Kealases – New engineered enzymes for the stereo selective synthesis of chiral

-hydroxyaldehydes Marco G. Casteleijn1#, Markus Alahuhta2, Matti Vaismaa3, Ritva Juvani3, Marja Lajunen3, Jouni Pursiainen3, Rik K. Wierenga2, Peter Neubauer1.1) University of Oulu (Finland), Bioprocess Engineering Laboratory, Dept. of Process and Environm. Engineering; 2) University of Oulu (Finland), Dept. of Biochemistry; 3) University of Oulu (Finland), Dept. of Chemistry. # corresponding author: marco.casteleijn@oulu.fi

Triose phosphate isomerase (TIM) is a glycolytic enzyme with very high substrate specificity. The wild type enzyme is a dimer, and each subunit has the classical TIM-barrel fold. By modifying the loops involved in subunit interactions in the dimer, a monomeric enzyme (monoTIM) has been created and characterized. It remains active, although less so than the wild-type. Current studies include attempts to change the substrate specificity of monoTIM. These protein design studies have now resulted in a new variant (ml8bTIM) [5] with a shorter loop-8 and a much more extensive binding pocket which has no affinity for the original substrate.

Our studies are focusing on understanding the catalytic properties of the Wild Type (WT-TIM) [2, 3] and applying this knowledge to our newly engineered enzymes (fig 1).

Introduction

1. Arkin M.R., Randal M, DeLano W.L., Hyde J., Luong T.N., Oslob J.D., Raphael D.R., Taylor L., Wang J., McDowell R.S., Wells J.A., Braisted A.C. (2003) Binding of small molecules to an adaptive protein-protein interface. Proc Natl Acad Sci U S A. 100, 1603-1608 (important reference concerning the capture approach of weak binders)

2. Kursula, I., and Wierenga, R.K. (2003) Crystal structure of triosephosphate isomerase complexed with 2-phospho-glycolate at 0.83Å resolution. J. Biol. Chem 278, 9544-9551.(about the importance of P168A)3. Kursula, I., Salin, M., Sun, J., Norledge, B.V., Haapalainen, A.M., Sampson, N.S., and Wierenga, R.K. (2004) Understan-ding protein lids: structural analysis of active hinge mutants in

triosephosphate isomerase. PEDS (submitted). (about importance of A178L)4. Lambeir, A.M., Opperdoes F.R. , Wierenga, R.W. (1987) Kinetic properties of trio-sephosphate Isomerase from Trypanosoma brucei brucei. Eur. J. Biochem., 168, 69-74. 5. Norledge, B.V., Lambeir, A.M., Abagyan, R.A., Rottmann, A., Fernandez, A. M., Filimonov, V.V., Peter, M.G., Wierenga, R.K. 2001. Modelling, mutagenesis and structural studies on the fully

conserved phosphate binding loop (loop-8) of triosephosphate isomerase: towards a new substrate specificity. Proteins 42, 383-389.6. Plaut, B, Knowles, J.R. (1972) pH-dependence of the Triose Phosphate Isomerase Reaction. Biochem. J., 129, 311-320.7. Schliebs, W. Thanki, N., Eitja, R., Wierenga, R.W. (1996) Active site properties of monomeric triosephosphate isomerase (monoTIM) as deduced from mutational and structural studies. Prot. Science 5, 229-239.8. Sun, J., Sampson, N. (1999) Understanding protein lids: kinetic analysis of active hinge mutants in Triosephosphate Isomerase. Biochemistry, 38, 11474-11481. 9. Thanki, N., Zeelen, J.Ph., Mathieu, M, Jaenicke, R, Abagyan, R.A.A., Wierenga, R.K., Schliebs, W. (1997) Protein engineering with monomeric triophosphate isomerase (monoTIM): the modelling and structure verification of a

seven-residue loop. Protein Engin. 10(2), 159-167.

References

Measured (based on reference [6, 8])TIM variant Km [mM]

GAP

Kcat [sec –1]

GAP

K’m [mM]

DHAP (10mM AsO4)

Kcat [sec –1]

DHAP(10mM AsO4)

WT Tb TIM 0.39 1374 3.6 (Km = 1.3)2) 507

WT P168A 0.017 28 1.561) 41)

1Ml1 TIM 5.6 1.5 15.6 0.0989

Ml1 P168A ? ? 4.3 0.0033 

Literature [4,7,9]Km [mM]

GAP

Kcat [sec –1]

GAP

Km [mM]

DHAP

Kcat [sec –1]

DHAP

WT Tb TIM 0.25 6167 1.2 1083

1Ml1 TIM 5.7 5 N.D. N.D.

MonoTIM 4.1 5.17 12.2 0.21) Optimal range of substrate concentration not yet established.2) Ki value of arsenate for WT Tb TIM is 4.6 mM [4]N.D. : Not Determined

Table 1Preliminary results of the activity assays regarding the P168A mutation in WT Tb-TIM and Ml1TIM. D-Glyceraldehyde-3-phosphate (GAP) and Dihydroxyacetone phosphate (DHAP) are the natural substrates for WT TIM.

Recent Wild Type studiesWild Type TIM is well understood, and many publications can be found on its catalytic and structural properties. However, some aspects are still poorly understood.

We are currently investigating the loop-6/loop-7 properties of wild type TIM (Wt TIM). Observations in a liganded structure of Leishmania TIM [2] showed P168A in a planar state which is a strained conformation, which could facilitate loop opening. These investigations, and of others [3], are of importance for the engineering part of the project, since only in the closed form the active site is competent for catalysis. This knowledge can than be applied to engineer more active variants of poorly binding (new) ligands.

We found that the catalytic properties of the Pro168 in Wt TIM and in Ml1 TIM are altered (see table 1). The affinity for both substrates has increased and the Kcat (turnover number) has decreased a 100 fold.

Engineering new enzymesOur focus lies on new monoTIM enzymes with an altered binding site, but unaltered catalytic properties (fig. 1). This knowledge is based on structures (fig 2.), and obtained by directed evolution and crystallography. Capturing new ligands to new binding pockets of engineered enzymes is challenging, but not impossible [1].

Our approach uses covalently bound suicide inhibitors, and MALDI-TOF mass spectrometry (MALTI-TOF-MS) and NMR to find (weak) binding ligands. Based on modelling and rational design we are currently investigating several potential ligands that may bind the newly engineered binding pocket. Known suicide inhibitors for the Wt TIM have proven to work in pilot experiments to set up these techniques.

Conclusions► WT TIM and Mono TIM are good scaffolds for enzyme engineering

► Proline 168 in loop 6 affects binding of substrate and dissociation of product

► Capturing new ligands can be done with the use of covalently bound suicide inhibitors

Figure 2Connolly surface picture of ml8bTIM V233A-I245A. The active site is at the yellow oval, the new binding pocket is at the green oval. The side chains of Lys13 (catalytic residue) and Lys239 (rim residue) are shown in transparent mode. Regions of negative electrostatic potential are colored red, and positive electrostatic potential are colored blue. In this structure loop-4 is in the shape of the active site in its active conformation will be different. This picture was made with ICM (www.molsoft.com).

L

Overview of the generated variants. ml8bTIM has been created in previous studies [5]. All mutants downstream from ml8bTIM have been created within the kealase project terminating in near future. The Wt TIM concerns trypanosomal TIM. Structural information is available from the variants labeled with *.

Figure 1