Towards new enzymes:

From Triosephosphate Isomerase to Kealases

Peter NeubauerUniversity of Oulu31.08.2006 (PDB2006)

Peter Neubauer

Introduction

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Norledge et al; Proteins: structure, function and genetics (2001)

• 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 inter-conversion of dihydroxy acetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (DGAP)

Loop6

Loop7

Peter Neubauer

Introduction

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Catalytic residues:Lysine 13Histidine 95Glutamate 167Ligand is represented as green densityLoop-6 closes upon ligand binding

Loop6

Loop7

Catalytic residues:Lysine 13Histidine 95Glutamate 167Ligand is represented as green densityLoop-6 closes upon ligand binding Reaction mechanism as proposed by Kursula et al. Eur.

J. Biochem (2001)

Peter Neubauer

Dimer to monomer

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Borchert et al (1993; 1994)

Thanki et al. (1997)

e.g. Wierenga (2001)

ml8b TIMNorledge etl. (2001)

• 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.

Peter Neubauer

Monomeric TIM activity

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Mode of binding of 2PG in wild type and monomeric TIM (ml1 TIM).

Lys 13

2PG

Glu 167

His 95

Loop-6

• 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

Peter Neubauer

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Tools for new enzymes

What?

• Design of new artificial enzymes

• Design and organic synthesis of new substrates

• Creating active enzymes

ChemistryWild type studies

•iterative process

(Random) mutagenesis

Selection of best mutants

Screen for activity

Pool of enzymes

TIM (variants)[inactive]

KealasesInput Output

Mass Spectrometry

X-Ray crystallography

NMR

Multidisciplinary approach of the biocatalyst consortium

Importance of chirally active aldehydes

Our Start: Our goal:

Wild type studies

Peter Neubauer

Recent wild type studies

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• 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:

► 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?

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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) • 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.

Peter Neubauer

Tailored ligands

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The Concept

• Catalytic head group stays the same

• Achor part contributes most to the binding affinity

• The anchor is optimized for maximum affinity, but can be cleavable

A possible Substrate

A Possible Inhibitor

(cleavable) Anchor Catalytic head group

Original binding site

Catalytic - S i t eExtended binding pocket

Enzyme surface

Peter Neubauer

A-TIM

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• 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

Peter Neubauer

A-TIM

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• 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

Saab-Rincon et al. Protein eng. (2001):

Poster: Non-natural enzymes: Directed evolution of a monomeric triosephosphate isomerase variant – a case study

Peter Neubauer

Recent findings

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• 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

Peter Neubauer

Recent findings

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Catalytic end

Anchor

OH OC

CC

OP

O

OH O

A R

New binding

pocket

Loop 7

Citric acid binding in

A-TIM

Loop 8

O O

C

OO

CC

CC

OO O

CV

A

R

Transition state analogue wild type

Substrate wild type

A R

OHO

OC

CO

P

O

OH

• 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

O

O

OH

OH

OH

O

OHS

O

CH3

O

O

O

OHOH

O OH

OH

Citric acid

Malic acid

MW-1

Mare, de La et al. Biochem J (1972)

Peter Neubauer

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• New A-TIM enzymes towards the transformation of new modified nucleosides

• 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):

WOZ

O

H 2

C

O

O H

H

O H

H

Y X

WOZ

O

H 2

C

OH

O

H

OH

Y X

O

H

O

H

H 2

C

H

O

OHX

Y

WZ

O

1

23

4

5

O

O H

OH

HY

X

O

WZ

O

-/-anomersO

OH

H

OHY

X

O

WZ

O

ribo

arabino

AV-TIMtransformation =

= Anotherpresentation

-/-anomers

Ribo or Arabinodepending on the stereochemistry of the C2=O group reduction

R et roa nalysi s; gene ra l Sc hem eMikhai lopulo I.A .

1 2

3

4 5

Spontaneous cyclization

O

O

OH

OH

OH

O

OHS

O

CH3

O

O

O

OHOH

O OH

OH

A possible application for A-TIM

Citric acid

Malic acid

MW-1

Peter Neubauer

Acknowledgments

Faculty of Science

Department of Biochemistry

Prof. Rik WierengaMarkus Alahuhta

Mikko SalinVille Ratas

Department of Chemistry

Prof. Jouni PursiainenRitva Juvani

Prof. Marja LajunenMatti Vaismaa

Dr. Sampo MattilaNanna Alho

Faculty of Technology

Bioprocess Engineering

Prof. Peter NeubauerDr. Mari YlianttilaMarco 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/

Collaborators

University of Kuopio

Department of Pharmaceutical Chemistry

Prof. Seppo Lapinjoki

Technical University of Helsinki

Dr. Petri Pihko

University of Antwerp, Belgium

Prof. Koen AugustynsProf. Anne-Marie Lambeir

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