Creating new enzymesDirected Evolution of Triosephosphate Isomerase
Mirja Krause1, Marco G. Casteleijn1, Markus Alahuhta2, Mikko Salin2, Ville Ratas2,
Rik Wierenga2, Peter Neubauer1
1
Bioprocess Engineering Laboratory, 2Biochemistry - Oulu University and Biocenter Oulu
INTRODUCTION
Triosephosphate isomerase (TIM) is a
glycolytic enzyme with very high substrate
specificity (fig 1). The wild type enzyme is a
dimer, and each subunit has the classical
TIM-barrel
fold
[1].
Several
loop
modifications resulted in a monomeric
enzyme (ml1TIM) [2] which remains active,
although less so than the wild-type.
Subsequently this has resulted in a
new variant (A-TIM) with a much more
extensive binding [3, 4]. Our current studies
attempt to change the substrate specificity of
monomeric TIM.
important a single point mutation for the
generation of a competent active site is [4, 5,
6].
Ala
Loop 6
Ser
r
Loop 4
Loop 8
Ile
Figure 2. TIM-barrel fold of A-TIM (grey). Starting
points are shown in black (A178L loop 6, S96P loop
4, I245A loop 8).
RESULTS
Figure 1. Wild type TIM (Wt-TIM) has high
substrate specificity, and only catalyses the
interconversion of the α-hydroxykentone DHAP and
the α-hydroxyaldehyde D-GAP. The transition state
analogue, 2-phosphoglycolate (2-PG), can be seen on
the right.
METHODS
Directed Evolution
The substrate binding loops of ATIM were
modified by site-directed mutagenesis to
obtain different mutant enzymes as a starting
point for directed evolution (fig. 2). In
former studies it has been shown how
Loo
p7
va
a
l al
s
gl
a
Loo gln
y
ly
p8 u
s
Loo
p4
Figure 3. TIM-barrel fold of A-TIM (grey). Targeted
areas for directed evolution are located in loop 4, loop
7 and loop 8
Figure 4. Flowchart showing an overview of the ATIM library creation, resulting in
16 different libraries
Directed evolution was performed in a fully
random approach (epPCR) and a targeted
approach.
The extensive structural knowledge
in this project [1, 4, 5, 6] has been the basis for
the choice of amino acids that were targeted
through Megaprimer PCR [7].
Library creation
Our aim is to build a platform of TIM
variants (Kealases) with a widened substrate
range. Therefore we established by rational
design a library of A-TIM variants (fig. 4).
CONCLUSIONS
The created libraries are used to screen in
vivo for different ATIM mutants which can
take over the reaction originally catalyzed by
other enzymes or catalyze novel reactions.
REFERENCES
1. Kursula, I., and Wierenga, R.K. (2003). J. Biol.
Chem 278, 9544-9551
2. Thanki, N., et al, (1997). Protein Engin. 10(2), 159167
3. Norledge, B.V., et al, (2001) Proteins 42, 383-389.
4. Alahutha M, et al. (2008) PEDS Advance Access
published on Jan 31.
5. Casteleijn et al. (2006) Biochemistry
6. Alahutha et al. (2008) Acta D.
7. Wu Wu et al. (2005) Nucleic Acids Res. 33:e1107.
ACKNOWLEDGEMENTS
This work is supported by the Academy of
Finland (project 53923 & 11784)
More information:
www.oulu.fi/bioprocess/biocatalysis
[Kealases and Biocat projects]