Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties.


Abstract

A monomeric variant of triosephosphate isomerase (TIM) with a new engineered binding groove has been characterized further. In this variant (ml8bTIM), the phosphate binding loop had been shortened, causing the binding site to be much more extended. Here, it is reported that in the V233A variant of ml8bTIM (A-TIM), three important properties of the wild-type TIM active site have been restored: (i) the structural properties of loop-7, (ii) the binding site of a conserved water molecule between loop-7 and loop-8 and (iii) the binding site of the phosphate moiety. It is shown that the active site of A-TIM can bind TIM transition state analogs and suicide inhibitors competently. It is found that the active site geometry of the A-TIM complexes is less compact and more solvent exposed, as in wild-type TIM. This correlates with the observation that the catalytic efficiency of A-TIM for interconverting the TIM substrates is too low to be detected. It is also shown that the A-TIM active site can bind compounds which do not bind to wild-type TIM and which are completely different from the normal TIM substrate, like a citrate molecule. The binding of this citrate molecule is stabilized by hydrogen bonding interactions with the new binding groove. Study holds ProTherm entries: 24486, 24487 Extra Details: citrate; monomeric TIM; protein design; suicide inhibitor; transition state analog

Submission Details

ID: sEUbBhv6

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:55 p.m.

Version: 1

Publication Details
Alahuhta M;Salin M;Casteleijn MG;Kemmer C;El-Sayed I;Augustyns K;Neubauer P;Wierenga RK,Protein Eng. Des. Sel. (2008) Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties. PMID:18239072
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2J27 2007-01-02 1.15 The functional role of the conserved active site proline of triosephosphate isomerase
2V2H 2008-02-19 1.18 The A178L mutation in the C-terminal hinge of the flexible loop-6 of triosephosphate isomerase (TIM) induces a more closed conformation of this hinge region in dimeric and monomeric TIM
4PC8 2015-04-22 1.55 Structure-based protein engineering efforts on the scaffold of a monomeric triosephosphate isomerase yielding a sugar isomerase
2VEK 2008-02-19 1.6 Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
2WSR 2009-09-15 1.65 MONOTIM MUTANT RMM0-1, MONOMERIC FORM.
1KV5 2002-03-29 1.65 Structure of Trypanosoma brucei brucei TIM with the salt-bridge-forming residue Arg191 mutated to Ser
3Q37 2011-09-14 1.65 Identification of Amino Acids that Account for Long-Range Interactions in Proteins Using Two Triosephosphate Isomerases from Pathogenic Trypanosomes.
5I3F 2016-05-18 1.72 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
1TPF 1994-05-31 1.8 COMPARISON OF THE STRUCTURES AND THE CRYSTAL CONTACTS OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE IN FOUR DIFFERENT CRYSTAL FORMS
5I3J 2016-05-18 1.8 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
5TIM 1992-10-15 1.83 REFINED 1.83 ANGSTROMS STRUCTURE OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE, CRYSTALLIZED IN THE PRESENCE OF 2.4 M-AMMONIUM SULPHATE. A COMPARISON WITH THE STRUCTURE OF THE TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE-GLYCEROL-3-PHOSPHATE COMPLEX
2X1T 2010-01-26 1.83 Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase
2X1U 2010-01-26 1.84 Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase
2VEI 2008-02-19 1.89 Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
2V2C 2008-02-19 1.89 The A178L mutation in the C-terminal hinge of the flexible loop-6 of triosephosphate isomerase (TIM) induces a more closed conformation of this hinge region in dimeric and monomeric TIM
2X2G 2010-01-26 1.9 CRYSTALLOGRAPHIC BINDING STUDIES WITH AN ENGINEERED MONOMERIC VARIANT OF TRIOSEPHOSPHATE ISOMERASE
2X1S 2010-01-26 1.93 Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase
5I3G 2016-05-18 1.96 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
2X1R 2010-01-26 1.98 Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase
2VEN 2008-02-19 2.0 Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
2WSQ 2009-09-15 2.1 MonoTIM mutant RMM0-1, dimeric form.
1TPD 1994-05-31 2.1 STRUCTURES OF THE 'OPEN' AND 'CLOSED' STATE OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE, AS OBSERVED IN A NEW CRYSTAL FORM: IMPLICATIONS FOR THE REACTION MECHANISM
1TPE 1994-05-31 2.1 COMPARISON OF THE STRUCTURES AND THE CRYSTAL CONTACTS OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE IN FOUR DIFFERENT CRYSTAL FORMS
2J24 2007-01-02 2.1 The functional role of the conserved active site proline of triosephosphate isomerase
2X16 2009-12-29 2.13 Crystallographic binding studies with an engineered monomeric variant of triosephosphate isomerase
5I3I 2016-05-18 2.2 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
6TIM 1992-10-15 2.2 THE ADAPTABILITY OF THE ACTIVE SITE OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE AS OBSERVED IN THE CRYSTAL STRUCTURES OF THREE DIFFERENT COMPLEXES
2VEM 2008-02-19 2.2 Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
1IIH 2001-05-11 2.2 STRUCTURE OF TRYPANOSOMA BRUCEI BRUCEI TRIOSEPHOSPHATE ISOMERASE COMPLEXED WITH 3-PHOSPHOGLYCERATE
2V0T 2008-02-19 2.2 The A178L mutation in the C-terminal hinge of the flexible loop-6 of triosephosphate isomerase (TIM) induces a more closed conformation of this hinge region in dimeric and monomeric TIM
5I3K 2016-05-18 2.21 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
5I3H 2016-05-18 2.25 Structure-Function Studies on Role of Hydrophobic Clamping of a Basic Glutamate in Catalysis by Triosephosphate Isomerase
2Y70 2011-12-07 2.3 CRYSTALLOGRAPHIC STRUCTURE OF GM23, MUTANT G89D, AN EXAMPLE OF CATALYTIC MIGRATION FROM TIM TO THIAMIN PHOSPHATE SYNTHASE.
4JEQ 2013-10-02 2.3 Different Contribution of Conserved Amino Acids to the Global Properties of Homologous Enzymes
2VEL 2008-02-19 2.3 Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
2V2D 2008-02-19 2.3 The A178L mutation in the C-terminal hinge of the flexible loop-6 of triosephosphate isomerase (TIM) induces a more closed conformation of this hinge region in dimeric and monomeric TIM
1AG1 1997-06-16 2.36 MONOHYDROGEN PHOSPHATE BINDING TO TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE
1TTJ 1995-09-15 2.4 THREE NEW CRYSTAL STRUCTURES OF POINT MUTATION VARIANTS OF MONOTIM: CONFORMATIONAL FLEXIBILITY OF LOOP-1,LOOP-4 AND LOOP-8
4TIM 1992-10-15 2.4 CRYSTALLOGRAPHIC AND MOLECULAR MODELING STUDIES ON TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE: A CRITICAL ASSESSMENT OF THE PREDICTED AND OBSERVED STRUCTURES OF THE COMPLEX WITH 2-PHOSPHOGLYCERATE
1MSS 1994-09-30 2.4 LARGE SCALE STRUCTURAL REARRANGEMENTS OF THE FRONT LOOPS IN MONOMERISED TRIOSEPHOSPHATE ISOMERASE, AS DEDUCED FROM THE COMPARISON OF THE STRUCTURAL PROPERTIES OF MONOTIM AND ITS POINT MUTATION VARIANT MONOSS
2V5L 2007-07-31 2.4 Structures of the Open and Closed State of Trypanosomal Triosephosphate Isomerase: as Observed in a New Crystal Form: Implications for the Reaction Mechanism
1TRI 1994-07-31 2.4 THE CRYSTAL STRUCTURE OF AN ENGINEERED MONOMERIC TRIOSEPHOSPHATE ISOMERASE, MONOTIM: THE CORRECT MODELLING OF AN EIGHT-RESIDUE LOOP
1TTI 1995-10-15 2.4 THREE NEW CRYSTAL STRUCTURES OF POINT MUTATION VARIANTS OF MONOTIM: CONFORMATIONAL FLEXIBILITY OF LOOP-1,LOOP-4 AND LOOP-8
1TRD 1993-10-31 2.5 THE INFLUENCE OF CRYSTAL PACKING ON CRYSTALLOGRAPHIC BINDING STUDIES: A NEW CRYSTAL FORM OF TRYPANOSOMAL TIM
2Y6Z 2011-12-07 2.6 Crystallographic structure of GM23 an example of Catalytic migration from TIM to thiamin phosphate synthase.
1IIG 2001-05-11 2.6 STRUCTURE OF TRYPANOSOMA BRUCEI BRUCEI TRIOSEPHOSPHATE ISOMERASE COMPLEXED WITH 3-PHOSPHONOPROPIONATE
1ML1 1997-03-12 2.6 PROTEIN ENGINEERING WITH MONOMERIC TRIOSEPHOSPHATE ISOMERASE: THE MODELLING AND STRUCTURE VERIFICATION OF A SEVEN RESIDUE LOOP
1DKW 2000-11-03 2.65 CRYSTAL STRUCTURE OF TRIOSE-PHOSPHATE ISOMERASE WITH MODIFIED SUBSTRATE BINDING SITE
4PCF 2015-04-22 2.71 Structure-based protein engineering of a monomeric triosephosphate isomerase towards changing substrate specificity
3TIM 1991-10-15 2.8 THE CRYSTAL STRUCTURE OF THE 'OPEN' AND THE 'CLOSED' CONFORMATION OF THE FLEXIBLE LOOP OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE
1TSI 1994-01-31 2.84 STRUCTURE OF THE COMPLEX BETWEEN TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE AND N-HYDROXY-4-PHOSPHONO-BUTANAMIDE: BINDING AT THE ACTIVE SITE DESPITE AN 'OPEN' FLEXIBLE LOOP

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Triosephosphate isomerase, glycosomal P04789 TPIS_TRYBB