bind
to the active site (seen in X-ray)
• Surface plasma resonance method to
screen for binders has been established
(Dr. Päivi Pirila)
• Citric acid as positive control binds in the
assay specifically
• Other compounds tested (with good
results):
O
BPEL
Citric acid
OH
O
OH
HO
O
OH
O
Malic acid
OH
HO
OH
O
MW-1 H C
3
Peter Neubauer
S
O
O
O
OH
5
W
Another
presentation
Z
O
H2
C
O
O
Y
=
H
1
4
2
3
X
H
OH
3
O
O
W
Z
W
O
O
OH
Y
-/-anomers
Spontaneous
cyclization
H
Z
O
O
OH
Y
-/-anomers
OH
arabino
Ribo or Arabino
X
4
OH
ribo
depending on the stereochemistry
of the C2 =O group reduction
X
H
5
R et roa nal ysi s; gene ra l Sc hem e
M i khai l opulo I.A .
Acknowledgments
BPEL
Faculty of Science
Collaborators
Department of Biochemistry
University of Kuopio
Prof. Rik Wierenga
Markus Alahuhta
Mikko Salin
Ville Ratas
Department of Pharmaceutical
Chemistry
Department of Chemistry
Technical University of Helsinki
Prof. Jouni Pursiainen
Ritva Juvani
Prof. Marja Lajunen
Matti Vaismaa
Dr. Sampo Mattila
Nanna Alho
Dr. Petri Pihko
Prof. Seppo Lapinjoki
University of Antwerp, Belgium
Prof. Koen Augustyns
Prof. Anne-Marie Lambeir
Faculty of Technology
Bioprocess Engineering
Prof. Peter Neubauer
Dr. Mari Ylianttila
Marco Casteleijn
Lilja Kosamo
This work has been supported by the
Academy of Finland (project 53923) and
Tekes (project Biocatnuc)
website: http://www.oulu.fi/bioprocess/
Peter Neubauer