595. Leishmania infantum Aldose Reductase: Expression with Molecular Chaperones, Purification and Kinetic Studies
Session: Poster Session: Travel Medicine/Tropical Medicine
Friday, October 30, 2009: 12:00 AM
Room: Poster Hall A
Background: Aldose reductase (EC 1.1.1.21) (1,2), a NADPH-dependent oxidoreductase catalyzing the reaction of a broad range of aldehydes, is implicated in the methylglyoxal catabolism (3), a toxic glycolysis byproduct.
Methods & Results: A candidate for LiAKR gene was identified by homology in the L. infantum genome, a trypanosomatid responsible for human leishmaniasis, showing 40% and 51% identity with yeast and human aldose reductase genes respectively. After unsuccessful attempts to obtain soluble LiAKR in E. coli expression systems, a yeast complementation strategy was adopted and enzyme activity was measured in protein extracts of a yeast null mutant for gre3 gene (coding for aldose reductase) transformed with an expression plasmid containing the L. infantum gene. Km values for methylglyoxal and NADPH and the specific activity were determined by following NADPH oxidation in presence of methylglyoxal. Being similar to the ones in L. infantum extracts the enzyme’s identity was confirmed.Soluble protein was only obtained by over-expression in engineered E. coli strains containing a chaperone system, DnaK/DnaJ/GrpE, DnaK/DnaJ/GrpE/ClpB, GroESL or DnaK/DnaJ/GrpE/ClpB/GroESL, which were coordinately co-overproduced with the recombinant protein optimizing de novo folding (4). The purified LiAKR revealed stable and kinetically active.
Conclusion: L. infantum aldose reductase expression and purification optimization will be particularly useful for further biochemical and structure-function analysis. As far as drug targeting is concerned, studying this enzyme is of utmost importance, not only to understand the methylglyoxal metabolism in this infectious organism, but also to unearth crucial differences relatively to its human counterpart.

[1]Vander Jagt, et al. (1972) Biochemistry 11,3735
[2]Vander Jagt et al. (1992) J Biol Chem 267,4364
[3]Inoue et al. (1995) Adv Microb Physiol 37,177
[4]De Marco et al. (2007) BMC Biotechnology 7,32
Lídia Barata, Graduated1, Carlos Cordeiro, PhD2, Gonçalo da Costa, PhD2, António Ferreira, PhD2, Ana Ponces Freire, PhD Professor2, Linda Schuldt, Graduated3, Marta Sousa Silva, PhD2, Ana Tomás, PhD4, Manfred Weiss, PhD3 and  L. Barata,
Fundação para a Ciência e Tecnologia, Portugal Role(s): Other, PhD fellowship source, Received: Research Grant.
European Molecular Biology Organization Role(s): Other, Short-term fellowship source, Received: Research Grant.
M. Sousa Silva,
Fundação para a Ciência e Tecnologia Role(s): Grant Investigator, Other, Post-doctoral fellowship source, Received: Research Grant.
G. da Costa, None..
L. Schuldt, None..
A. E. Ferreira, None..
A. M. Tomás, None..
M. S. Weiss, None..
A. Ponces Freire, None..
C. Cordeiro, None., (1)Faculdade de Ciencias da Universidade de Lisboa, Amadora, Portugal, (2)Faculdade de Ciencias da Universidade de Lisboa, Lisboa, Portugal, (3)European Molecular Biology Laboratory (EMBL), Hamburg, Germany, (4)Instituto de Biologia Molecular e Celular (IBMC), Porto, Portugal